1

CHARACTERlZATlON OF A MONOCLONAL ANTIBODY RAISED

TO THE HUMAN LYMPHOKINE-BLAS TOC; EN 1 C FACT{)!{

A thesis by Chungyee J. Leung

Department of Microbiology and lmmunology

McGirl University, Mpntreal, Quebec

February, 1984

A thesis submitted to the Faculty of' Gradu"ate Studies and

Research in partial fulfillment of the requirements for the

degree of Master of Science.

~ Chungyee J. Leung 1984

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Abstract

Blastogenic Factor (BF) is recovered from a 48 to 72 hour

allogene1c m1xed lymphocyte culture supernatant. It has been shown .. to restimulate cells primed in allo~enelc mixed lymphocyte cultures

independent of the original stimulator cells. In addition to

eliciting the proliferation of human cytotoxic lymphocytes, BF can

also coopera te in·a mouse thymocyte co-stimulator assay, as weIl as

sus tain both mouse.~nd human 1L-2 dependent cell lines. Ta further

characterize BF. its target cell, and the BF producing cell, we

attempted ta make a monoclonal ant~body to BF using eonventional

hybridoma techniques. A Biozzi high antibody responder mouse was

inununized with .BF and its spleen çells were fused with the Balbl c P3X20

my~loma. We presently have a monoclonal IgG l secreting clone. The

produc t of this clone (AC4-I·I-B5) can recognize human and murine IL-2.

o It can inhibit l mixed lymphocyte culture reaetions, stimulati.on by

T cell mitogens (Coneanavalin A and Phytohemagglutini~) and BF 1

stimulation of 20 MLR cells. It has also been shown ta inhibit the

, 0 --generation of, cy~olytic lymphocytes (CTLs) in a 2 MLR restimulatidn:

. The AC4-II-a5 ~gG'l i,s 'not spec.ific for a serum component or for a

lymphocyte or RBC surface membrane component. However, AC4-II-B5

lacks the potential to retain BF/IL-2 on an immunoab$orbant colu~n.

Initial ELISA and fluorescent an,tib09Y binding studies suggest that

this antibody can localize 8F/IL-2 produced in sérum free conditions,

and possibly the BF producing cells.

,----------~_._-------

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'" " ResUme'

Le Facteur Biastogenique (BF) est obtenu ri partIr du surnageant de

" '\' ,. ",

cul tures mlXtes lymphocytalres aiiogeniques apres 118 a 72 heures. Il a' ete

;" / "-- /' , demontre qu'il pouvalt restimuler des cellules ayant ete prealablement

, .. senslbillsees par une culture mIxte lymphocytalre et ceCI lndependamment de

.. la nature des celulules stlmulatnces utlllsees en premler lleu. Non seule-

,. ment le BF lndult la pro,llferatlon des lymphocyte,s cytotoXlques humalns

ffia"Î',5 encore, , ,

il peut cooperer a un essaI de co-sÙmu-latlon des thymocytes

de souriS et peut également maintenir des llgn~es cellulaires dépendantes .-

de l'IL-2 chez la sourlS comme chez l'homme. Pour mleux caracterizer le .-

BF, sa cellule cible et la cellule productrice de BF, nous avons tente de

" .-fabnquer un anticorps monoc1onal dlrige contre le BF par les methodes .-

traditionnelles d~.hybridation. Nous avons donc· immunise une sourlS de 1

BlOZZl bonne repondeuse en antico~ps et fusionne ses ce Il ul es sweniques , . . "

avec le myelome P3X20 de Balb/c. Nous possedons actuellement un clone.

secrétant une IgG, monoclonale. Le produit de ce clone (ÂC4-II-B5) peut

recon~attre l'IL-2 humaine ainsi que celle de sourlS. Il peut inhiber des

c~ltures mixtes . lymphocytaires de première intenuon, la stimulatlOn des

cellu.1es T par les "mitog~nes (concanavallne A et phytoh~magglutlf1ine) et la

, " stimulation par le BF d'une reponse secondaire dans une cultu~e mIxte

lymphocytaire. ,,. -"/",". Il a egalement ete demontre qU'il pouvait inhiber l'appar-

tion cIe lymphocytes T cyt<?toxiques (CTLs) lors d'une r~ponse II alre dans

une reaction mixte lymphocytaire. '" " L'~gGl AC4-II-B5 n'a de specificlte pour , - ..

aucun composant du serumi il n'est dlrige contre aucun composant de la .. ,

surface cellulaire des lymphocytes ni des hematies. Cependant, AC4-II-B5

... ". l , ne possede pas la propriete de retenir le BF/IL-2 sur une colonne immunoab-

.. sorbante. ,Les premiers travaux suggerent que cet anticorps peut localiser

le BF/IL-2 produit dans des conditions de~culture sans s~rum et peuta:'~tre

m~me les cellules productrices de BP.

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Table of Content~ j,

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Introduction 1\

\

• ~~~_LH_~Ié!ç.ure Review

1.

1.

1.

1.

A. The Mixed Lymphocyte Response, Historie Background

B. Hodel of the HLR

C. Ioterleukin 2 and Other Lymphokinès, Historie Aspects i

i) T cell derived soluble factors-overview

ii) IL-2, alloantigen stimulated IL-2 production

iii) IL-2, T cell mitogen stimulated IL-2 production

D. Conditions Regulating. IL-2 Pro.tion

i) The stimulus

'ii) Accessory cells and ,IL-l

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page

12

14

14

17

19

20

26

28

30

30 ;

32

iii) IL-3 induced maturation of th~ pre-IL-2 prodŒ~ cell 38

1.

iv) Drug modulation ~nd serum factor regulation of IL-2

production

E. Characteristics of the IL-2 Produeer Cell-,(,)ve~view.

i) Murine IL-2 producer cells

ii) Human IL-2 producer cells

iii) IL-2 produeing cel1 lines

Iv) IL-2 production leads to the IL-2 produeer eell death • 0

v) The human ~-2 gene

vi) Kinetics of IL-~ producçion ,

F. Characteristics of the IL"2 Responder Target Ce,Ha

,i)

11)

• Cells expressing the IL-2 réceptor

Functional biologie!l éharact~ristics of the IL-2

responder target cella

..

39

40

41

42

43

44

45

48

51 . ,

(

1.

1.

• l' 1

'1

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, lii) Characteristics of tht> 11.-2 dep('nd(>nt'cell linl'<;

G. Conditions of iL-l InteractIon ~~th tl1,'·II.-.' 1<,'\ l'plot.

~) K1net1cs of receptor expression

ii) IL-2 - IL-2 receptor interaction

.' H. Physicochemical Characteristi.rs of 0 11.-2

J., Puri ty of IL-2?

56

58

59

62

Chapter II General Hethodology of Tissue Cultures, 11.-2 P!:(~dUC~lOn

and Assays for Lymphokine Activlties,

II. A. IL-2 Production

1) Human IL-2 production

11) Murine IL-2 production

Iii) IL-2 produclns cel~ lines, the ueD 144 cell line > :

lI. B. Assays for Lymphokine Activities

i) BF/IL-2-.,ssay.a

a) t~ 20

HLR celI proliferation

b) o 2 MLR cytolytic,ac~iviti~s stimulated by BF/IL-2

11), M~rlne' co';;'stP11ator ··aasay

Iii,) IL-2 dependent and IL-:2 expanded celI Unes

Iv) IL-l 'Assay'

Cha ter III Imm

III. A. Haterlals and Methods

i) Source of antigens ,/

li) In vivo 'immunizations .(Bal~/c. (C57Bl/6 x C3H)F1)

111) In vitro immunization ~lb/C)

'-hl In v~vo 1 .... uni •• tion <,0"1 mieo)

64

64

64

6·6

67

67

67

67

69

70

71 ,

72

74

74

74

75

75

770

.-- -_ . ............-~-- ------------------------------------

(

-)-

~) Adoptive transfer of immunized rell'i (Kio(zzi mIre)

vi) AntülUdy !>CLH't 1111: cell .1S'i.1y!>

a) Plaque assay'i for immunoglobulin secreting cells

b) An tigen spec if 1< plaque f orming ce 11 asc;ay (PFC) 1

c) Ouc h terl ony type immunod i Hus ion

vii) Hybridoma fusion and cloning

a) Fusion procedures

b) Cloning

viii) Screenlng of hybridoma supernatants

a) Screening criteria for wells/clones positive for

71

7R

79

80

81 .. 81

82

83

irnmunoglobulin productions and 'anti-BF' activities 83

.i b) 'Anti-BF' biological screening assays

Ill. B. Presentation and Discussion of Results 85

i) ln vitro immunization to specifie antlgens, BSA and OA 85

a) ~ntigen specifie antibody producing cells (PFes) 87

b) IgM secreting cells generated following in vitro

immunizations 87

ii) Balb/c in vitro immunization to BF/IL-2 88

lii) Adoptive transfers of immunized cells (Blozz1 mice) 90

iV)

v)

ln vivo immunJzation, Salb/e and (C57BI/6 x C3H)F1 mice

Biozzi~ouse in vivo immunization tO,BF/lL-2

91

97

III. C. Tables and Graphs 101-123

Chapter IV the AC4-II-B5 Clone. PurificatiQn Qf the Hybridoma

, Products

IV. A. Materials and Mcthods

1) Maintenanc~ Qf the AC4~II-B5

124

124

-124

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-' ... ii) Ammonlum .,Ult,ltt' pre( ipit.1l1<l!1

Il i) ln trOodu( t ion 01 Hr" hvlH 1 Jorn .. l:d

l,V) Serum free preparatlon,> of II.C4- 1 1 -R '}

V) H3 leucine biol,lhplllng ,)1 H Cl J~(,I

vi) Physioc~l("al analy~l'" (lI .\(',.- 1 1- li', pr\H!IJ('t'i, Ol-.AE

ion exchange chromatography

vii) Physicochemical analysl~ 01 A:C4-II-H'l product'i,

Rabbit anti-mouse 19(, ImmUnO.1t 1 llllt)' ,olumns

a) Preparation of affinity column by Rlutaraldehyde

activated gels

b) Cyanogen bromide activation of gel~

v'iii) Isoelectric focusing

iX)' Radioimmunoassay

tV. B. Presentation a~d Discussion of Resuits

i) Ammonium su~fate precipitated, 20X concentrated

culture supernatant

ii) Serum free and ITS medium growth of AC4-II-B5

iii) Ascites growth of AC4-II-B5

iV) DEAE ion exchange column

v) R4bbit anti-mouse IgC immunlafflnlty column

vi) Isoelectrofocusing , 't,'

3 ., vii) H blolabelling of B5's IgC I

viii) Rad~eimmunoassay for IIGl

content

IV, C. Tables and Graphs

12')

1 2 '1

126

127 '

~

128

129

129

no

131

132

133

134

134

136

137

139

141

142

143

146-156

v.

v.

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c ..... h ... a ... pu.t"'c.J..r--!\_' _..!.A~l .,- 11-.]) fllo1oglcal Activitles

v ,\ 'Ill \'r-l 11" ilnd Mt' thods

1) ACI.-II-85 Inhibition of BF stimulated 2° fiLR cell

11) /tltl-BF ilssays. InhIbItIon of human 2° MLR-CMI. ,

Ill) Inhlbltlon of the 1° MLR

IV) Hyhrldoma 'Jupernatant' s effeot On mitogen stimulated

cells (T and B cell mitogens)

V) B5 IgG, InhibItion of the murine ~o-stimulator assay

V]) Hvbrldoma supernatant' s effect-on IL-2 expanded cells

and 11.-2 dependent cell lines

Presentation and Discussion of Results

1) gl<l!oglC."l] responses influenced by B5 t s IgGl

1') 7

l S 7

157

, ') 7

158

158

160

160

161

161

° Il) 2 MLR. cell viabilities and blast cell transformations

influenced by B5's IgGl

lil) 2° MLR. CHL responses influençed by B5 IgGl

Iv) Inhlbit ion of 10

MLR by the 85 IgG1

v) IL-2 dependent and IL-2 expanded cell lines

vi) Inhlbiton of the co-stimulator assay by B5's IgG1

vii) Responsiveness to T and B cell mitogens Influenced by

C. Tables and Graphs

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163

164

165

166

168

169'

171-192

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Çhapter VI AC4-11-B5's 19G I Ab<,orptton and B1I)èl1ng Sl~l~.l~

VI. A. Matèrlals ,1nd Methocls

i) Inununoabsorbant columns

ii) Immunoabsorbant matrlx of (ross llnked 11\1111,111 ',('film

proteins ll)/,

i11) Appll.cation of pre-lncubated B5 Igel

Ollto a preLaIlbrated

P-60 column

iv) Fluorescence antibody blndlng to ('('lIs

v) Enzym~ Link~d Immunoabsorbant Assay

VI. B. PresentatIon and Discussion of Resul ts

i) Absorption and bindlng studies

ii) Is IgG I specifie for a human serum component?

iii) B5 IgG l immunoabsorbant column

iv) Th~P60 gel precalibrated column

v) Fluorescence antib~dy binding studies

vi) Enzym~ Linked Immunoansorbant Assay

VI. \Y. Tables and Graphs

Conclusion

References

H5

196

197

199

199

200

200

201

202

203

205-212

213

224

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--/-L1St of TabLes

Tables

In vltro 'lmmunlzatlon to ovalbumln (Olt) and Bovlne

Serum ALbumln (BSA) 101

II ReslduaL BF/IL-2 actlvlty recovered from culturt' sup-

ernatant foLlolllng ln vltro lmmünlza!lon 103 ,

III BaLb/c ln V1VO lmmunlzatlon-Hybrldoma Scrpen1ng 105

IV BaLb/c ln V1VO lmmunlzatlon-Clonlng rcsults 107

V' Inhlbltlon of 20

MLR ceLl proLderatlon-Representatlve

results from 6 clones 109

VI Inhlbltlon of 20

MLR ceLl proLlferatlons by 10x

ammonlum suLfate preclpltated cuLture supernatants 110

VII Inhlblt lon of 20

MLR cell prol derat lOns by asc~es

f LUl ds \ ( on 10

MLRs and B VIII Effect of hybrldoma supernatants

111

celL mltogen stlmulatlons 113

IX 810ZZ1 ln V1VO lmmunlZatlOn-SCreenlng Results 115

x 810ZZ1 ln V1VO lmmun1zat1on-Clon1ng ResuLts 116

XI InhlbltlOn of BFIIL-2 st1muLated 2 0 MLR cell pro-

llferat10ns (c~ude cuLture supernatants) 118

XII Bl0zzi ln V1VO 1mmunlzat1on-Inhlblt1on of 20

r1LR celL

prol1ferat1on by serum free hybr1doma supernatant 120

XIII AC4-II-BS cLone-2° MLR prollferatlon lnhlbltlOn by

vat10us hybrldoma preparat10ns 122

XIV Rad10lmmunoassay for IgG1

content 146a

XV 810ZZ1 in V1VO lmmun1zat1on-Serum free 1nhlbltl0n

data 147

XVI DEAE lon exchange chromatography 149a

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Tables

XVII Fractlonat1on of 85 on Rabblt antl-mouse IgG 1 , IgGZ

lmmunoafflnlty columns

XVIII IsoeLectrlc focuslng InhIbItIon results

XIX SlolabelLlng of 85's I9G 1

XX S10ZZl ln VIVO Immunlzatlon-CLone testlng-InhlDltlon

of 8F/IL-2 stlmulated 2° MLR cell proLIferatIons

(Crude cuLture supernatant)

XXI a AC4-II-BS clone-2 MLR prolIferatIon InhIbItIon by

varlOUS hybrldoma preparatIons

XXII and XXI II

Two dlmenslonaL neutraLlzatlon assay of 85 hybrldoma

and BF/IL-Z as measured by IL-Z dependent stImulatIon

of thymIdIne uptake

XXIV Klnetlcs of 85 I9G1

addItIon-InhIbItIon of 20 MLR

praL 1 ferat Ions

XXV Effect of AC4-II-8S on murlne 20

MLR

XXVI Effect of BS on cell vlabll1tles, blast cell trans-

XXVII

XXVIII

XXIX

XXX

XXXI

XXXII

XXXIV

3 formatIons and H T uptake

Effect of AC4-I1-85 on the generatlon of CTLs trom

a stlmulated 20 MLR

Effect of 85 IgG1 on the 1° MLR

Effect of B5 on murlne IL-2 dependent ceLls

Effect of 85 on human IL-2 expanded ceLl~

Effect of 85 on the co-stlmulator assay

Effect of 85 on mltogen stImulatIon of human and

murlne cells

Pre-absorpt~on of BS IgG, onto target cells

Recovery of BF/IL-2 from immunoabsorbant columns

page

1 S 1

154b

155

171

175

..

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178

180

181

183

185

187

188

189

190

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Tables pages

xxxv BloLoglcaL actlVltlCs recov0red tram the P60 column 209b

XXXVI En7.ymf" Llokerl hnmnno.I"'i.1Y of V.lrlolls "antl-IL-2" 211

l're'l'.r<lt!.lone

L l st ofF l gu r e s

F19ures

MadeL of th~.rnurlne MLR

., Standard eurves for RIA

., Prof l Le of 85 on DEAE lon ex change eoLumn

" Prof l Le of FCS on DEAE 10n exchange eoLumn

') Prof l Le of 85 on l soe Lee tn e f oeus 1 n9

Il Prof l Le of 85 On pre-eaLlberated P60 eoLumn

page s

18

146b

149b

149c

154a

209a

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Abllrf'v lat lOns

AEF •

ALLogenelc effect factor

Ant 1 -SRBC Ant lbody dHected agalnst SRBC

Antl-BF Antlbody dHected agalnst BFIIL-2

[3 cell Thymus lndependent, Bursa or bone ~arrow derlved lymphocyt~

BF BLastogenlc Factor

BCGf B cell gro~th factor

BSA BOVlne serum albumln

BS The AC4-II-SS antl-SF monocLonal antlbody/hybrldoma cLone

C Centlgrade

Curle

Carbon 14

ChromlUm 51

CML Cell medlated lympholyslS

Con A Concanavalln A

cpm counts per mlnute

CRSC Chlcken Red Blood Cell

CTL Cytatoxlc T Lymphocyte

DEAE Dlethylamlnoethyl celluLose

DMEM Dulbecco modlfled medlum

DMSO Dlmethyl Sulfoxlde

DNA

EMEr~ Eagle's modlfled medlum

FCS Fetal calf serum/fetal bovlne serum

9 gram

G75 BF BF semlpurlfled on G7S column chromatography

HS Human serum (pooled)

HEPES N-2-hydroxy ethylplperazlne N'-2 ethanesulfonlc aCld

!..

(

HGPRT

H3

IL -1

IL-2

• -11a-

Hypoxanthlne phosphorlbosyl. transteras(

t r 1 t 1 um

Interleuklne-1, LAF

1nterLeuklne-2, BF

IL-3 Interleuklne-3

19 1mmunogLobul ln

19G" IgM.. 1mmunoglobulln of the subc Lass of G1 , GZ' M, A, E ••

1EF 1mmunoelectrophoresls

LD

m

p

M

MAf

MHC

'1f

MIF

MLC

MLR

,.'0 OA

P3x20

PBS

PEG

PHA'

PHA-S

PFC

PWfiI

RPMr

Llter

Lymphocyte deflned, Class II antlgens

m' II 1, 10-3 , ml , mg, m Cl

mlcro, 10-6, pl, pg, ~Cl

Malar

Macrophage Actlvatlon Factor

Major ~istoco~patlblllty CompLex, HLA ln man, H2 ln'mouse

Mitogenlc factor

Macrophage migrat10n lnhlb,tlon factor ~-

Mlxed lymphocyt~ culture

Ml xe.d lymPhOCyt\ react ,on

Mac rOPhftges

Ova lbuml,n

'\

HGPRT myeloma used ln hybr l:doma fus lon

Phosphate.buffer.ed sallne

POlyet~Ylene gl~col

PhytohemaggLutlnln

PHA stlmulated human tonsll cell supernatant, IL-2

Plaque torming cell

Pokeweed mitogen

Roswell Park Memorlal Instltute tlssue culture medlum

-----_.----------

R oc. ~1 G,

R 0( '1 G2

seIF

so

SOS

SRBC

Staph A

T ce II

TeM

TCGf

TrlS

TRF

TSF

LPS

{.

-1 11.-

Rabblt antl-mouse IgG,

Rabblt antl-mouse IgG2

Secondary cell lnduclng factor

SerologlcalLy defined, Class 1 antlgen

~odlum dodecyl sulfate

Sheep Red Blood Cell

Staphylococcus Aureus CDwen 1 protelr A

Thymus dependent lymphocyte

Thymocyte condltioned medlum

T cell growth factor,IL-2

Hydroxylmethyl amlno methane, C4H11 N03

T cell replacing factor

T cell stlmulated factor

Llpopolysaccharlde

•

( 1 li "

, . .. • > .-...

1.

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la. 1", ,Bat. ~ Low ••• t.ia (1) ob •• "e. tllat, " ..... lyapkooyt.. of

"Y. a oellal" l'dlif.l'aUy. .. •• t "a. .... oJaar .. t.d .... ..,. IlIA

syaùe.l., .. 1 .. & o.u u ... fOZll&Uo ..... o.U cliYhio... It" ..

... a 1004 la Yitd II04Iel to "tnaia l'.aot).Yity tawal'da fOl:.1 ..

traa.plaata&10 ... ti •••••

SI.ac. thea. tU ... ba. ..... ".11 o"l'an.l'ia.. 1. tem. of-

~

'Jpal., ad tlle 1' •• VO_i •• o.U .Ü •• t ... fiaed '-7, •• nlo.ioal ...

flUlCtioaal "iol0.1oal .aa. (~5I.25J.44 •• U,S'). ne bIJol't.... of

l,....oki... la the ... wa. "1".& ..,. tU fiJuliq. tllat-. 1)

L~otiDe. 0&& l'.plao. tll. l'.q.ir ..... t. of yal'lo.. 0.11 ~.

' ..... t\\l fOI' .. "C l' •• ,.." (260,35) .JUl ~) LJ'II1'ukJ. .... alo .. ou

.ti~.t •• Ub •• tl of r •• va"'i., 0.11. 1. th. 2- ILR (3S,37,~'2).

Tb. .ia of thl. tll •• i, i. ta pl' ..... t wo~k 1. whioh ". att .. pt.d

to pl'od1l.oe a ao.ooloul uUbody to th. h.u l,..,ho~ .. Iat.d.1Ikia-2

<n.-2) • I.t.d.1Iki ... -2 11 ••••• Ua! fol' .. JILC r •• po..... CoJUlitio ... . '

which re.ul t ia th. laok of IL2 prod.oUoa wUl .... ~.t. . w.ak 01'

.. ,lilibte Ja,e re.poa.. (1,2'1,~21). Id ••• IL-2 u. b ••• aJacnra ~to

.,.oifioa!ly Itiaa.tat. th. pl'iJIa. c,.totozio lY11Jhooyt.. (C'!'L) la th.

2· Ja,R.. F1I.tul't applioaUoa. of .' aoaool.oul' ,a.Ubo'" .ireotecl .... i •• t

n.-l •• ,. be to ohar.oteriS. u.. IL-2 prodac'r •• u -.4 th. n,-l •

l'e.poader oeil with ai.. of t~.ial oaJoff tlla ILR b1 re .. l.ti.. th. ~ , 1

\---- - --,-----.

1 \

• 0

.. ~

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a.allabtll~ of IL-l. ~ ta YiYO oirowlatlal 1 ... 1& of IL-2 ~aa .1.0 .

ba "t.lllo.. bJ da .... of a ... alU •• r.cU,ot..luao •••• 7. It wo1Ll4 b • ..

JO •• ibl. to clet •• t ,ùaal.. la otro1LlaUal U.-l te.e1. wllioJa._7

lMi •• t. tJlII JOt .. tial 'ft aotl.aU.. priMd oytotosiG l,-p1looy:t. la

raj.otlo. o~ tr ... plaat •• tl ......

..

1

(

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Ckpt., I

Q

1. A. ft. MiD4 LJllPlaooy-te ... po.... Historia Baeklroud

tlt.bllâ.4 Uaat .. ..C nspoll". ela.raeteduel b,. blast ceU

1 bsai.. I .... U.U,. caU. boa 1 .... Uo.ll,. aiai1ar niaals .. el h_u

ari.s, dbl!., ••• cI f.U,. .. ber. ,.hicl • poor ILe "'POIl". B.èlt..!l

.al. (3) atui'd tlJe " .. tic ",ab' •• st. for .tilialaUoll la •• ~

r .. po.... IIOti., iJt.at stblal.Uo.. la,. eoa .. aie .iee "hicll dUf.r iD

Uae l ,.,i08 (12-I) 0' th. ILS 100.a r ••• lt.cI 1. ,rol1Ie,atioll of (k,

r .. pollAl., e.ll. (4.5). IJ..J",OOI.l aJt.o..d tlaat h __ IILA-D relioa

dilt.S' .. e" • "Ul atl~'alat' ,roHf,S'.cloll la a ~1· .:oC relpos"

Ctrotbai aU BnueS' iD 1974 .Jr.ow.d û..t

cytotode l7llflooyt,. ".r ...... rat.cI fo11o.l., IU.alaUo. of ela •

.... po1Ul.' e.lh i. u -.c b,. .110 .... :1.0 0.11. or t..o, o.Ua (14).

BU. .Il...,.. ad otM, po.,. • • ...,.eI Ü.t tJt.. _ t.r •• t ... ti.... for , '.

O)'totosle l7llPlo~.. n~d ia .. .... '".r. d1r.ot,4 a-'.l.d tll •

.. U, ••• oode. for br tlIt 12-&: ... re.io... Td.aalli.d n.l1. (10).

IlJn'oolet. Gnaa.t U. Al •. (la.l'.20) .... cI ~at 'tU .... 'flaiTal.at .,

of 82-1.», tJae BLA-~.B.C ",10.'_ 004. for tIl. tar .. t •• U .... '<

r •• opia.. 'b7 tU , .. lM. cn... Juli.. Nk, '.II.~t.cI Uac ILl-A. S. C

0' 12-1,1) (ola •• 1 .. ti ... ) .. tll.ate 41tf.' ..... alOM •• ,. taoapabh

of .11eta, • 'd, .. S'Y.... 4 ...... or ~-I .(ola .. II .. ti ... ) r'll.a

dift.reao •• nu b. " •••• t •• i. or_Z' lOS' a 1- &c pc.l1f.~.&i ... , t~

\

---------- - - ---------._- ----------

1

rb

1

•

..

1

'-15-

re.poa •• to be seen alon, with the .ub.eque.t le.eratioa of CT.Ls. The

C'D.s w.re specifie for th. Cla.. 1 a.ti,ens. Furthenaore. B.ch' s . (

»&~~~~~~~~ __ ~l3.16) showed th.t the cla",II aatilen •• nd

01... l anti,... did not n.o •••• rily h.Te to b. on the ....

" stillalatial c.ll. \

It 11 now .oo.pted that in • 1· MLI.. BLA-D L • or 1Il-1 re,ion .

. diffeZ'e.o.. will .ti.ulaU th. olouaI exp.nsion of alloreaotiTe T

helper c.11. to prollfer.t. (21.3.64.65) aAd JILA-A.B.C. or Bl-E.D

re,ion. (2l) will sti.ulate th. pre-crL. to prolifer.te and

differe.tiat. to priaed CILs. The,aajoritor of prollferatin, oeil. in ,~

th. 1· JILJl .re the T h.lper oella. T h.lper eel1 proliferatio. and

th. .ü •• queat IL-l prod.ction .re .... ntial for pre~. to

pZ'ollferate and diff.r •• tiat. into priaed CIL ••

lIow .. er. i. tlI.. l. lIlJl whlch la th. r .. tiaalaUo. of pd .. ed

aeaory o.lh. ola .. II anti, •• diff.~e.oe. alou wa. .affioieat to

Viller ,. l· JILa oharact.rhed by a IIOre rapid and, latea .. \

" pfoliferatiT' r.apon .. with ~peotfio ... ory (~4.25.26.27.28.2'.30.31) •

.

• "'Olfio ."orr retalD,84 wa. tk. abil~t7 to ly .. ~.11. espr ••• in,

da •• i •• 'tlle.s of th. orill .. 1 1- .... tiatlaUa'l oeU.. n •

"JOI.'itor of' r •• poa.dhl o.l~. ia th. 2- lL2 a~. tll. pd, .. 4 C'D.a 1

apoifla fOI.' tll. ola •• 1 .. ti ••••• apr ..... o. tll. 1· &Jl .t1aalatia. J

a.lb (32).

Lat.r work perf0ftl84 by IIU1er ai&d IIi.JaeU (36). Oppeû.ia 11 .&l.

(33). Üa.u ad Cohea. &ad VoUl • .I1.al. (3 •• 35) 1 ... 4 that tJa. , ~ ,

o.11a1ar r.q.~~ ..... t. la ILe a •• -. r.plaa •• by a ... aifia ooa.dltiau4

.. 41_ •• perutsat. Mla'1'.at o.U_ .l'.qair.4. ia a 1- allol.~la ...

-~- ~---.....---.. --

""16-

ca. be reptaced by the lymphocyte activation factor (LAP. or

I.t.rleukin-l) derived fro. a 2" hours old cul tue .'upe,rna tut of LPS

stiautated adherent cells. Stu4ies yith purified IL-l have su".sted

that IL-l caD ~eptace the adhereDt cells iD a l' KL2.

'a,.er aDd RolliDJhoff. a.d Uotila 11~. (35.37~ hav. show. that

soluble factors derived froa th. su~rnatut of aD allol'Deie KLR yere

capabl. of stiaulatiDI a 2' XLl, in th. ab.eDce of .tiaulatinl e.ll. or

BLA (or &2) a.ti, •• s. Studie.~by Go~doD and MaeL. an in 1963 (38) and

D .. eroa. IAdependent r •• ~arch.rs h... shown that factors derived fr~

allo •• n.io c.ll stiaut.ted and aito,en .tiaulated culture suparnat .. t.

ca. Itiaulate l' ILl. ceU proliferation iD th. abs.nce of the

r.stiautatin. cell. &LA (112) •• ti •••• (83) or aito .. ns. 7he.e lactors

y.re .. ti. •• nlcall,. DOu,.cifio Rd yill stiautate r .. poDod.r ceU.

oa •• tia~at. l,.-phooyte. dariyed froa aaoth.r .,.oi'.' i ••• rat. Rd J

atee. Tk ••• lactors ar. coll.ctively taowa •• Int.rl.atiD-2 (3'). It r '

is b.U ... ·.d th., D.-2 il the obli •• tory si,..l .Uoit.d b,. th. T

.. ooU sipal to the pr.-cD. wJaicll Il •• drea4y ' .... ' the IlLA-A.B.C.

-,. ali ... · 1 .. ti •••• "UI expre.. ta., ree.ptor for n,-2. Cla.. 1 ~

_ .. ti ••••• ad n,-1 to •• tker yi11 tri ••• r tb. 9r'-cr.L. to dilf.rentiate

&ad prolif.r.t. laco prt.ed CIL. "i~ .,.011ioity to 1,. .. tar,.t c.l1 •

• spr •• ain. th. atia1l1ati •• 0.11'. cl ••• 1 a.ti'I!.. (3.40). la a 2'

.... n,-1 alo .. wJaetller' 1t la pr04aoed as a r •• .t c 01 BLA-D or )12-1

stbnlaUo. or •• ppUed ezo •• AOas1,.. la •• IUd .. t to trill.r C1'L

~ .... _ .. -t'~ .. _ ,

•

, .

-17-

prollferatiou (8,46).

n. rupondiul celh in an JILa have b •• n char.ct.rized both by

their fuaotio .. l biololio.1 .cti ... ity, aud br •• ro10Iie.l ....... Th. T

h.1per ceU whlch pro duce • R-2 are LyT 1+, 2-, 3-, (110 ... ) or 01.1' 4+,

3+, &Dd L.u 3.+, Sb+, 1+, 4+ (h .... ). 1b' aytotoxie lyaphooyt •• Ar.

" LyT 1-, 2+, 3+" OD." ,3+, 8+, .. d L.u 2a+, 1+, 4+ (41,42.43.44.45).

A. thi. th •• il. il priaarily ~ov.c,rued yith IL-2 .. d it', roi. ia

th. !.aue re.pon ... w~ yill be oOIlO.ntr.tin, .siAly on n.-l. the n.-2

prod.c.r cell. IL-2 rupolUl.r 0.11, aud condition. le.dinl to IL-2 il'

prod1lCtion~ BowaT.r, th.r. Ar. a ... ro •• oth.r 'factor.' deri .... d froa ,

th.· ne Iv.peraataat wUeh. wUl be' bri.ny di.cu ... eI <Sectioa II).

lü •• d c1lltllr. 11lper1L&taata cI.rb.cI ira. IlLe •• ito •• aic. or .pecUia

&.atll.nie ·at1a1llaUo.. cont.in a' ... ari.ty of ' factor.' capable of

Il.ctor. whloll aicl ia 1,.,llocyt.

cliff.r .. tiatio.. ..taratio., &ad pro1if.ratio. Ar. pr •••• t. LikeYi ••

tIl.r •• re factor. ybioll .s.rt th.ir intl ... o •• oa .. pri8ecl and pd ... cI . e.ll. ia 'ploifle ... ao •• ,.clfie aaaaer ••

l. B. • .. 1 of tU ...

'11le followu, .od.l ha. b ••• prOJH) .. cI as • worki., Ilypothesil for

tlle JBJL It il a .odel nU t an.-.l n,-2 •• aD. obu,.tory 1i,..1 for

oell proUf.r.Uo., cel 1 lat,r.etlo. aD.CI n,.t .. l .eti .... Uo. of tll •

• ~cific aytolrtic l,.,Jaocyt... _. ybla to ill •• tr.t, tIl ..... aU.l

co.trollial th, ~ ... re.po .... )

-18-

briDe MI'

Pr.eur.or IL-2 produe.r 20. SDII (-)

\Cla •• 112-1

Stia1llatinl C.ll

/

II. LD -----)

Cla •• 1. sn I121:.D

~

l (-IL-l

1 ... 1 1 1 J . , . Il IL-l / @.-",,,, ,,'-

IL. 2 ~ . ( ~:;~+»)--------------------~-> ~

, Pria.d en. Lyl-2+3+ ! ~'

(la th. Ju_an 1ILa. th. cl a .. II uU,.al Ar. th. IILA-D anti"u

aad th. clau 1 uti,.al ar~ BLA.-A.B. C .. ti,.a ••

proclue.r c.ll. Ar. 01'1' 4+. ancl th. pr;"'d CTL. ar ... 011' 8+).

Th. IL-2

The "'''.0' of 'Y.nU l.adinl to a positive ..... ar. a. fol1ow.:

1) D.-3. Inted.ûb.-3 (to b. eliseu .... d in •• oUOIl I-D-iii) will

o.... th. cliffer .. tiatioll of a pr.cur.or IL-2 proeluc.r c.ll into a

.. ,tllZ' D.-2 proel.e.r c.ll (L1'T 1+,2-,3-, or 01'1' 4+). B.t thi. D..-2

proclu.r 0.11 do.1 Ilot .xpr... a r.e.ptor for IL-l. Pr.eulorl of

IL-2 proel.c.r c.ll. clo .ot .zpr ••• th. '1IZ~.tio activity of 20 alpha

h1'ûozy at.roid d.h1'uol.aa •• ·• Upoa D.-3 .ti*1ll.tioa into a .atUrf

IL-2 proclao.r c.11, lb ••• c.ll •• spr ••• 10. SDB aotlvit1'.

1) Alloi • .,ie 01 ... II "U"., ca. at1ll1ll.te th. n,-2 proel.o.r ceUs

ellr.ot11' (likewh. "0 c •• T 0.11 aitol.n ••• oh as PlIA. CollA. and th.

aoaoo1o_~ &aUltocly OlT 3). 1

3) A. a co."'lItne. of at1ll1llatio •• th. D.-2 proda.c.r c.ll provides a

.i ... l to th. aacropha,. to i.itiat. IL-l prod.ctioa. Th.re il

1 -19-

evidenoe to IUlsest that a direot cell to cell contact is required t6 ,

tr iller D.-l production by the macrophagos (lib" ho •. lywaphokino s , are ~ ,

produoed "hich ean eAhance D,.-l production). At the same Ume tho

IL-2 producor coll acquires the expression of an IL-l receptor • •

4) Upon rocoivin, tho no sipals. a) class II alloantiaon, and b)

IL-l fra. tho Ilacrophaaes, the IL-2 producor cell "rolifer.tes and

produoes IL-l and other lymphokines. This includos MAF "hich' can

~ance D,.-l production by .acrophs,os.

5) The D,.-2 respondor colIs (in the disgrma of tho,MLR it is dopicted

" as a pre-CIL, but thooroUea1ly in an OJllJoinl iJIIIDDJle response. ono c.)j'I

.ho1lU conaidor tho po ssib ill ty th~O n.-2 ro spoDdor coU can bo •

T suppressor cell. T holper 0011 for the B coll ro.ponso as weIl as a

pre-Cl'L) are stimulatod by tho class

these colIs expross an IL-l reeoptor.

l antilons. Upon activa,tion,

Thorefore the d-2 rospondor 1

coll requires t"o signaIs for evontual activatioJl and proliferation;

a) the cla.. 1 snU,ens, b) IL-2 producod and secrotod by the

activat~ . IL-2 producer coll. Follo"inl activation by the two

sipals. the' D.-2 ro sponde r cel 1 underloes differentia tion and .

prol iferation. In the case of CIL" there "il1 bo a alonal

proliferation ,of eytolyt1c colIs which can lyso cells expressins the

class 1 antilons of the stiaulatory cell type.

'6) AlI priaed IL-2 responder cel1s "il1 express the IL-2 roceptor snd

can be subsequontly stimulated by IL-2 alone in the absence of further ... antiaenie stimulation (i.e.'2' XLi.).

, 1. C. lnterleukin 2 and Other Lymphokines. Historie Aspects

1

, -20-

Since the initial reports by Gordon and MacLean, and ~asakura and

Lowenstein in 1965 (38,47), there have been subsequent reports of

soluble factors derived fro. cell free cul ture sup.rutants "hich are

capable of stiaulating lymphocyte proliferation and differentiation

independent of a stilliulating antigen (35,37,121.122,123,124,36).

Studies by various researchers in this field have characterized a host

of factors. The se factors have been ,iven various names based on

their antigenic specl f ici ty or nonspe ci f icity, the ir biololical

functions, mode of factor productions, and the- .ssay systm.s used to

characterüe the factor s discussed.

The interest of the present "ork is on the nonspecific

lymphokine, Inter! oukine-2, and other lymphokines (specifieally

interleukines 1,3) "hich can influence IL-2 production. Following the

Second International Lymphokine Workshop in Ermatinler, Switzerland in ,. 1979 (39) it ".s asread to catagorize the various nonspecif~c

lymphokines Into groups of Interleukine 1,2, or 3. The basis for such

catago'rization "as based on the lymphokine' s biological activity, it' s

cellular origin, and it's target cell type.

1. C. i) T cell derived soluble factors - overviews

Interleukine-2 has bcen kno"n by numerous n&me. based Initial1y

on the Assay systems used to characterhe the lymphokines

(35,36,37,121,122.123,124). IL-2 "as or iginally mo"n as the

lIlitosenic factor, blastogenic factor, T

C(Mst illlui a tor. thymocyte stilllulating factor.

ce Il growth factor,

thymocyte mitogenic .t

factor, killer activation factor, and ~ndary cell inducing factor. •

•

1 -21-\

AlI those factors havo in co .. on se?eral charactoristics. 1) TheY'are

aIl produced by a lIature thywu. dorlved T cell. 2) Tho taraot coU.

of all th... lywaphokiJlu are thywaus dori.,.od T oeIl s. 3) The tarie t

cell II1Ut bo an anti,e~. or T coU lIitolon pri.aed coU. and 4) AIl

-0 ,

tho.o factors have si.ilar physicoch .. ical characteristics il thoy are

produced by the s .. o species (i.e •• urine or huaan or1,in) (12').

In 196'. Gordon and KacLean diseo?ored that huaan peripheral

lywphocytes activated in a t"o "ay IIbod lyaphocyte out ture "ill

relo •• o a lIito,onic factor "hich is capable of tri.,eru, blast cell

transforaation of sinlle oeIl cultures. 1t "a. suh •• quently shaYn by

the ~ .. e Iroup that alloleneically different lyaphocytes. ospecially

cella differin, in the BLA-D relions. "ill .ti.111ate blastolonie

factor/llitolenio factor productiou and tho subsequent recovery of

those factors froll tho spent( cul ture superna tant. Uotila.!.1.!l. have

subsequently shaYn that the peal: rocovery of bhsto,onic factor "as

.. betyeen 48 to 71 hours (35) •

Althoulh tho initial "ork by Gordoll and JlacLean suuestod that

blastolonie factor cali. sti.ulato sinlle cel1 culture •• it is DOW

acceptod that bhstoloDic factor. or any of the ~ ~ctors belonlinl ta

/ the ll.-2 class of IY1Bphokines. "ill peferentially ati_utat. snU,on

pri.od cells. (Bowever on. csn DOt ruJ-e out the pouibility th.t s .. e

cells in a sinlle cell culture ha"e already been 'aAti,en priaed' in

vivo prior to trusfer t:o tissue cul ture., or that colIs "ere priaed

by a co.ponent found in tis.ue culturo .ediua).

The roquirellont for priaod cells as n.-1 tar,et cells is lfest

illustrsted by ~~other 'll.-2' previously bo .. ~·the Co-stau1ator or

1 -22-

thyaocyt. sttaulatin, factor (TSF). DiSabato ~ Al. (123) lad Paetkau

.!.1..Ll. (121) f01Uld that the T cell aitolen Con A stiaulat.d aouse

Iple.a c.lls to prodllc. a loluble factor which can 'help' thyaocytel

to r •• poa.d to low dos.s of aito.ea.. It is bo"n that "itllia the

aou.e thywUI ther. are fo" B colIs. f." .. cropha,.s. Lad f." .. ture T

ceill. The thyaocyto populatioll lacks aaturo T helper cell. "hich are

capable of producinl n-l. ConsequantIy thyaoc::ytu "UI aot respoa.d

to a low do.e of T coll aitoleas, or aay aaU,eaic .tiaului. P.etkall

.!.1.!.l. 026.127,128) .hoYed that the additioa of the loluble factor

(teraed the Co-stiaulator) to thyaocyte. It1aulatod "ith low doses of

ConA "ill read,r these cells rosponlive ta CanA. Co-stiaulator. alone

"ill aot sUaulate thyaocyte. "hich have aot beea priaed by the

aito,ens. DiSabato aad Chea studied the thyaocyte stiau1atial factor

(TSF) "hich showeci siaUar charachristic. as the Co-.tialllator.

Ka ther the adnl CoDÂ .. the aitolen. DiSaba to aad Chea stucUed the

abUity ot m ta proaote thyaocyte respon ... to PUA (123,129).

Al thou,h one of the first publ ished reports aD n,-2 "u the study

of an n-l deri'Ved froa an allo,euic JILlt -stiaulaUo'D of luIaaa

lyaphocytes. others have showD. aisUar results u.ia, auriae

lyaphocytu in JIIJl (37). Sjoberl in 1971 (130) shcnred th~ T c.ll

ai to,oal caa Itisa1 a te sploea c.111 ta produc. fi.-l al.o.

Furtheraoro. quaati taU.,ely IIOre n,-1 caa b. reco.,.red froa aito.en

stiaulated spleea cell thaa froa KLR culture supernataats. Gillis.!.1

Al. (131), and Kor,an ~ Al. (132) siudied plant lectin .tialllated

n,-1 production. The factor they have charact.rized. T ce11 ,rowth

factor. i. ba.ed oa the ability of this factor to lustain the

!...

\ ,

\

-23-

prolif.ratioll of aito.ell or alloUlti,.1l staulat.'d lyaphoo1't.a in 1011'

t.na cul tu... It b.c... .Tid.llt that th.s. 10a, t.aI cul t1U'.~lls

prolileratioD. IVpoa yer. totall1' d.pend.llt 011 TCGF for surYiTal and

f coatiauous oult1U"s ia th. pr"'DO' of IL-l, tla.s. c.lla b.o_.

aar,spollaiT' to th.ir ori,iDal anti,'Dlc stiaulu ••

retaiud th.ir ori,iDal biolo,ical fUDct!oas. Th.s. D,.-l cI.pead.at

c.11 lino. haT' proT.a ta b. aa laTaluable too1 in th. studi" of IL-l

ancl th. IL-l dopead.at tarl.t c.11s. It has also proTid.d Us with a

rap1d a .. ay for the pre •• nc. of n.-l aino. th.s. fi.-l d.pead.at c.1la

yill di. if not luppl ••• ut.d yith IL-l 'TOry 14 hours.

It il ,ol&ib1. that ' n,-l' ref.rs to a ' coll.otion' of anti,.a

sp.cilie lyaphokin.. (133), produeed b1' th. T helper c.lh upoa

stiaulatioa (134). 1f. can DOt ooac1ade tla. 'IL-l' la oa aoheular

'DUty (u ••• ctiOD OD phy.icoch •• lcal charact.ristic. of n.-l). nor

.xclû. th. pouibility that th. 'D,.>nl aetiTity' h •• dlat.d by

uv.ral l}'IIPhokius. Iad •• d in lGa. of th •• a 1',1 ier ca t.,orisa tiOD' of

lyaphokia.s. th. T 0011 r.placial tactor (TIF). yhich r.plac.s T c.11s

iD a B c.ll antibody respoa... had b •• a attribut.cl to th. n.-l

cat.,ory (13'.136.137.138. 139.141). Bow'Ter 1U.r p'Vitle.Uoaa of

D,.-l haT' d.fiAi tiTely separated nF actiTity froa th •• ohcular p.ak

~r .. poadiD, ta n.-l sotivHy.

T lyaphocyt •• proclac. aot oaly aati,.a aoaspecifio factors .ueh

as fi.-l or 'l'I.F. but they at.o produc. u1I8.roua satil.a specifie

lyaphot.in.. with th •• acropha,.a. B &Ad T c.ll. as tarlet c.lh.

Althouah this th.,ls ls a stucSy of IL-l, oth.r T e.l1 lyaphokiD" yi11

b. bri.f1,. , •• atioud. Th. fo1lawin. lyaphokia.s Ar. cI.fiait.Iy Ail of

-24-

th. n,-2 cl... b.ud 011 th..ir biolo.ie.l speoifioity, alld

pkysieoeh._ical •• p.r.tioll froa th. a.jor n,-2 peak aeti.,.ity 011 ,.1

fil tratio ••

Lawr.llC. 1.1 .Il (140,143) r.port.d a 'tr .... f.r factor' cl.rh.d

fra. .. tl, •• prta.d h.aa .. peripkeral ly.ph.oeyt... B%traot. y.re aade

frOia periPJaer.l lyapkocytes of iD.di.,.idul. yh.ich. uh.ibit.d posith.

delayed type 1lypers.ll.itiTity ta th. allti,en t •• ted (l... PPD, or

tû.rculill positbe iIldiTidula). 8%tract. of th... lyaph.oeyt ••

coataill ail .. ti,.a .pecifie traasfer factor yhich. e.1l traa.f.r

specific uti,en r.actiTity to all otherw is. 1III.%'e.eti..,., or

noataaUllized indi.,.idual. Tr&Jlsf.r factors .re of s.all size aol.cul ••

yhich. are reco •• rabl. ill th.e yaUr dialysa t. of lysph.ocyt. .stract ••

Truufer factors Il... sa fÀr b.ell r.port.d ill h.aa&Jl alld n •• lU

aOakey.. Specific tr"lfer factor. eûibi till, i_Ulli ty to b.cterial

&ad fUll.al utl,en. h. ••• allo b •• n r.ported.

Tr .. sfer factor. are Ilot consid.red IL-2 silice the acti.,.e .oi.ty

is not ,.cretad Ilor reeo.,..rabl. froa th. cul tu. .uperna taat.

Physieoeh. .. ieally it i. auch. sa.ll.r th.&Jl ,IL-2 and po ••••••• allti.enic

.pecifiaity yhich. n,-2 laaks.

B •• id •• th.. anti'.1l specifie traasf.r factor. th.~! .r. Ilaa.rou. . (' ------~ ,/

report. of uti,en specifie factor. yh.ieh. aid ill t~ c.U ~body /

rupoll'.. Fel_an and Buten III 1972\ (142) reported a T cell cl~ ti,.n .pecifie .olûle factor yh.ick coopera~e. yith &Ilti~1l priaecl B

cell. to procluc. antiboclie. to b..pt.Il.. ~.ria.llt. Yere perfora.cl ill

yh.ich. ILB-carri.r prt..d T cel1. w.r ••• paratecl froa DNP-haptell prt.ecl

, B c.lh by a ••• ipera.abh a .. br ... into wo •• parat. ch. .. b.r.. Botb.

\ . - -.. . - ". - - ----- - - ~ .. _----

-1$-

o.U population .... re r •• U.ulat.d .. ith n.JI-DNP. and a 'poIlUv.

respoDH wa. ..ea iD t.ftI,.~ of Ultiboely proeluotioD elireoteel. to th.

llaphD DNP. Bowev.r if th. T o.U. "'1" r_oy.ct or il T oella priaed

to a diff.r.at oard.r w.r. 'ILS. el. DO aatiHcly procluoUoD by tla. B

0.11 ••• 1'. I •• n. Sino. th. 0.111 .'1' ••• p.ratad by a c.l1 Û8peftl8abl •

•• braDe. th.1l any ulp that T e.ll provide. to th. B 0.11 _at b. via

a •• brana ,am.able .oluble factor. UDlib tla. T o.U replaoial

faotor .hicll il Ilon-.,eoific. tlaia Il. 1,. l' factor la UlU,.n .peoifio.

Orili_lly F.'ldll.a .t.1.11. suu.ated tllat, tlaia r 0.11 Ilel,-r faotor " ••

part of th. anU •• a reo.ptor OD T oell.. n.y f01lll4 that anU-II

anth.ra CUI. l'_ove tlala Ilel,.r faotor (144). ~0.JJ..Al. ia,1"",

(145) aael Crou .u.l1. ia 1971 (1"') .... est Uae tla.lr UlU •••

• pacifio Ilelper faotor for th. B 0.11 la DOt ~ollobalia 1!kt.

ratla.r it po....... .C-1I2 anU..... ne k.l,.r factor they h .....

oharaot.riz.d is of a .. aIl .al.oalar ".ilkt of 50.000 elàltoaa which

i ... all.r UUl ~uao.loballD.

'Ill. 1'01. of the allo ••• de· .ff.ot faotor (AD) 11 qu.tio_ble

(147). Biololieally AEP ha • ..., of tla ..... okaraot.riltio. a. IL-l.

1Iow ..... r proelllodo. of Al!.F i. via a "OOMary r.n1a1llaUo. l'aUer U ..

.• pri .. ary stiaulatioa with allo .. ti..... To laiUat. A1lP produtio ••

1t r.q.ir.. .. ia YiYO Rriaary Itl.-1.Uo. foll •• d by .. la yitro

.. co.ary reaU.alaUo ••

I.ptmatant followial •• ooaclary r •• tia1llaUoa.

9trllotua11y. AD il a lar.er 'agleeû. tla.. IL-l. JJ!P h a

.lyooprotda aDel i. O_PONel of two , ... Ua of "'0.000 elalto •• uuI

10.000 to 20.000 dalto... thi, factor allo "ara th. ra and J2 ,

._- ------ - ----- ----~-.---,------

(

.'

-26-

aioro.lobulia d.t.~l .. at.. ABP o.a .ot lit. 7RP la repl.oi •• r 0.11.

l •. Ul .. Ubocl, ,~oclucial I,.t.. it o •• ItillalU. prbeel cn.. ad

aitOI'. Itill.J.t.cl tkyaoo,t... .ad ~ •• tor. CIL .... ~.tloa fro- T 0.11.

ok.r.ot.rhtio of .tillal.U •• th. prolif.r.Uoa of .. priMel r o.lla

"ith th ........ it1l.cle of prolU.raUoa .. oth.r r o.U aito •• a. IUla

•• CoAA aacS PlIA. ne •• thorl of Alta .. aacS Elta adaU tla.t _Ul AD

oaa lt. futh.r p~ifl.d. ou o.aaot r1ll. nt u. po •• iltUlty o..t AU

l. ooapo .. cl of •• ?ral faotora of "laiola 'IL-2' il 0 ...

1. C. ii) IL-2. Alloabti ••• Itill.J.t.cl IL-2 prod.otio.

IL-2 oa. lN' r.oo?r • .t boa o.Jt'U' ..... nuat .. t of Jn. .. - au

avi....... Altllaa'" Cok •• U4') la 1915 r • .,rtecl tIl.t ... o.lt~.

nperM tut. oaa ...... t tll. "Ipo." of .... T IJllPlaooyt ••

Itt.a1.t • .t b7 allo .... io filtroblaltl. Pl.tl la 1916 (149) llaow.cl ~at

-.Il .. ltvl ... ,.natut oa. pr.t.re.ti.ll, Itillul.t. CIL .... r.Uoa.

I .... r .l1.Al. (1) t01lllcl tllat lLJl ni tUI IU,.,..tut. fra. a-I

rilio. iaoa.patiltility. " ••• fflotl? la .tt.alati., tll. ,roilleritioa

ot 82-1:.D priMel CILI. B. teauel tllls faotor tJa. ..001lda1."7 C'D.

ia4uia. faotor (SCD') .iac. tIli. f.otor Jla4 littl. or ao .,f.ot o.

_,rialcl ,rJ ... q JLa oelli.

Buli.r "ork 1ty Baola .I1.aJ.. (3) bd .11 .... tlaat 12-1 clil,.rity

le.d. to tu IUa.JaUo. of th. _jar pr,ol1l.raUTe r •• po ... ia ,tU

ILL ni.... .la1Jor.teel tutIL.r to ..... at tJa.t, the .jor

,rolUlratia. 'o.t'la' la th. 1- ..... 'ltia1alat •• t~' prolU.r.te .,. '12-1

r •• io. dilperity. b.1081 to a ."ala •• 01 LtT 1+, 2-,3-, T 0.11. "Jalck

"

(

(

(

-27-

are bell ••• 4 to b. T ke1,er 0.11.. ~l .... &1.tl0. of 0.11. aoat.! ••

fn if ..,. tJLat elqtr... oyto1,.tl0 a.p.o!ty (3). Y.paer.u. ü. •

...... '.4 that tbe 12-1 r •• ioa .. tl ••• (al ••• II .. ti •• a) .t~al.t •• T

ul,er 0.11. la ~ l' ... to pr..... aU •• or.t. SCIP lato tlae

nltv ..... n.at .. t "', 1.50). z..a..11 If tU,. .. laull for LyT 1+

.ple .. 0.11.', aU .t~.tàte4 Uia popùaUoa "ill .110 .. U •••• i ...

..... or "lth th. alto ••• CollA. '004 r.oo •• ~i .. of SCD ".r. obtai •• d

froll th. oaltllZ ••• ,eraatut.

faotor fra. kaau ILl valtv. .~raat .. t "kiok o.. Itt.&l.t.

IJ'11Pllooyt •• to ..... r.o bl •• to ..... i.. Uoti1a J.1.Al. (35) pUluli the

flaer ok~.ot.ri.tio. of tkil .olabl. T 0.11 faotor "kiok ".. t.Ea.d,

tJa. Bl •• to ••• io ,.Gtor (.). SU .Jac,.,.d tJr..t ,e~ipJl.ra1 lyapliooytu

froll IILA-D 4iff.rat l"i'Y14 .. 1. 1.a4 to ps'04..otio. of IP i •• 1'· JILL

SJaU.r to ü. 112-1 i. th. avlM .C. IlLA-D r.,io. 41.,.rlty "U! , .

1 •• 4 to tl&e .tiaal.tlo. of T hel,er 0.11 prollferatl0. i. th. ILl. .&4 -ao ... q ... tly lb. ,r04.otio. of If. St .. l.. of BLA-D ld.atlo.l

~lbU,.,. o~ f_Ui .. 80t oa,1,. .11.1 .ot .t~1&1.te p~olif".r.tio. i •• li

.... aeltJa.r ".r. tIler. pr0411Gtioa of D. '

The .... y for" .0U.lty b .taU.r to tla ••••• ,. for SCIP i. tlle

aulM .,...t_. ., o.. It~al.te prilled C'l'L. la • 2' 1LIt, aacI IP la •• . ao .ff.ot 0& .. priM. l' 0.11. Or ... t1a1l1.t.4 b •• 1i PBL.. IN.e4 tll.

, • r

.tllral.tloa of priM. cn.. by ., .how" tk. . .... UaeUo. ..d

lat .. llty of r •• po.... .. r •• t1asl-atloa 1»,- tlae ori.iaal '.Ua1&1aU ••

a.lla. IP .~lblt. !t'. ao..,.oillo1ty by U'. abllity to atiaal.te ,

'--~-~~---------~_.----------------------------------------~~~.--------

)

(

, ..

-21-

~ •• po".S" • 11 ( o ••• ~ ... -...~~._.

Botll UotU. ù .Al •• aU y .... ~ ud 201 U.pof f <37,!.SO) .llow.d 1

\

ta.t BP ... SCIF. \ ~ •• ,.otl ..... 1y, ~ .. 1IiS' ... t.t.ot ,~ot.l. 1tlo.,..atll.th

•• olaui_ la Hclei fo~ tJa. p!:odutl0. aad ~a1.a.. of D.-2 f.ato tll. ILK

•• ,.~t .. t. Tn~ta •• t of tJa. ~ •• po".~ o.U. i. tll. 1· -.a "lth

p~otai. 1tlo~Cll •• :h lûi1tito~, ( ••• U ... J~c.yoiA) "Ul a1t~o,.t. th.

P~0l!!!!.tto. i. ..... ao. ...... r aa t.taot DNA ,yatk.tl0 •• ohaat .. y ••

ut Mo •••• r11y nq.ir.d for th. r.ocr ... rr of Bl'/SCJll 1_ th •

•• ,.nat .. t followl., ILC ,tia1LlaUoa. Tlli."u .hOWil by I-irr.4iat.cI .,.,.

G.U, "llioh .oald pI'Gd.c. BP if th. c.U... "er. _pproprlattly

.tia1Ll. t.cl.

"Ch.. aael Gordo. la_cl oo.flDl.cl that IF/n.-2 r.,U •• l.tl0Jl of

priMel cn.. "a, IlOt clu to aUo .. U.,... r.It ••• cI iato th. Gal tur •

•• ,.r~t .. t.. y .... r.ad Ro11iApoff ,Ia.",.cl tJaat SClF " •• Dot alterad

fpUowia, prdDOÙaUGD "i ta Aip li tr. of uU-J12-1: or .. ti-l •

•• tibocll... Th.7 ,.".,t th.t SCIF/n.-2 40e •.• ot c.rry' Ile .atl.e.i.'

cletUlliaa_t ••• cI therefor •• tia1l1.tio. of pri •• d CTl.. "a. Dot ... la the

MIe •• u •••••

1. e. iil) IL-2. T .el1 81toiaD .tia1Ll.t.d IL-2

Gllli. 11~. (121) yorklD, "ltJa 8url.. .,I.Di. 1y.pAocyt... .-cI

Mor.a. (132) "orki., "lth h .... 1'8rlphera1 1yaphooytl' llacl •• t.b11Ihecl

tll.t T , •• 11 .ito,.... ,ull •• ooac.u ... a11a A aacl pAytola .... ,l.tlat •• .. . oa •• tta1&Ïat. T h.1,.r 41111. to proliflrat. aadl procl.o. IL-2. 8111i.

01'1.1 .. 117 l'lhrr.4 to thi, lyapllokiu •• the T c.U ,rowtll faotor

---<.----------~--

..

"t b •••• o. th. all11U,. of tll. Tt"GP to ._at.la th .... ialliUty and

\ ,rolif.ratio. of a oytol"tio odl li .. "Ilick bel b •••• aiatala.eI lA

IL-2 cIe,. .... t ,rowtll for 10" teaa 01lltu... lJLtu.:,ti •• ly it ka.

b ... aJaow. 1Iy otlr..r ,rot,. ,,«Ua, "itlr. Il ... l)'llplaooyt •• U.t two B

0.11 .ttol.... pob" •• el .ito.... a T 0.11 eI.,.acI •• t i o.U .Uo ••••

aacl Prot.ia A ".r. botll oa,abl. of .t~ati.. IL-2 proel_ctio.. Th •

• xact •• 0Ja .. ia. of th. B 0.11 .UOI •••• U.aJ.at.el n,-l ,rocllft:tioa.

Coaclitio.. "llicJa. r.~lat. IL-2 ,roel_ctioD followl., .itol •• .. • ti_aJ.at!o.. ar. .iaUar to ooaclitio.. r.plati., allouU,.. -.a

.tiJnlateel IL-2 proel"ctioa (... Hotio. I-D). a .... ti.ll,. _tU'. . . , th".. eI.ri .... el T c.11. "ill ,roelu. IL-l (51). x-atu. tla,.ooyte ••

o.lh fr_ .th71lio O:.'.t aioe. aacI T 0.11 clephteel oeil popaJ..Uo ••

"Ul aGt proelu. IL-l (1'2). À4Ja.reac o.lh. or th. IJllPlaoki .. D.-l

are r.ttir.el for IL-l proclaotioL Ivilleel T c.ll •• Ul AOt ,rocltc. "

Pby.icocll .. ioally th.ra Ar. ao eliff.r.ao •• b.tw.aa IL-2 ,roeltoeel

aUo, •• , allouti •• a or t.-or .. u ••• .tlatlatiou •

P.rla.,. thi. rafl.ct. tha atl U,h' 010.. ati.aletecl by .ito •••••

ratllar th.. th. r •• triot.eI 010 .. 1 .x,p&a.io. fo!lawi.. alloaRtl •••

• tiaal.Uo •••

IL-2 40 ••• Ilow .,.oi •• eliff.r •• c •• ( ..... ctl0. I-B). Bow ..... r it

i. "el1 •• tabliab.c1 tllat lL-2 cI.ri .... cI lroa Il .... 0.11. c ... t1aaJ.ate

IL-2 t.r •• t oella' frOll .,.ci •• 1 •• r o. tla. pllJl0 •••• Uc .oal.. i ••.

rat. aacl .io •• .

• ...... r. II1IZ'W n.-2 ou ut .U.alat. la ... IL-2

-{

t

,-

-30-

1. D. Co.4itio •• a.lUl.tinl IL-2 Production

i) The Stillalu.

U~tt.al.t.d. or ü.a&ture T celll will Dot produce IL-2 (157). T

cel1 aito,enl 11Ich al CODÂ uut .PIIA. aad T cell d.pend.nt B c.11

aito,... luch a. PWJ( ad Prot.in A wUl IUalllah IL-2 production

(212). Prot.in A i. beli.Ted to Act ••• n .nti,.n in Itiaut.tin, IL-2

rel.... (220). Purified aatil.n. luch •• PPD .nd tet&JI1U tomi4 will

'stilllLlate IL-2 productio. only if th •• nti,en. are pr ... nted ta the

IL-2 prod.lIcer c.1l. on the • pprapr i. te .. croph., ••

(157.155.158.160.161.162.163,164). B cel1 aitosena luch a. LPS .nd

PMA C.lUlDt stiaul.t. D.-2 prodllction (153.1").' Cell. Itiaal.te4 with

exo ••• iT. CoDA will r.11I1t in th. proliler.tio. of T luppr ••• or 0.11.

ud th .. cI.cr •••• IL-2 prod1LotioL

50111111. aatisen. .10118 will Dot .tia1L1.te D.,-2 producUo ••

wll.r ..... craph.S.' pre-plllaed yith the .ntls ••• will Itiaal.ta D.,-2 \

prad1lGtiOL 'lUtharaora. u int.ct IILA-D uUs.n (or aD 111-1 uUs.n

O ... lM oelU (01 ••• II QU, •• ) O. Ua. uoraphaS. il r.q.irecl for

uti,.. pr .... t.Uo. ta the T oell. &Del ltiaal.tiO. of IL-2

prad.ctioL It il •• tablished th.t reoo .. 1tio. of for.i .... ti •••• ~

the T hel,er o.U la by rec:opiUo. of d. ford.. aatis.. i •

... ooiation with th. IILA-o (G-I) •• ti.enl Oll tJa.a .. c:rOfh.se ••

hperillentl weI'. perfazae4 i. w!aioll th. ......-0 IUlti.... of the

a.oropu,.. w.r. bloobd by • h.terouU •• r_. or .aJlOGloaal

aatilNlclie.. Alth01LP th ••• oeIl. w.r. Tiabl.. .IUI war. pre-plLl •• cS"

[~~

-31-

yitll th. soluble antil •• " they did not .xpre .. the BLA-D &nU, ••••

and co.uquently did' not ,tialÜato IL-l productiou. nereforo

,ti_alation .f D.-l prodution by ,olubl. &nti,o., ~e_, to have tho

.... requir .. ent. a •• tiaal.tion of T helper coll, "hich aro b.li.yod

to b. the D.-l prodllc.r cella.

Stiaalation by an allol.ndc da .. II anU.e. il I1lffich.t to

tri".r D.-l prodllctio.. "hor •• , tho clat' 1 a.ti,.ns are pOOl'

,ti_alators of IL-l production (3',37). lt is ".11 .,t.bli,hed that B

ceU, and _acrop.al.' expre .. _oro clat' II anti,.n, on th.ir coll

,urfac., than tb. T c.lls or other noa-lyaphoid cells. Consequently B

c.U,. and •• crophal.s are ,ood ,ti_lÜstor, of ll.-l prodllction by

alfolenale cls., II anti,on. Blockin, th. .xpr.ssion of th.

allo •• neic cl... II &Atllon "ill abro.at. ,ti_lIlation of IL-l

production. Purifi.d T c.ll, _ith rel.tiyely 109 o%presion of &LA-O.

12-1 aatilons .re poOl' ,ti_ulators of_IL-l production. Solublo cla ••

II &Ati,e.s, cell ... brue u:traots, or coll frapents. evo. in the

pr .... c. of a synl.noio .aeropha.o, "UI not ,ti.alato IL-l

produotio.. IAd.ad ,ti_ulation of ll.-l prodllctio. by al10.0 •• ie ee11

require. aa iatact viable c.ll aa a ,ti.lÜ.tor.

It has b •• n ,h09n that laapte. eonjlllaUd allto10'ous colIs ean

,ti.alata IL-l prodllction,' perbap. by a .echani.. s!aUar to .. a110.0.eic cell 'ti.alation. ne la.pten _odifie, th. synleneie ola ••

II aati,en sufficieatly so that it il recopi:ud a. • ' ford,n'

anti.en. Tr&D.foraed tuaor 0.11 lino, (eith.r yira11y or ch .. ically

trustoraed) aro allO lood ,ti.lÜ.tors of n.-l prodll~ction .Uher by

the pr.sontation of DoW tuaor anti,ans. or by th. reooJD.ition of viral

r

"

t -32-

or ah .. ically 80dified cell surface .nti,ens.

Finally. th.r. ha.,.e been reports recenUy that th. aonoclonal

antibody Orr-3 can Iti •• ht. cell proliteration and n.-2 production

(165.18-4) • It appurs that 01l'-3 can Act lib th. T cdl .ito.ens.

and ca. coapetit!v.ly block the ConA and PRA receptors. It has been

...... t.d that 017-3 can recopize and bind to the T coll .it0len

receptor. and thus triller the cells to respond. Boyover, in vie" of

the pos.1bility thst T ce11 .it0lens can b1nd via nuaerous receptors,

it il difficult to conclude that Orr-3. a 1Il0nociouai antibody. "ill

recopize ~ UDique .1ta,enic receptor. The physical effoct of Oll'-3

croIS liDl.:inl several ce11 surface antilens .ay be sufficient to

tri"er the cell. to re.pond and produce IL-l.

1. D. li) Acc ••• ory cell. and IL-1

Kacropha,e. or Adherent cell. are an es.ential cell co.ponent for

IL-l production (32,153.15~). It has been shoyn by nu.erous Iroups

that r"0'9a1 of adherent cells by nylon "001 col Dans. carbonyl iron

clepleUon. or adb_vnce to plastic "ill abrolate production of IL-2

followiAI ait0len, .oluble antilen or alloantilU stimulations.

Addition of 1-2' Adherent aells into purified but stilllu1atod T ceU

populations "ill restore n,-2 production. Addition of nOn-llonocytic

Adherent cells such a. fibroblasts, cannat r.estore tL-2 production.

Bow..,er. .acropha,.. do not produce IL-2~_ Sti.u.la tian of p1lrified

.acropha,es does not l.ad to the recovery of IL-2 iA the aplnt cu1ture

•• perua tant (167). Bowever. another lymphokine. orilinally kn..o"n as

l~hocyte activation factor (LAF). based on It. abillty to stimulate

:.

1 -33-

thymocytes to proliferate, yaa recovered frolll the supernatants of

activated lIlacrophages. LAF, or n,-1 15 produced by activated -----

lIacrophages, and its target cells are T lymphocyte (107). Oppemeim,

in collaboration yith Smith ~ Al. have shawn that n,-1 preparations

prodll0ed by purified activlted Dlacrophales do not contain Iny IL-2

activities. Mb;el li li. (109) and Smith and Lusson have shown that

purified n,-1 can prolllote IL-l production in a purified T cell system.

It appears that IL-1 can replace the role played by the Adherent cells

or lIlacrophages. Larsson has shawn similar resul ts of TI..-l repl cement \

of lIlactophale using IL-1 produced by the WEHI 33 celL line. Purifiod

IL-l dono can not stimulate ll.-2 production. Also purified T coUs

(alter romoval of adherent lIlacrophages and B cells on nylon yool

columns) yill not rospond to lIlitogons, antigons. nor d10antigens in

the absence of macropha&es. Boyever, stimulation of purifie..d T colls

by lIlitogens in the presence of lIacrophage derived n,-l doe. lead to

cell proliferation and IL-2 produc'tion. Addition of increasing

UlOunts of tîIL-l yill lead to a corresponding increaso in IL-2

product ion.

BOY does ll.-l regulate IL-2 production? Pal.cios and Moller

------(157) have shown that non-activated T cell.s (human PBLs) will not ~

absorb solnble n,-1 in th. supernatant_ whereas aitosons, antilens. or

t~~ alloanUgeu' stilllniated T cells can ab.orb IL-l fr.,. culturo

supernatants. It wa. suggested thon that uon-activated T colIs do not g ,

express the IL-l receptor yhereas T colIs, up04 activation yill

express th. IL-l receptor and therefore absorb soluble IL-l. In

paraUel " experiments, it YU shawn that colIs yhich can .bsorb IL-l

-34-

can .lso produce IL-2, ... hereas cells ... hich c.nnot .bsorb IL-l c.nnot

produce TI..-2.

lU togen stimula tion of IL-2 product ion is media ted by direct

stimula tion of the ll..-2 producer ce Il. ...... itagens can stimula te the

IL-2 producer cells to express the ll..-1 receptor. and at the s.ae time

stimul.te macrophages ta produce ll.-l. The IL-2 producer cells must

recoivo t .. o signals before it ... UI initiate IL-2 production. 1. e. the

mitogen stimulus. and the euhancing offect of IL-l.

Since it has been previously shawn that the HLA-D (82-1) class II

antigons are involved in .-ntigen .nd .lloantigen stiJllulation of ll.-2

production. it .... s questionod ... hether the class II antigons pl.y a

role in TI..-l production. (by the HLA-D or H2-1 positive macrophages).

or ... as it involved in trillcring the expression of the IL-l receptors

on the TI..-2 producinl cells. Blocking the HLA-D antilens on the \

sti.ulator cells (by anti-HLA-D antiser.) will prevent the sti.ulati~,

allogeneic cells or antigen pulsed macrophales to stiJllulate tlle IL-2

produoor oells to express their IL-1 receptors (155.157.158). Without

the expression of the IL": 1 reoeptor, the ll,.-2 producer cell doos not

receive the IL-l si&nal ... hich is required for ll.-2 production.

nen IL-1 ... as ex08eno~sly .dded to a cul ture of ll.-2 produoer

cells stiJllulated by alloleneie cells or soluble antigen pulsed

macrophages, previously treated ... ith anti-HLA-D antibodies, one could ~

not restotoe IL-2 production. nor could oue trigler the expression of

the IL-l receptors on the ' respouding' ce Ils. Addit10n of ll..-l to

cultures treated yith a control .utibody. or .n untreated culture yill

lead to enhanced IL-2 production. Therefore. this sUllests tllat the

'.

t -35-

HLA-D antilons are involved directly in stiaulatinl the erpression of

the IL-I receptor.

In 1 i,ht of the above erperiaent "here ll.-1 cannat restore an

anti-HLA-D blocked re.ponse, and incre.sinl IL-l addi~on ta activated

cells resulted in increased IL-2 production. it would appear that IL-I

plays an .. plifyi.J1l· role in IL-l prodnction. Al thouah TI.-l is a

cruoial co.ponent for ll.-l product ion, it ha. no stiau1a tory

capabilities by Hself. ll.-l cannot stiaula te IL-2 product ion in the

.l:) abaence of anothor stiaulatory silllal, 1. e. aitolen., alloa.n,ti,ens or

soluble antilens.

By blockillii the expression of tho HLA.-D allti.en on the sUanIator

cell. 110 stiaulatory anti,enic silllais "ere deliTered ta the IL-l

producer colIs, and the aacropha.os. Thu., thore "as no IL-l

production sillce the aacropha,e. "ere not .tiaulated, &Ad no IL~2

production since the n.-2 producer cells did not receive either

.i~als I or 2 (ie. HLA-D allti,en •• Ad IL-I). Sillce the IL-l producer

coll did not receive a si ilia 1 1 trOll the aJlti-BL.A-D blocked

. stiaulators. 1t " •• thorefore .121 stianIated ta express the IL-l

receptor. alld thus it did aot ro.poDd to oza,enously addad IL-l.

nere Jare .eTeral reports of T cell deriTod tuaor coll lilles

"hich a.y produce ll.-2 consU tutiT017. or follow in, aito.on

sti.ulations <169,170,171). IIost IL-2 producilll tuaor cell lino. do

DOt requiro Adherent cell. or IL-l to .,tiaulate or eùuco IL-l

production. The EL-" .ubUne "Uch la .n TI.-2 producer follow inl

aito.en stillulations doo. Ihow a r.quir .... t for phorbol ayrlatato

aco ta te (PlIA) "hich can replaco IL-l. Upon Itiaulation of EL-4 by

1 -36-

ConA or PKA alone some IL-2 yaa produced. but li,nificantly aoro IL-2

ya. producod il EL--4 was si.ul tu.ously .tiaulated yith PlIA and CoDA.

nat is the 1'01. of PlIA aAd hcnr do.. it fuaction as an IL-l

replac.aent in atiaalatinl IL-2 production? PK! and CanA tOlether can

al.o pro.ote IL-2 Iyntheail by .ou ••• pleen cel1. and huaaA POL ••

Ro •• nstreich and Mizel in 1979 (172) reported that aacropha,u

and IL-l can be replac.d by PI(A in the pra.otion of ly.phocyte DNA

Iynthesi. and cell proliferation. llovera.tl li. ill 1980 (173) shcnred

that PlIA proaote. teminal differ.nti.tion and accumulatiou of cells

in the SI. sta,e. Lib.he. I:iIuel .ll.!.l. (1 H) alla showed the aeLa

cells yere blocked in the G1 phase by PlIA and the "pas.a,. al thes.

cells throu,h the S phase yaa slowed. fillal1y St.dIer 11..!l. (175)

Ihowed tha t PlIA and ConA s Uaula tod huaan PBL. will produce IL-2 in

the G]. .ta,. of th. coU cycle.

Therofore the aode of action of PK! .ay posaibly be to prolon,

the SI pha.. of the cell cycle. IJld thu. prolon, the period

correspondin, to IL-2 product ion. It can al.o prev.nt pas.a,. of th.

cells inta the S phase which correspond. ta the IL-2 utilization

pha... Al thou,h PlIA can replace IL-l and .. cropha,e. in lel.cted

ayst_s. it is alao mown ta be able ta diroctly Itiau1lte IL-l

production by aacropha,es. aAd IL-2, production by T helper cells.

There i. no evidence ta .u"ost that PK! is an exact a .. lOlu. of IL-I.

or that PlIA and IL-l act by U .... e •• chaAi .... Thore la al so DO

• vid.nce ta sU'l.at that IL-l can arre.t IL-l producin, cells in the 1 •

G1 ph.... or th.t IL'-l can black p .... ,. of cell. into th. S phase.

Th ••• ch&Jli~s of IL-l .. plificatioD of IL-2, production are uaknown.

-37-

Pal acio. h.. .ho.n th.t COIIÂ .tiaalat.d IL-l product io... b,.

.. croph .... r.quin th. pr.s.nc. of OlT-04 positiT. c.lla. n. IL-l

produc.r is also b.l leT.d to b. aA On"-04 poai th. c.ll. Studlea

~rfora.d w1th Ul n.-l producia, c.ll li .. (lPla. uuI CFla3) ahow.d

that th.a. clo .. d On"-4+ IL-l prodaci ... , cell. w.r. cap.bl. of helpia •

• acropha ••• aad th. P388Dl .. croph& •• twaor c.ll li .... to prodac. IL-l

upon .ito ..... sti.a1l1ation. Th.r.fore th ..... c.ll whicll produc •• 0.-1

Uld .xpr..... the D.-l r.c.ptor w.. al.o capabl. of helpia •

•• cropha... to produc. 0.-1. Pr.l iainar,. .xp.rlaeata perforJMcI by

lb.Palacioa .uU.st that th. IL-2 produc.r c.ll .x.rt. a h.lper ~ff.ct oa

•• cropha ... throulh a dinct c.ll co ... tact aad i t il not •• 4iat.cl by

th. ni •••• of a .oluble factor.

Macropha.. conc.atraUoa. al'. orucial to IL-l pradlLct ion ia

tia... c1Ll tu.s. Co.c.ntratioaa of 3-1(" .. cropha ••• wUl .Ù .. o.

IL-l productio •• wh.r ••• addition Df ,r •• t.r th .. 10. .. oroph •••• ca •

• clepreu th. productioD of 0.-1. n.r. il .ca. eTiUDC. that

•• cropha.e •• ppr .. aiou of IL-l production .. ,. b. ..diat.d b,.

pro.t&.laD4iJL. (176). ID .xp.riaeut. pedona.4 i. which iDCloaethacia •

. Ul iùibitor of proata,lULdin. synth.sia waa ad4ed. a partial

abro,ation of .. croph •••• uppr •••• d IL-l pr04uctio. wa. a •• D. Bow'T.~

sipificallt •• ppr ••• ion r_.iud .t .. cro,ha.. conc •• tratio.. of

.r.ater th.. lOtI r.larcU... of th. pr •••• c. or aba •• c. rof '",)

iadoa. th. c iD.

U.refor. low COIlO'Dtr.Uon. of .. oro,ha... al" b ... ficial to

IL-l productio. Th th. aynt.huis of IL-I. AUitio. of illor.aaill,

.. ouata of purifiecl IL-l wUl had to all l.eu ••• ill IL-l ay.Utah.

-31-

It il lUllitely that .acropha,e auppreuiOD ol D.-2 prociactioD is

•• cllateel by the .YDÛuis of too auch IL-I • Proata .. laDcli.. froa

.. craph.... play a _al 1 role iD the •• ppruaioD of IL-2

prolifer.UOD. h.cnreTer the exact .ecllaa.i.. of ucroplla,e •• ellatecl

auppr ••• ioD is 1UI.bowu.

1. D. iii). D.-3 ~ac.cI aataratio. of the pr.-IL-2 proclacer cell

IL-3 i. a lyaphokia. proelacecl followiDI aitoleD. .oluble aati,eD

or a11oaBti,'D .ti.1l1atioD of uture T cella <178.180). CODelitioD'

for D.-3 prociuetioD are ideDtied to D.-2 procluetioll. BoweTer. D.-3

do •• Dot billcl to a DEAE colaaD wh.rea. IL-2 biads to DEAl cellulose

aDei caD b •• 1ut.cI with a .alt coa.ceDtratioD of 0.11M NaCI.

~i.i .. lly D.-3 waa ideDtifieci ba.ecl OD ita .bility to iacl.ee lOG

h:yclro~.t.roicl clehydrolea.ase '%pre.si'oD ia spleuic lyaphocyte. froa

athyaic •• /uu .ie. (17'). Th •• zpr ••• ioD of 20G SUI is b.li.T.eI to b •

.••• 0ei.t.eI with lyaphocyt •• ataratiou. Byclroeortisol •• uaitiT' c.lls.

aa.ociat.eI with th. J..aatar. c.ll. clerb.cI froa the cort.x of th.

tllyaa. are lOG sœ ulaUTe. wherea. the .atare cortiaou r •• istut

ceUs are lOG SDII poai tl'n. Tou, athyaic DaI ••• OU" cella are 10.

SDB DllatiTe. BoweTer. iacubatioD of Du/llu sple •• oells with IL-3 for

a .iDia_ of six hoars will iadac. the .xpr ... io. of 20. sDIÎ. Ali T

h.lper ce 11. (Tlay 1+, LyT 1+,2-), pre-m.s (na,. 1+, LyT 1-. 1+) 1

baaophil ••••• t cella aad utaral killer celh are 20. sœ po.itho.

"cropha.e., B colis, .atare ,r .. alocyt ••• aacI .rythroicl cell. are 10.

Sm! DlI.tiTe. BowlTer oaly basopllil. aacl ... t oells s._.d to

retaiDeel the reqair ... at of IL-3 for ,rowtll. CIOaiD, e%p.riaeDt. with

.- - --_._-----~ 'f -

, IL-3 la •• ri •• to ba.ophil •• Ad salt o.ll-lite cultur •• (111).

Exp.rluatl ".1'. p.rfora.cl "iUa IL-l cl.riT.ct froa T he1per cell

li.1 au 1EIIl 33 ooaditioucl •• cli_ UIO). n •• IL-3 ".a .clclecl to a.

lAiU.Uy ny 1- oultur. (l ••.•• , ••• ph •• c.lla). 1t ,rOllot.cl th.

cliff.r •• tlatio. -to 'Illy 1+ c.ll. ..cl both IL-l ,roct.c.r aaAl IL-l

r.apoa4.r 0.11a. ~

lt "oal4 .ppear that IL-l i. r.4air.4 for th. 41ff.r.atiatio. a.cl

aaturaUo. of a 'l".1ly 1-. hyclrocorti.ol a •• liti ••• lOG sœ u.ath.

ta.&tur. T c.ll i.to a 'Illy 1+, hydrocorUaol r .. htut lOG sœ

poaith. ceU. n. lOG SDII po.iti •• calla al'. th. ,r.ouraors of T

Ilelper 0.111. n.-l ,roelacer T cella .114 ,re-cn.a.

l. D. l'Y) Dru. ao4alatlo. u4 .. na faotor r.adatio. of IL-l

proctsctio.

." r •• po ... to IL-2 u4 th •• aba., ... t ".rall ~ 1' .. 10 ....

c,.clo.poria A o.s lùibit tIl ..... r.Uo. of cn.a au oaa 1Jr .... t

allop'aft r.jaoUo. aacl ,raft yara.. .at clb.... UI2,183).

CJ'clolpoS'ia A b. Ma. ùow., 1) r .... S' ~ .cUyat.ct T oell.

ur •• 9O •• h. to D.,-l. 2) to clecS' •••• IL-2 pS'ocIsctio. by S'eUeri_. tk.

IL-l ,roctao.r c.Ua au: .. po.ah. to IL-I. aU 3) to 1aIli_lt tJa •

.,..atJulia of D.,-l b7 _crOJlla ••• by ,rrn.Ci .. th. OU' 4+ plI trOll

cooperatis. "itla th ... cro,.a ••• to pS'Ocl1lO8 a-l.

CoS'tloo.t.roicla U15,18') 0.. ~ ... at th. D.-2 ,S'Mao.r oella

caaaot restor. D.-l ,roel.otioa "JlicJa b. H.. ..PPZ' .... ct by

- . --- --------

f -.<

\ /

4.xu.tlLa.o ... Bow ... r deUII.tIl •• OU treat.'" 0.11. oa. abao~b U.-l.

n.~efœ. 'tll.~. la IL-l ~.o.ptor .zpr ••• io. b_t a o.llala%'

'"", -' aar .. po •• iT..... to IL-l. Coriiooat.rold. oaa at.o deor.... lL-l

Pre1i11i.u~ .q.~u...t. perfome'" by Budt .t1.IJ.. (177) Jaa ••

Gl00 oa. iûlb1t R-l aoU.ity. Palaoio •• how .... tllat a faotor foud

la k ...... rua c •• al.o lûibit IL-l prod_otio. by tIl. IL-l prod_ci ••

0.11 li ... - lPlaa lad CPla3. n.~efo~ ... 1û1bito~ fo'" i. IIOnat

.. ~ua .. y ~ ... l.te th. t-... re.po ... by oo.trol11 •• tla. ~ .. po ... to

&ad ,rod.atio. of IL-l.

1. 1. U Cliaract.rhtlc. of tll. IL-2 p~04 .. r 0.11 - ".nin

t..at1lJ:. T oaU.. ...U.alat.'" T o.U.. or stiadate'" r •• U ••

c.lb will ut pro"'"" D..-2 (1$3). 'ollowi.. .ito.... sol .. l.

.. ti •••• or al10 .... .1o stia&lat10.s. tU IL-l pr04_cer o.U will --" _ 1 • a:p~ ... tJae IL-l r.o.ptor. D..-l p1'od.otio. la .. ,. ...... t o. Ul •

~ ... ac. of D..-l ... tIa. u:p~ •• s10. of th. IL-l t'ao.ptor o. tll. lL-l

pro41&c.~ ce 11. " ... r o.. .. D.,-1 pr04 ... ~ oeil la acU •• t •• to

prM'" IL-l. it do •• ut ~ ••• in tke orla1aal .U.al .. o~ U,-l fo~

tke oo.t 1 ... 1 p~04ut 10. of D..-l.

prMU.~ o.lb U ••• 1· ... 0.1l1r.~. U,-1 p~04u.~ 0.11 U ... ) ar •

.......... t oa tIa. stillal ..... IL-l for ....... t r.st1aalaUo. to "

a" •• ". of aay .tillal .. ).

( -u-

JL-2 pr04.otio. 1Jy aay ~ of o.U do ••• ot réq.ir ••• aoth.

DNA .yath.tio •• o ... i ... Kita.yol. C.tre.t.4. or 2000 ra. 1I'r.4i.t.4

0.11 •• Ul pro •• o. JL-2. 1Icnr ...... r 3000 rad lrr&4i ati o.. 07010 •• sialü . aM prot.ia ayaih.d. iaJailtltor •• Ul abrolat. IL-2 pr04actio ••

1. 1. Ji) Kuiu n-l pr04.c.r oelh

G11l1 • .t1 & aJUI .... 1'0 •• oth.r Iro.p' ...... âft. t •• t th. n.-l

prMa.r 0.11 ia • th.,... deri .... 4 n,. .1+ 0.11. Hoa T 0.11. of

lyapko14 01'1,1. U... B odl.) .111 aot: pr04.0. n-l. aeith.r o ••

• 0MOyt.. BOl' filtrobla.tt. 'uih.~. oal,. _tu. T odla .Ul

prMace IL-l. "tu. T 0.11. frGa .11 1189.014 or' ••••• pl.... 1~

..... peripll.r.l bloocl. to •• il ••• "Ul pr04.c. IL-l 8pO. appropriat •

• tt..l.tioa. T.byaocyt •••• 10. oo •• l.t of 8O.tl,. ~t1&l" T 0.11 •• 111

oal,. pr04~ 1-21 of IL-2 aoraa11,. r.caT.raltl. frua .. tar. .pl •• ie T

0111 01&1 t'ar" U.7) • ('l'Ilyao07t .. ' .1.0 holt .. oroplt..... .Jalo1l oa.

prMa. IL-I ........ tlal OCMQO ... t for B.-2 ,r04aUo •• ) S,.clflio

.. 1.ctl0. of oortl.o.. r .. 1at .. t tlly.ooyt.. .JaioJa al" th. ..tu. ,

~,..,oyt. pop1&1atio •• U1 âow dpUloaat1,. 801" D.,-2 p~04utioa tllu

..tr.~tio .. t •• tJayaocyt •• tiaa11,.. 00 .... 10 .tJayal0 .a/ ••• ~o.. d ..

to th. holt of • tlL.,.io. 1ro ... t. cio IlOt po ....... t1&l" T 0.11. &ad . 'th .. ~ lyapJLoi. oell. derlT.4 frua ,.na. a1&/ ••• 10. '1" ut oapabl. of

• n.-2 .... pro ... Uoa. 1Iaw ... r tlt..~. al" r.port. tJaat a,.. ..,.. .10. <,r •• ter Ua 12 8O.th. 014) .Ul pr04... IL-2 if appropriaul,.

.tiad.t.4' .itJa alloeatJ.... ... .lto.... ua.). 'utJa'l'IIOr. tJt.a

1 .. ,1. of n,-1 proclao •• lty th. Da/ ••• 10. are .tall.r to dl. l ... h

fo'" i. aonal aaJ.IIal.. TlLe pr04utloa of IL-l lt,. a •• d .. 1_ .ie. la

(

ct.,eu.at oa a 'l'hy 1+ "LyT 2- 0111 ,,1&1011. ... I, •• t. tJ&at tJ&h pa~ti01l1aS'

IL-2 p~octu.~ o.U .oa. cle •• 1ope 1. tla. appar.at ab .. a.. of a th,..! •

• aYiro .... t. ~r. al.o Ippaar to b. a .... tio r •• triotioa a. to th.

~oclutioa of IL-2 by a,.ct aa/a. aie. 11Jl4. aot all .t~aia. of a./a. 1

aie. "il1 pr04&O. IL-2 01' '%p~'" n, 1+ •• U. a:poa I,ia,. la Ti." of

th ••• azo. Ulle atJaJ'llio al0 ••

U.i., lpeoifio .. ti •• ra. it UI N ••• atabUù.ct by _.1'0".

~O." that n •• aria. D.-2 prChl1lO.r 0811 la Thy 1+. LyT 1+. 2-. ,,- ,+ aa4 (7+) Ua,). Caator &lUI Boyc. Jaad' ori.iully '''II •• t.ct th.t U. T

laelptr od! la th. &Il la 1 LyT 1+. 2-.3- 0.11. PUlr.k!.J.1.t.L. i.

1910 UJO} ....... t.ct tJ&a~ tIl. T Il.1,.1' 0.11 tor tIl. en. ZOtlpo ... la a

----LyT ,+ 0.11. PvtJa.1'IIO~'. tIl ....... t.d tJ&at LyT 1 &ad LyT 7 apptlr

oa th ..... o~lh 4_ ta tlt.izo laolt of oo.pl ••• tatio ..

nu.~tfor •• tIae IL-2 prCHIu.r la n,. 1+ LyT 1+.2-,3-,5+.7+,' ...

biolo,ioal1T tkil 0 .. 11 !&a. be •• ~lioat.4 a. tlt ... jor prolif.ratias

o.u la dl. 1- &L &ad il'beli ... 4 ta b. th. T ll.l"r .0.11 of tll. en.

r •• po ....

SJai1I~ 1' •• &1 t. k... beu aIlOira ila t!&. rat. Al tlao.p tJ&. rat

q.t_ laou th. .q.iTal.at of tkt LTT .. ti .. ~. to d,htia,ai.1& tla.

a ••• 'a of T o.U., li !&a. ben lia .. Uat rat IJ1&PkoOJ't.. deriT'.

frGa th. 1,1 ••• wl11 pr04.oe IL-2 ,,!&er.a. ~tv.r. rIt tJ&yaocyt.1 ar.

, 1. 1. 11) ..... IL-2 '1'04&0'1' 0.11.

A. la tll. auiat qlt_. tU Il ..... IL-2 pr04 ••• r 0.11 il aU,....

( .... ,-d.d . .,.d T c.ll. Bepl.tio. of T celh (1... slBe ro •• tUal> "ill

.brol.tl U-l prod.atioa. Stia1Ll..Uo. of B o.lh ." IICUlOoyt .. "ill

IlOt produ. IL-l.

T 0.11. 1.01.t.d fro. periph.ral blood or th. to •• il "ill le.d to

produ.r ••

th. IL-l prodao.r o.Jl ka. b... d.fla.d ph.aotypio.lly bY

0Il' 4+ c.U ka. be ... n.blith.d a. th. 't: hel,-r oell lA th. ILL na.

L •• 3.+3b+ c.lh .1". th. pat.U.,. T h.lperl iadu.r c.l h. Mitol ••

stia1l1.tioa of 0I.'l' 4 .ariched c.lh wUl r •• ut la 100d D.,-l

produtio •• "h.re •• GEr 8+4- c.ll. do .ot prodac. D.,-l.

1. B. iil) IL-l produlal cali Il ...

la hope. of obt.!ai.1 1.1"1' aad ooa.i.te.t qaa.titi •• of IL-l for

puif!o.Uoa pupo .... r •••• rch.r. b'i •• to .tady T o.U t_or c.l1

li ... which .iJht prodac. IL-l. Att .. pt. "'1" •• d. to prodaa. T cell

hybridoa •• by fudoaa of IL-2 prodacial T o.lh to &.1l .. hopt.ri ..

prod:aoial hybridoll.. Bowe.,.r. th.n are r c.ll d.rl.,.d tuaor 0.11

11 ... which prodace dth.r h1laall or .aria. IL-l ooaaUt.ti.,dy. or

followial .tlauatio. by .ito.e ••• r Mo.t of th... D..-l prodacial cell lia.. reqair. .r49th ia th.

pr .... o. of '-lm. fetai calf .el'lla. llarthuaor.. they "ill prod1l.01·'

IL-2 oaly apo •• tl.al.tio. by T cali .itol'.' 1. the pra ••• ce of fetal (

c.lf •• rua. th. LIaM 33, alld th. lurkat cell lia •• prodac ••• uri ...

· ( /-~-

aJUI h_u. D.-l r •• pectiTe11' <169.171). n.s. cell. au.t b. Irown lA

t-1~ PCS &Acl cio DOt proclnc. IL-l cou.titutiT.l1'. Bow .... or upon

.U . .a1Ila1:ion b1' 1. PlIA. Ir.at.r th&ll 1000 ui t./al of IL-l are

prod,1UI.cl. n.r. 1In. b •• n .a.. reports of IL-l pS'ocluct iou b1' PRA

.ti.a1llat.d, cell liu c.ll. ill '.1'_ fr .. couitions. G.uraIl1' 1 ...

D.-l aotiTit1' .a. reaoT.S'ecl in .eS'u. IS' •• ooadition.. Th.S'o wa. allo

a report of ou D.-l procluoinl oell liA.. th. Gibbou oS'ilin IILA 140i

.1Ii01l aan ,row &lld. pS'oduce IL-l aou.titutiTely iA appro%i .. tol~ l. FCS

' .. di,. (191.).

th.S'. al'. a f •• c.ll lin •• b •• id •• t1l. MLA which aaA produc. D.-2

coa.titutiTe1y, iIlal'udin, Tariou. suhlin.. of EL-oi. Bowe ... or. all

'aoa.titutiTe1~' n.-l procluciJI., cell liA •• are iaclucibl. by T coU,

altO'.Il' to prod1lCo a laS',.r quanti ty of D.-2. Porhap. th •.

'coa.titutiTe11" pS'oclncial c.ll HUI' al'. con.t&lltly .tiaulated by

l '1

•• ru. or tl .... culture aito,.nia factor.. Al te ma tiTely, th ••• coll ,l'.~

u ..... y repr".llt a furt1l.S') tez:.iIlally cliffer.ntiated c.U .hich

dq •• uot r.quir. a .t1.1I111. to iAitiat. D.-2 procluctiou.

1. K. tT) D.-2 pS'ocluction l.acl. to th. D.-l procluc.r aell death

It 11 •• b •• n .hawn that .o.t IL-2 produciAI 0.11. and, t .. oS' ce11

liUl. aallllot b. rep.atedly .tiaulat.cl ta pS'ocluce D.-l. C.ll d.ath

u.ua11,. ocour. follawba, iu:ractiou of D.-2 biosYllthui. aAd D.-l

r.l.a •• (192.219.168).

Bow ..... r Palacio. report.d OA two D.-2 Rr0duai •• c.ll liA ••• 'aplaa

a.d 0'2&3 .hiah could b. np.at.cll,. sUa1Lla t.d by PlIA to pS'ocluc. D.-2

proyid.cl that iA b.tw •• A cOIl •• cutiTe PlIA .ti.a1llatio... th. 0.11 ••• 1'.

-·45-

w .. h.d aud r .. uspe~ct.d in frash aedia. (176). It il qu.estiouble

wheth.r the s ... n.-2 produciu, c.U wa. n'p. a tedly sUaula ted sinoe

foUawiu, each PlIA stiaulaUou. coU proliferation occur. "hich aay

replac. tho population of dyiAl n.-2 producial cells. Th.r. is no

evid.nee to sUllest that th. PUA sUau.latld proliferatiul c.lls are

necessarily the n.-2 producer cells also. Ind •• d it is possible that

foUawiA, aitolea sti.ulations. not all D.-2 producinl c.lls w.ro

indueea to produce IL-2. Subpopu.latious of thes. D.-2 produc.r •• y b.

indueed to proliforate without induction of IL-2 production. It il

lenerally b.lieved that D.-2 production teads to doath of the D.-2

produce l' ce Il.

1. E. v) The h~an D.-2 lene

Bleackly ~ ~ (1983). Gillis 11 ~ (212). Efrat 11 ~ (217)

and Bans.n ~ ~ (216) hav. shawn that th. D.-2 .RNA •• xtraeted froa

variolU D.-2 produe1nl colIs (activat.d EL-". ud .Ta.rkats) can b.

trandated .lA yitro. Incl.ect D.-2 can bo .ynthesheet d. nOTO iA a

unopus laeT1s oocyte and rabbit re ti cul oey te c.ll freo protein

.ynthosis ..,...t_. 1101" rec.nU,., th. 10 .. ~or t.llo h-.. D.-2 bd b •• n

cloud and !t. ..q ... nc. &Aal,. .. et (211). A cDNA w .... cie fra. ~

.xtraet.d fra. Cod .ctiv.t.d la.rbt c.lh. n. D.-2 cDIU ha. b •• n

id.ntifi.ct 11,. ih abUit,. to hybricUa. to ta. n-2 IIIINA. Sequllc.

aaal,.d. of th. D.-2 cDNA "u cleUmiud by r.striction .adoll1lchas.

di, •• tlon "niA.. n. IL-2 cDNA h., tho pot.aUaI to cod. for 153

-iaoo acids nd al so co.tai.. tla. .ù.ryot. iai tiation codo.. n.

k .... IL-2 •••• i. locat.d o. ckroaosoa. 4.

-46-

AD IL-l plu.id DNA ..... constructed with the n,.-2 cDNA inserted

into the peE-ll pl.s.id and this w.s us.d ta transfect cultured monkoy

cos c.l1s. Biolo,ically active ll.-2 w •• recovered froa th. COS cell

speat cù tu.re .edi ••

Al thOllah .tt_pt. are b.in, •• de ta insert the IL-l cDNA into •

b.cteri_, preliaiJaary work in this .spect h.d be.n unsuecesaful •

(Bh.cUy, p.r.onal cO_1lD.ic.Uon). ne difficul ty wu due to th.

inability ta in •• rt the co.plet. n,.-2 cDNA into the b.cteriam

(howeT.r, inco.pleto short fr .... nts wero in.ert.d).

It la coaceiyable that nontually an 0.-2 producin, b.cteriam

... ollld b. aT.il able. lith ,en. tic enlineerinl, it would also b •

po.sible ta dllplicate th. IL-2 lea.e and produce cells/b.cterium

e%prusinl .ultiple n.-l ,enes, thllS ait uliaited supply of n,.-2.

With the 0.-1 cDNA, it wo.ld also be po.sible ta probe the existence

of the n.-l ,ea. w i th in the h_an ,.nGae.

1. B. yi) Elne tics of IL-1 prodllction '.

The kinetic. of IL-l production differs fros IL-2 recov.ry which

i. the otten llsed •• thod ta us.y for' D,.-l productioD. Specifically

the .o .. t of n.-1 recoy.red doe. n6t Dec •••• rHy ren.ct n.-1

prod.ction •• ince iD • hulk culture. n,.-1 i. b.inl used up. ~b.orbed,

or de.troyed .s qllicUy a. the n.-1 responder cella are Itiaulated.

Ali n.-1 prodllcilll tissu cul tu.re., wh.th.r 1t be aitolen or

.. ti,eD .tiaul.ted .pleen celh. PBL. cr tUllor coll lin... ex.hibit 1

,.Û D.-2 recoyery batyeen 16 to .. 8 hoars' followin, Iti.u1&tions

(35.37,169.170,171) • Boweyer, the iJaabil i ty to d. tect IL-2 in culturo

-<47-

superna tants a t an earI1er tillle may merely reflect the insensi tivity

of the ASsay systellls for IL-2 activity. It is possible that IL-2

production is initiated allllost ilDlllediately upon stimulation. ho"ever

it lIay tako up to 12 to 24 hours befon sufficient IL-2 have

accuaulated to levels which are "ithin the lower lilllits of the

available Assay systelll for n..-2 activity.

IL-2 producing cell lines (LBRM 33 and lurkats) folIowinJ lIlitogen

stilllula tions will maintain a high level of IL-2 recoverabIe in the

cul ture superna tants. The earl lest time in which n..-2 is detectable

is approxilllately six hours fo11owing mitogen stimulation (169.171). . ~.

Detectable level s of IL-2 production at 6 hours occurred prior t'a

detectable DNA synthesis and cell division.

In, normal spleen cells or PBL stimulations. peak IL-2 recovery

was reached at 24 to 48 hours (35,37). However, beyond the peak

periods. there "as a drastic decrease in recoverable n..-2. This was

partially erplained by the activation of T suppressor cells and

pre-CfLs which require IL-2 for growth and prol iferation. Wheraas 1

with the tUlll~r ~ell lines which produce IL-2, high levels of IL-2 were

lIaintained. since fe" LL-2 1I01ecules were absorbed or utilized by the

tlQllor cells as they do not express an ll.-2 receptor. However there

are reports of ll.-2 producing ceU l Ines which al 50 express the IL-2

receptor and are dependent on IL-2 for gro"ta (see section I-F).

I. F. Characteristics of the IL-2 responder target celIs

By definition. the IL-2 responder taraet cell woald express an

IL-2 receptor and util he IL-2 to initia te cell prol Heution and

-48-

diflerenUa tian into a funct ional cell. The pr iaed cn..s have always

b.on conSlderod the ll.-2 taraet cells since this population of cells

proferontially absorbed ll.-2 to their coll surface recoptorl. and as a

resu! t cytolytic acth'Hies yore erpressed. Bowever. in viey of

rocont findinls that T suppressor colIs as yell as T helper cells caD

oxpress IL-2 receptors on thelr cell surfaces. and the maIntenance of

T helper aDd T suppressor coll lines in lonl tera cultures by IL-2. it

bec .. e evident that the IL-2 responder tarie t co Il 1S.aQ.t rostrlctod .......... ta the CTLs alone.

Thorefore. the characteristIcs of th. IL-2 responder tsrget cell

yill be definod by 1) tho erpression of the IL-2 receptor whlch .ay or

may not lead to cell proliferatIon aDd dIfferontiation 2) the

tunctional blologicsi characteristl.cs e%prossod by the activated IL-2

target cells. and 3) the cell surface pheDotype of the various IL-2

rosponder cells.

1. F. i) ColIs exprossing the IL-2 receptor

UDstimulated T cells do Dot erpress the IL-2 receptor and

thoreforo cannot absorb IL-2 fra. cul turo supe rna taD t s

(35,37.155,156.157). Cells of other lr-phold or non-lYlllphoid linoago

do not erpress the ll.-2 receptor. Dar utilize IL-2. ThIS Includes B

cells •• acropha,es, monocytes, erythrold cells aDd fibroblasts. Some

T cell tmaor cell liDes will absorb IL-2.

Colls activated by T coll mito,ons such as CODÂ and PRA yill

express the ll.-2 receptor yhereas stiauiatlon by the B coll lutolen.

LPS yill not load ta ll.-2 receptor erpresslon. Sinee tho monoclonal

a.a.tibod1' 011'-3 can coapete with ConA a.a.d PIlA for th ..... b1ncliAI

sit •• OA the T cell. it i. net surpr1siAI ta f1ncl that OEY 3 caD alao n,-

triller IL-2 r.ceptor expressioD.

Alloanti,.A stiaulated c.lll such as iD a 1· KLK or 2· KLR wore

sti_alated to express tho IL-2 receptor. Incl •• d Ootila ~ ~

iJli tiall1' showocl that praed cells iD a 2· IILI. w111 absorb (aAd

ntilize) exoloAona!y appliod IL-2. Ihereas iA a l·.MLll the inability

to recovor sipificant levels of ll.-2 iD culture supernatLD.ts beyoDd

.8 to 72 hours was attributed to the absorption of ll.-2 by the ll.-2

respoDder tarlet colIs. The same phenGaenon was seen w1th auriA.

MLlb.

Soluble antileDs, 1f presonted on a functioDaI IL&cropha,e will

sti.ulate the expression of IL-2 receptors on IL-2 tarlot colIs.

Hoyever, purified T cells (doploted ol IL&croph.,oS) callAOt be

st illlul a tod by sol ublo antigens aIone to express the IL-2 recepto~.

Likew ise antilon pu! .ed lIIacropha,o s pre-treatod w!th a.a. anti-HLA

antibody calU10t Itilllulato ll.-2 receptor expression. It i. ,enerally

believed that the HLA-D, Hl-I aDti,ens are crucial ta antilen

• presentation to tho respondiDI T cells, aAd the subsequent induction

of the IL-2 receptors.

Contrary ta tho ll.-2 producer cells, the IL-2 rospoDder tar,et

coll doos Ilot necesurily have to b. a •• ture T cell. As .entioned

baforo, tyo assays for the presence of ll.-2 iDvolve the use Dl

b.aature T colIs (126,127,128). The a •• uaption i. aade that if a cell

can respond to IL-2 iA tenu of proliferatioD, it aust also expross

the IL-2 receptor. Paetbu .!.1 ~ haw. shown that thyaocytes will

r \

-50-

r •• poad to ,xoleo.ous11' .upplied IL-l 1f th. c.U. Ar. coacoaitaat1y

et!.1Llat.d with a aitol.ll. Libwis. Illla.rous Iroupe hu •• howa th.t

~ 1'oual Ilu/au spl •• a c.1ls which aora.lly 40 IlOt respoDd to .llo~til'De

call b •• tiaal.ted b1' .110 .. til.IlI iD tk. pr ••• llce of IL-l.

ner. hue b •• 1l reC.llt r.ports that D.-l call stia1l.1.t.

prol if.r.tioll of fr.shl1' iaol.ud but .nU"1l or aitolell a.altiau1. tcd

sple.a c.Us froa UU/DU aie. &lld aoraal aice (19-4.195). Th.I •• uthors

a.de the SUII.ltiou th.t uoa-.cti .... t.d cells cau express the D.-l

r.eeptor ud relpond to IL-2 in • pro1if.r.ti ..... "&Dar. Dow ..... r. it

i. d!ffieult to ••• ua. th.t fr •• hly i.o1at.d spleeD c.lls .re

'uolr'.cti .... t.d' . AIl le t.l cal f s.ru. cOllt.ius so.e aitolellic

acti ... ity for 8OU .. and hUllan calh. ID th. proc. SI of isola tilll

lyaphocyt ••• the c.lls could h ..... b •••• eti .... t.d b1' th. licoll hypaqae .. or oth.r eoapoullt. uot IlOraally f01Lllll ill th. .erua or the sph •• of

u &lliaal. Finally. .tudi.. of th. .atolo,oul .ix.d lyaphocyt.

r,spoll" (AJLll) ha.... .howll that autololOU& B cell& ud 8Ouocyte.

(which .re BLA-D .nd JU-I po.ithe> C~Ul staal.te .utolo,ou& T ceÙ.

to prolifer.te .Ad .xhibit çytolytic acti ... ity. Th. basic requireaent

to I.Derat •• po.iti .... AMLR w •• to ch"l' th. Ilqra.1 ratio. of B c.lls "

(aad aOllOcyt •• > to T cell.:

Can th. ~ b, • r.aSOA that a.afr.etiollAted .pl ••• c.ll. expre ••

th. D.-l nc.ptor? If thi. la tt'ue, th.A purified T c.ll •• whieh are

1.eU.I ill B e.lls .nd ao.ocyt •• tiaal.tiOlls, .hould Ilot .xpres. th, ,

D.-2 r.e.ptor s. Palacio. &lld F.rnand.z <ln) ha .... SÀOWD th.t IlYlOD

woo1 purified T c.lls will Ilot r'lpoad ta eZDI'ao •• IL-2. whar.ae th •

.... populatioll of a.afr.ctiouud cell. Ixhibit lood proUf.ratio. ta

-51-

.%OI,IlOUS D.-2.

Not ouly do prt.ed CIL. and aito,.1l .tiaalat.d thyaocyt ••• xpre ••

tlI.. IL-2 r.ç.ptor. both th. pataU •• T u1per eel1 ud. th. T

• "ppru'Ol' c.ll .xpru. th. D.-l r.c.ptor. E ... ide ac. for thi •• a •

deriY.d froa T a.lper Uld T nppr ... or c.11 Hus .hich an. ba.a

•• tablish.d ia D.-2 depaad.llt ,rowtla.

Na..ro1l. ,ro1lp' h •• e pre •• llt.d .... idellc. th.t ODe a.eh .. i .. of T

.uppr ••• or cell action ••• by ab.orbiA, .11 tha D.-2 (191.199.187) .0 th.t oth.r c.Us (1. 1. T a.lper &Ad cn..) .ill aot r.cei •• the IL-l

sipal. ExperiMnt •• ere perfora.d iA .hich iaer ••• illl a .. bers of &Il

I.tablished T luppre •• or cell 1~1le w.re added ta a caltare coat.iailll

aito"1l .tiaulated IL-l produeia, e.lli.

oeil additioll. 'a decr ••• illl "OaDt of

tryp.i. tr •• tad T suppr.s.ors .er. Ilsed •

With iner ••• illl T luppr.s.or

D.-l w.. recoT.r.d. n'Il

(tmsia c •• r_cn-. the cali

• urlac. n,-2 rec.ptor.). DO deer.... of D.-l r.co .... ry, ••• "ail.

n.r.for. T 'Ilppre •• or cell. 40 .xpr ... th. n.-2 r.c.ptor &Ild a_y

pouibly ha ... a aip.r dfiaity for D.-2 thu th. receptor .xpr.ss.d.

ail T h.lper or CIL cell •• Bow ..... r. olle callAOt nü. out th.

poasibility th.t .... ppre •• or factor .. y dir.ctly .ffect the r •• pon ..

of n.-2 r •• poùar t.r,. t c.1la to n,-2. Fur the ftlor. • a .uppr ... or

f.ctor. wh.ther i t be '%pra.sed on th. T sllppre •• or cali lufae., or

be .. creted by tha T ."ppre •• or call diractly iafllllaea IL-l productioa

b,. tha IL-l prod1lca l' ee11.

I. F. il) F .. etioul biolo,ie.l ch_ractarhtic. of the D.-2

r •• poù.r tarlat cel1.

-52-

It i. w.ll .stabli&b.d by th. dilf.r •• t ,rotp. of Uoti\a 11 ~,

Y .... r 1.1~, &ad Brv..aaer ud Cerotti.i tla.t th. prt..d en.. Ar. the

tar,.t c.ll. for IL-2. Additio. of IL-l (BP. SelF) to a l' KLR wlll

r •• ult i. c.ll proll'.r.tio. &ad .... r.tio. of aytolytic T c.lls. Th.

cytolTtic .cti'Yity , •• rated i. directly corr.laud to the late.dty

uul UuUc. of IL-l iadte.d cell prolHer.Uo •• Ph •• otnieaIIT.

It ha. be.. .how. that 1Ulder coll4i tioas la which a ".ak C'Il.

re.po ... " •• ,eurat.d < •• ch a. ~iti Bl~.D i4e.tic.1 .tr.ia. of aice.

\ or with BLA-A.B.C ideatical iAcH.'Yidu1s) r •• tialliatio. i. a l' lIl.a bT

IL-l .1.0 l.ad to • poor prolil.rati'Y. r •• poa.. "ith littl. or

M,li,ible C'J't'olytic .ctbith~. - Altlaotp 80.. CIL proUleraUo.

w.r ••••• i. ateh .,..t.... th.T ".r. probably due to CIL prolil.r.tio.

wllich ".r •• tiaul.ud by • aoa-.C atiatIll ••

ReUiall1.Uo. by IL-2 .iD • l' JILK ahow. tllat proUl.ratio. àad

peak cytolTtio .ctbity coi.cide.. 1 • ..ri .. JLJl peak prolH.r.tio.

ud en. .ctbity occurred 14 hollZ. followia, IL-l r •• tiaul.Uoa. la

IL-

r •• poa •• "aa .... UT .t 41 hour. (32.200).

cella cleri'Y.d lro. allouti,ea ud IL-l .Ualll,ted '\

r. alao cytolTtic I,-pJaocTte.. 'l'llyIIocyt.. froa

C57Bl/6 w.r. atialll.ted "ith PlI' tuaor e.ll. ia th. pr •••• c. of IL-l.

Sp.cifie C57BI/6 aaU-PIl' CIL, w.re reco'Yer.d aft"r fi'Y. claTa.

Yithollt th. additioa of IL-l ... ,li,ibl. en. acti'Yiti •• ".re reco'Yered

froa th. thyaocyt ... or w,ith all/all aple •• eeU ell1tur .•• (137.1'6).

Yith th. ree •• t u'Y.lopant of ,rowi., IL-l d.pead •• t e.1 la iD loa,

t.ra cultur ••• it h •• b.ea po •• ibl. to ,row cytolTt1c c.lla of 'Yarioll'

..

-53-

.peoifioiti... CIL c.l1 Il ... k ... b •• a .a1atala.4 la IL-2 4.,ea4eat

.. ti •• a •

.. ti •••••..• ) &ad 0.11 li ... la .... b ... 4.riy.4 froa aor.al aJUl .Ü.,..ic

a./a • • ie ••

T k.l,er c.U, aJld T .a.pp~ ••• OI' c.U, .caa r .. pou ta D.-2 ia •

T helpel' cell. loI' th. B cell uUbo41'

r.apoa .. , aa4 for tll. en. r •• po ... Uy. b •• a •• oe ••• lully ll'owa .a4

010 .. 4 by IL-l. Fulk.mor. aa n-l pl'oel.ell' c.ll lia. (b.lley.el to

• ~ r

pl'0111eratloa i. loal t.ra calture.. Thlr~_.re al.o r.port. of a T

•• ppr,.,01'·cal1 li .. yhich y., .. latai .. 4 ia IL-l 4.pea4lat IrowtÀ .. d

prolif.ratioa (201.102).

Yiü. iAcrea.ia,ly refia.d t.ehalq .... '1~ 1. po •• ibl1 to ,hot that

.lao.t aIl ,Gb •• t. of T c.l1' yi11 r •• poad to D.-l ta t.~. of ,rowth

aa4 proliferatioa tll loa. t.na oultar ••• RoweY.r B c.ll ••

.. oropk..... ba.oplails. ...t c.lll, fibrobla.t, &a4 NE c.l1a al"

pl'obab11' DOt I.pport.. br n,-1 ia ,rowth .. d pro li ferati ail.

Pre11aiaary reports that B c.ll. a..s NI c.U, yare ,tiaulateel by

'IL-l' cal tu. a.4laa .. y la '.ot b. 4 .. ta th. B c.l1 .rowth factor

(BCGF) aacl iaterferoll actiyity l01lllc1 ia .oat cruel. prlp.ratioa. ol 'l

D..-l. Libyia. D.-3 yhick al,rat .. clo .. l1' ta n.-l oa S.pha4.z G200

"7 be re.poll.ibl. fol' .appol'tia. IrOYtÀ of ba,aplail. aad .a.t celll.

1. P. iii) Cbaractlri.tic. of th •. D..-l d.pelldea' cll1 li ...

Thel" al" maaero., report. of D..-l 4.pe~'llt calI li ... ykick al"

-54-

oytolytlo c.lla. A11 oytolytio 0.11 liua .ùibit .C r .. trict.4

cTtolyd. 4ir.oU4 towarda U.! ... ~i,i .. ll1' .U.al.Ua, allo .. U ••• or

.odifl.4 .110 .. t1 ....... t..or .. t1' ••••

Gl111.' (134.151) CILL-2 cytol1'tic 0.11 li •• ka. b, .... iat.i •• 4

t. D,.-2 4.,. ..... t arowtk for ',..u.. ni. cell U .. r.taia.cl iu

ori,isal cytolytio .pecifioit}' 4ir.ct.4 t\.r4' th. PBL-3 l.abais

tu,.t o.u. ~ CloAiJl' ual,..i. of U. ori,lu1 C'ILL-2 •• 0.14 that

th ••• clo ..... re rl.tricUel for th. ~el .C re.io. sd 'pecific for

th. nL-3 trudonaed t~r c.U. n. CILL-l c.U U .. la total1,.

cla,. .... t o. U.-2 for aro.th &ad prolif.ratios. It biC'"

~ •• po •• iyl to .ito,.. .t!aalstioD .d .arpri,ia,l,. 40.. aot r •• poad

Uu.. C1"l;,L-l cio •• Dot r.qv.ir •• te.d.r sor fUler c.U for arowtll.

It 40 •• rlqair. to b. f.el eT'ry 24 to 48 hoar •• tth • fr •••• ..,11' of

D,.-2. ae.OYal of D,.-2 for 14 hoar. al tlilst.l1' r •• 1ll t. ta o.U cIIath •

.... win aG.t n,-2 cl.,..4 •• t c.ll 11 .... tUy ucl.rao • 'criais peri04'

,.ri04ic.ll,. •• ch.r.ct.r1&.4 by decr •••• d cel1 prolil.r.UoD .. cl

r.pl4 c.11 ela.th.

~n,. .. broob" L3 cytol1'tic c.ll U •• i. cl.peu.at o. n,-l &ad •

f111.r 0.11 1.,..1.' fo~ ,rowth ... p~olif.r.tio. (203). th. fill.r 0.11

"y. or .. ,. aot b • .,.. .... ic .itll th •• U.1ll.tcw la tIl. ori,lu,l ...

Ira. •• ioh th. L3 U .... , cIIriT'cl. na.. fill.r c.ll .ppear. to •• ppl,.

• 'o.1"c, 01 1. azolltriot.cl aA.r •• t c.ll pr_otia. eff.ct. Myl0. -wool pvifi.cl c.U. ca .... t b .... 4 •• fUlIr oella as ~y wUI aot

•• pport L3 c.ll ,rowth la th. pr ••• ac. of n,-2. n. U c.lh .re n.,

1+ LyT 1-2+3+., aacl tt i •• pecifio to..rda tIl. l1D4 .110 .. t1 ....

r"

-55-

.,.oifio for ü. _1. KY .. U.... ..d,.e.. of Ü. .Uor

ki.tooo.patibility ooapl.z aad .azoio ••• izo •• &ad ok .. ioal1,. .adifl.d

(204.203.205.20'>. .

• Gla .. brooke J1 ~ ka"e al.o e.tabliak.d a àoa-oytol,.tl0 T k.l,er

oeU lu.. wkiok il ",,.u..t o. IL-2 ... a .. S .tillalaU •• teeder cell

lar.r for lZo.tk aad noliferatio ..

na. D.-2 ete,. .... t. D.-2 ,I:od.ol •• U T ut,.r •• 111 oa. r.plao.

IL-2 la ... iati., tlae L3 o1'tolyUc oeIl. to po. .... kill U. 13

.,.oifio tazo •• t C.UI. l .... d. ..,e&'Utaat. of .. S .U.alated 1.2

oell. co.taia D.-l aoUYity wkiok ar. ca,allle of stiaüati., tk.

,rolil.ratio. of &Il D.-l cIe,..cIe.t C7tolytic 0.11 li .. , ooope~at.,yitk

.ito •••• la .tiaalaU •• aonaal '1"1 ••• oeil ,roUleraUo. &ad .utaia

D.-l ........ t o.U U ... i. Irowth ... ,rQUfIeZ'aUo ••

ne D.-2 prod~d .". tla. U oell li .. follOlrl •• ILS .U.alaUo ••

1. i".tioat to D.-2 deri".d fzoo. .ito, ••• 01: alloutl, •••• tiaalat.d

.,1 ••• 0.111. n .. uOl of D.-2 ,rH.otio. b7 üe 1.2 0.11 li •• ar. ,

.iIIUazo to tllat of &Il lLIl or .1to •••• tiaalat.d U.-2 pzood.ctioa. na.

1.2 0.11 U. ezJaibits peak D.-2 reGO •• ~ st 24 1Io~. fOllowi., ILS

.U.alaUo.. Alter.' Mus. ~" ya. a clra.Uc "or.a.e 1. D.-l

r.oo..&'a"h 1. tk •• .,.rutut. Da,zoadatio_ aM _tU taaUo. of n,-2

b1' tk. 1.2 c.lla ac~_ted for tka .. or.... Ua r.oo".rabl. B.-2.

Out 1aa1ated 1.2 o.U. oaaaot nodsoe n,-2.

de,. .... t o. n.-2 fOI: p'owtk aa4 ,rolUeratio ...

-'6-

o.U •

... N .... l Û .J.k (202) il tlle fir.t IL-2 d.pe.d.at e.U U •• wkiok 1

.ùi1tu ... pre •• or fwaoUoa •• w.ll .. asti, ••• peolfioity. Thi. T

.~ •• ao~ 0.11 11 ...... t ••• 70.000 daltoa •• pp ... ~ factor wkicll

oa~ '1IPP~... t.k. ulp prOYl48d by T ulpera to i~.O' B 0.11. to

p~0411Oa astUto4y 41r.oted a.ab.t tIl. SIBC ,l"oo,llori ... U,... n.

.appc ••• or f.otor i. 1.- as4 1-1-. Yitll tla ••• '.bli .... at of tlais

.. ~ioa1.~ 0.11 11 ... it •••••• t. tllat tla.'rol. of T •• pp ••• or oall •

. • çpr •• ~. oa. p~04.oe as uti... .oifio ."PP ..... or faoto~.

"eT.~. tllia T .app~ ... or oal1 ... 1». aai.tai .. 4 by D,-2 la 10a. '.111 odtv.. .2) r .app ••• or o.lla oas at.o fDOUO. by .ûiblt1:a. D.-2

f~ oa.1 tv. • .... 1:11& t .. t ••

.... r •• aor f ... tlo .. oo-.zi.t.

1. G. Co_iUoa of D.-2 lat.raoUo •• ltll tll. IL-2 no.p'tOl'

u n .. u .. of r.oaptor .apra •• lo.

na.... ar. "i"ao.. to ...... t tIl.t D,-2 • • iat.~.oUo. .i tll ~

•• tbata. T o.lla 1 •• i. as IL-2 r.oaptor. Bapr ... loa of &Ja. IL-2

noe"or ~ oo_lUo_l .poa, 0.11 a.ti.atlo. wur... ..Ua&1.' •• •• U. 40 .ot .spr ... tU D.-2 no.ptor. WU.ll,. It w •• .u.. tll.t

alto .. a or &atl •••• tillal.t.4 T •• 111 .Ul ".orb IL-2 fr_ ... Uu.

•

-57-

aoti.at.d c.l1. i~oapabl. of ab.orbin. IL-l. Th. a •• -.ptio •• aa .ad.

Uat trypain r .. ~.d th. IL-2 rlo.ptor.. Both aotl •• t.d .iabl. c.ll.

or .11ltaralùla,-ù fiz.4 c.11 •• ill abaorb IL-l. Sua •• 11ltarald.h,.d.

tr.at.d o.lla Ar. ..tabolicaUy inaoU... U .oald appear Uat

abaorptio. of IL-l do ••• ot nq'llir. &a .as,..tia .... t as 10.' a.

tll.r, 1,. '%pr •• do. of tIl. IL-2 no.ptor. n. futh.r fiAdia.a Uat

IL-2 abaorptioa i. tta.,

.tr ••• th ••• tla. b.llet that n.-2 &ad 0.11 lIlt.notio. i ... diat.d by a

.peaifl0 0.11 ... br ... r.o.ptor.

SLao. oal,. aoti •• t.4 T 0.11 •• zpr ••• th. IL-l r.c.ptor. it .aa of

Ut.n.t to .t"" U. u..tia. aad r.platio. of U •• zprudo. of tIl.

U..-2 no.ptor III U. Iaope of ud.rataadia. tIl. IIUJclaeai •• of T oeil

soti •• Uo.. St .. i.. by Lar •• oa ... Co.tialao (lOi) .... GrolWik aa4

of t'Wo to fou koua, foll •• 4 by tIl. r_oyal of clat .Uo •• aio

atiaalu r .... r •• tIl .... otl.at.4 T oolla r.apo •• i •• to ':&OI'ao •• n-2

ta a cio ... peAde.t ..... ~. T 0011 •• Moll •• r. CollA pal.04 for h ••

tIl .. t'Wo llou. 41. ut rllpo" to .so •. aoa n~2. Siaoe fou uu. Cod ...... '.ti.aa1at •• T 00111 0 .. r •• po" to

.so...... IL-2. tU ...... Uoa .s. ._ dast foer Itou Cod

pal .. 4/sotiYaU4 T oelh .Ul '.Epr ••• clat IL-2 r.o~,tOl'. 'uU.naor ••

o~o T 0011 •• oro .otl.at •• by .ito •••• to '.Epr ••• tJa. IL-l r.eoptor.

tIae ori;!_l .U..... (aito ••• ) .a. DO lo ••• r r.q1lir • .cI. S.aoq •• t . -

.0U ,rotU.raUo. la i ... ,. .... t of tU pr .... e. of tJa •• Uo •••• ,

r.Ô.r it il cIe,. .... t o. th. a •• UsJUllly of D,.-2. no '%pr ••• ioa of

~ D,-2 r.o.pt~ by fou Iloar. fol1awiaa ati.al.Uo. ocou, prior to

.. "

-51-

4.t.otabl. DNA ~th •• ia aad c.ll 4i.isioL

llill.r .u .AL. ha.. • .... at.cI that 12 JaoU's 014 allo .. tl •••

at1aa1at.cI 0.11 •• il1 dso upr ... th. B.-l no.ptft. n. clilfereae ••

b.w •• a tk. U •• Uo. of n,-l ree.ptor .spl' ••• ioa follo.ia. aUoeati •••

01' aUo... .tilnLl.tloa .. ." r.fhet tk. aoaoclo_l .UI .. pol,.010_1

-.t'U'. of th. sU.-.1... lt la d.o posaible that 4iffu •• t .ab .. ts of

'T 0.11. aa." .~ibit .iff.r •• t U .. tics of B.-2 r.o.ptor .spr •• sio ••

o.c. a c.11 aoq1lins tJ&. B.:-l r.e.ptor, il r_aiAI r •• po .. 1.. to

B.-l. Aoti.at.cI nt r .. tiAI c.U •• (i ••• 1<4 .. ,.. 014 'le ... c.11.)

al tJao.p AG loa •• r bl.st-lib prolU.rat1A, o.U.. are f1Lll,.

r •• poasi •• to B.-l ... 'Dibit tJ&. n.,-l r.c.ptor. 0a1,. .0 .... oU •• t ••

T o.lla .... trypsi. tre.t •• T c.U_ .il1 ut .sprus tllt n.,-l r.c.ptor

(_a T o.lls cio _t .spn.. lA n.,-l r.eeptor "'.a if tll.,. •• 1"

.oti •• t •• blast-lib 0.11., ~ ••• LPS .ttaslat.4 • 0.11.).

1. 8. li) D.-2 - n.,-l re.tptor tat.racUoa

ne stadi.. o~a tJ&. (B.-2 - B.-2) r.o.ptor iAt.raotio. ".. .. ••

po.sible by tk ••• aU.bllity of .. iAt'.naa11,. r..tio.cU •• lab.ll.4

B.-2 (20'). ..... B.-2 pro...... " toIl. lubt oell \U.. • ••

.. ' • .oceset .. l,. labeU •• "Ula 135 _ûioai.. ... B3 leui". Alt.r

pu-UioaUoa. tlle pe.k r .. ioaoU.ity oorr •• po" •• with th. D.-l

biol0.1cal1." .cti •• fr.otio •• o. ,.1 filtr.tio ••

n. lab.U •• D.,-1 llao... Uat .cth.t •• T c.lla. I"ch as IL-l

........ t c.ll li ... (boÜ T Il'el,.r ... c."tolyUc c.lll), aUol.a an4

alloaatil.a acti •• t •• aari ... pl.ea c.ll.. üy.oe,.t •• aa4 h .... PBL.,

.spr •••• eI th. D.-l r.e.ptor. n ••• r.sal t ••• 1" coalht.nt ,,1th the

-,9-

clata .llieJa ...... t tJaat olLly .otb.t.cl T c.11. "Ul .baorb 0.-2.

PvtlLeDIOC. Robb .l1 .IL. ûow.cI tlaat h_ .. 0.-1 (J'uut) "UI biad to

D.-l reM,tor •• çr .... cI o •• eti .... t.cI _a.riAe c.l1.. Al.o 1I.IÛ.b.ll.cI

Il .... _ito •••• tiaalat.cI IL-l ••••• 11 •• r.t .pl ... toa! iad.c.cI 0.-2

.UI o ..... te for the lab.lbcl luut U.-l biacli., to IL-1 reo.ptors

.zpr ..... cI o. lnIriae c.u.. n ••• r •• ù t. coat iraecl th •• pec! •• cro ••

r •• oti ... it! •• of 0.-1.

lAter.ctio. of 935 or B3 taMU.cI 0.-1 "ith • T h.lper ... d a en.

D.-l cI .... cIe.' o.U liu aJaowed th.t .t 37'C a&zialla lnd. of D.-l

.. tab •• r. ob •• rT.cl .ithi. 15 ai •• te.. At "'C .atu.ble IL-l 1lptab

occvr.cl .ft.r 60 aillat ... It .a. aJaow. that 0.-2 had • r ..... raibl.

a .. ooiatio •• ith the nceptor. At l7·C. 5a. of the lab.llecl IL-l

cUlSooia'eci fra. the oella "ithi. 40-50 ai.at... lAt.ct cU •• ooiated

n,-1 ••• recOTereci. 1Iow ..... r. th.re ••• al.o .. .UJII&tic cI.pend •• t

clearaclation of rece~tor houcl IL-l. Alter t'wo hoars .t 37'e. ,reater

th .. 7a. of th. cliuociatecl r.clia.cU .... n,-l."a •• ot ... oci.tecl "ith

Tel pr.cipitabl. prot.i.. (co.trol TCA preoipitatio. showed th.t

ar •• ter tha. 97'- of th. r.clio.cti..,. 0.-1 ".s _ pr.cipi table). Bowe..,er

at 4'C UtU. or DO cle,r.daUo. of lab.lled ll.-1 "u ob •• rv.d.

n.r.for. IL-l receptor int.r.ctio. ".. char.cteriz.d by rapid

... ooi.tio. (15 .i •• t •• at 37'C), ~ slow re..,ersible dissociation of

batact 0.-1, (40 to 50 ainutes at 37'C), . a.d an .Il%ymatic, te.p.rature

a.d ti •• dep.nd.nt de,r.dation of receptor bOUAd IL-2.

1. B. Physicocheaical characteristics of 0.-2 --

Physicocheaically thore are slilht differences b.t1reen IL-2 of

4iffer •• t speeios. AlI n,-l of aurb .•• h1l8&D. or othor speci •• or ilin

ar. prote... MDai U.,.. (1. o. trypdn. Chy.otrypaiZl. subtil Isin.

leuiu _iaopepticlase). n.,. are DOt .. n.iU .... to Ilucl ..... (DN ••• 1

RN ... > 1101' utU_iai4a... AlI D.,-l. teat.4 so. f.r b ..... a pli stability

ben ••• pB l.l to 10.0. - D.,-ls Ar. st.bl •• t 70·C for 1~ ain.tes, th.,.,

.r. stable in 0.1~ SOS aad stable in 6.0M uze ••

lhziAe n,-l bas • IIOleeular w.lpt of 15.000 da! tODI, i t c.n b.

eluu4 fre. a DEA! ion nch_le ru iD • t pB 7.4 wi th a 0.185)( N.CI

salt IrecUent. D.,-l ean b •• laUd froa carboxy •• th"l •• ph.ro.e at pB

6.0 with 0.050J1 N.Cl. lIurin. n,-l 4.rh.d froa CollA Itlaulated spleen

-cells laas CYo aolec1ll.r speei .. with iso.l.ctrie POirS of pl 4.5 an4

5.4.

Mo.t la_&JI D..-2 hal a aoheular welpt of 15,000 dalton. with a

pl at 6.5. It C.1l b •• l1Lted froa DEAE ion .xeh&Dl. resiA .t pH 7.4

with 0.050 )( NaCl, allAi .l1Lt.d fra. Carboxyl &eth,.,l sopharo •• at pH 6.0

with 0.ll5 )( NaCI. H_an n,-2 can show thre. dilf.r.Dt specie., a, ~.

l. b .. ed OD th.ir delrees of ,lyco.ylaUon .Dd sialylation (lI0) which

are refloc~.d in difforont iao.loctric points.

A. roported bl' Moehizuki (211) n,-la prodllo.d bl" n,-2 produe.r

t'a.or coU lines h • .,.e sliJhtly different isoel.ctrie points. The

Jubt la.an ll.-2 h •• a pl at 7.75. The aous. LBRII 33 cell line

producod an additiou.al n,-l spechs with a pl betw.eD 3.9 .nd 4.2.

Biolo,ic.11y t.or cell lin. produc.d IL-2s are iDdistiDluishable

frOID IL-2 produced by aitoseD sU.lllatod spl •• n c.lls or PBL ••

Gillis II AL. have deve10ped a SDS-PAGE purification of IL-2

which laiaht h.lp in und.rltandinl th. aol.calar het.rolen.i tl" withiD

-61-

'IL-2 proparations' (212). Murine LBRM 33 tumor coll lino n.-2 "às

purified by sequential gel fil traUon. ion oxchahge ehromatography,

and preparative flat bed isooloctri\ foeusing. 'fhen the various

.peehs of n.-2 (pl 4.5, 5.4, and 3.9\ to 4.2) were applied to a )

SDS-PAGE, seven to nine protein bands ;lro rosolvod by eoamassi blue

stainini. Howovor only a fo" bandsC,,;ro associatod with signifieant \

IL-2 biologiea! activities. For the' pl 3.9 to 4.2 mur ine IL-2,

biologieal activitios "oro assoeiated with protein bands of MW 25.000

and 21,590 daltons. For the pl 4.5 IL-2, activities "ere assoeiated

"ith MW 27.4100 and 28,000 dalton and for the pl 5.4 IL-2. on1y the

15,000 dalton band possessod LL-2 activity. A minimum of sevon

protoin bands were assoeiated ~ith each pl speeies. Therofore a

minimum of five protoin bands did not correspond to any signifieant

IL-2 activitios. (Al torna tively, their bio-Iogical activi tio s wore

destroyod on purification.)

Siailar rosults woro seon wi'th tho Jurkat LL-2 of pl 7.75. Seven

protein bands wore seen botwoen 10.000 to 20.000 daltons. of which tho

13,500 da1 ton band corresponded to IL-2 b io10giea.l Act ivity.

Ba .. d on the Aboye data. it is ovident that 'IL-2' in the form

and preparation which is often in use eontains protein bands which do

not necessarily correspond to signifieant IL-2 biologieal activities.

It is unknown "hother theso non-IL-2 active protein bands are

inaetivated, degraded IL-2 or do they represent another biologiea1 <1>

function. However. IL-2 activity does correspond to several pr6tein

bands whieh may refleçt diffe~ent degrees of g1ycosy1ation or

sialy1aton of the same proteine

-62-

1. J. Purlty of IL-21

Pryltawsky.!.1.Ah (133) roc.ntly pr ... nt.d eviel.nce that aost

conveu.tional IL-2 proparat~ou.s ln us. contaln aore than ODe lyaphoklU..

_hich cau. influence aonocyte &rowt.ll and functlon. This conf lnal th.

data of Ely tl .!.h. (213), Sabel li .!.L. (2H) and Schrur and ree.

(215) • At least 10 blololle actlTltles attrlbuted to soluble factors

_ero found ln the cloncd IL-2 prOdUeln& L1 cell llno culture

lap.rua tant.

lnter! eron.

The lywphoklnes fOUlld 1fere 1) ll.-2. 2) IL-3. 3) À.

4) laF, a factor _hlCh lnduced la e xp r e s s 1 0 n by

peritoneal maeropha,es l.A Tltro, S) A ractor .hlCh au~cnts Fc

roceptor aedlated pha,ocytoS1S ln Tltro. 6) KIF •• acrophale al,ratlon

inhlbltlon factor. 1) MAF, a.cropha,e actlTatlon factor, 8) BCSF, B

cell Itl.ulatinl factor _Iuch .. y be Il.Dari) ta BCGF, B c.ll ,rowt.ll

factor .hloch can lupport B cell ,ro-th Hl lonl terlll culture, 9) CSF,

colony Itlaulatin, factor. and 10) A factor capable of prolonllonl.A,

the Iyu. the 1 i sand •• cr et 10 n 0 f the .e co n.cI. and four t.h co.po ne n t 0 f

coapleaent by ,ulonea Pli peritoneal .. cropha,el.

Th. tlA. cours. of varlOUS lywphokloAe productlon Tarus. Peak

D..-2 actlovlty .as recoyered betyoen Il to 24 houri. Peak ll.-3 and CSF

rocovery .a. betyeeu 24 to 48 hours. and peak la e11hanclni factor

OUltF) , NAF, and À. lnterferon .a. obtal.ned aiter 48 houri. As

expected only the level s of ll..-2 deerease .1 th tl.e S l.nce the Ll ce Il

line i. dependent on D..-2 for Iroyt.ll and proliferation.

Even a cytolytu: cell lln., 1.3 •• al fouad to produee factors

_hich predolillnantly lnf 1 uenee .acropha,e fuet Ions. The cy toI y t 1 c L3

•

-63-

cell liu. has b •• n shown ta prodllce ). Interferon, 1lIF. 1lAF, IUIF, and

a factor wh1.ch elÙlaace. Fc .cdla ted pha,ocytoll •. ne 1..3 c1'tolyttc

cIo.. ia ~pendent on ll.-2 Uld a f 111er ce Il layer for ,r01Jt.h aJui

prol iferation. neretore J.t 18 Ilot snrpriainl tllat L3 cella •• creU

ly.pJloun.a wh1.ch CUl hI.llu.llce th. filler cella to pro~l4e the

aupportuli ,r01Jtll condlt101l for L3 cell ••

CO~.lltI01l&1 COIl .tlaulated IaOUM or rat .pleell celll also

COlltUIi ll.-2, CSF. BCSF. and ll.-3. B01Je .. er, Ùe 1 .... 11 of each

In cOllalderat1.01l of the heterolellous cell populatI011

1t~1l1atltd by COIlÂ. It 11 con.cltlyable that IJlhlbltor. of l}'11pÀoklU

prodllct Ion aU flmct 1011 w.re al.o trI"ered. It 1. al.o po.uble that

.0.. l,.-phok1.lle. wer. .ubsequ.elltly • uuillced' br D..-2. S J"Ace the L;

c.ll bu. 1. a ha.o.enolll clODed cell lua. lt w01l1d appear that the

1 ..... 1. of lyJapÀoklne. prod.c.d by L2 rcflect the prOd1lctl0n of

lyaphok.1.lle a per ce Il. Olltil • purlfled ll.-2 1. a~a1.1abl., aa.n1'

blolo,lcal actl~ltl •• aasocaatod wltll 'n.-2' .. y be du to another

.ore speclfie: factor (allch aa BCGF .tiaulatlll' antlbod1' producan, B

cell., or lnterf eroll supper t llli NI: ce Il ,r01Jth) . 'll.-2 '

preparatlolls are b1' far DOt a ha.o,eIlOll •• olecular apeClea .

Chuter II

(Jf'!lrr~1 "f'thorlolo~v of Il .... UI' lulturf''', 11--.: lrodurlien ,llld

A. dl.eu ... d ln Chapter I, IL-2. are prodneed by IIIUIll1e and haaan

aetlyated aature T lywaphocyte •. Thl. chapt e r co ne e rns -the se thoc1. 0 f

ll..-2 productlon whleh we u.ed. aDd the yarlOnS assays we cho.e to deteet

the .,t'elance of Il..-" Gelle raI teehZll que 1 ln tlSSue ctlltl1rel of b.naan

and IaUrlne lywphoeytel are al so dlleussad.

A. LL-2 productiol1

l! Hua.11 IL-2

Huaan IL-21 were recoyered froa culture sU])erl1&tants of aUOal1tl,en

or alto,en It.1anlated perlpheral blood lyaphoeytes (221.222).

,HuaaD perlpheral blood collected by veDO~S p~cture wa, deflbr!n&ted

by lel1tle S1IrlrllD' to proaote flbrln clot YOr"aatloo aroUl1d ,lasi bead •.

AlterDAtlvely. blood wal colleeted ln heparlU. Theu appro%l~tely 3~ to

.0 al of a 1 2 dllnted deflbrlDated or hep.rloaed "hole blood was

layered onto 15 al of flcoll-h.,."aqu.e wlth a specIfIe ,rulty of l.073,

Fo11owln, a 30 .Ulute centrlftl,atloo at rtoteaperattlre at 1.500 rpa.

lY1llPhocytes aDd aonocytes were~ reeovered (rom the f leol1 Interphase

(223) •

AlI hua.n celIs were cnltured at 37·C. 5' COZ ID lOto huaan serlDll ln

RPMI 1640, The haaan sera used were pooled. autolo,ous or AB dODor

le r1llll • Occa'slol1&11y frcsh senJa was heat lll&ctlvated at 56°( for 30

aUllte •.

Alloutl,ea stiaul.t.d BF/IL-l prodllct iOIl " •• ' .st.b lish.el by

co-où tutal PBL. frGa t'1ro allol.ll.icaIly diff.reat doaors., L,."Iocytes

".r. oo-ollltlLr.d .t • 1:1 r.spoû.r ta stia.letor cell retio .t 1 x 106

c: .11. pa r al iA 1'" BS-IPIlI. or !Il 1.. BSA-IPIII. Spellt c.l tlU'1

s.perllatut. ("hiclL OO.Ulll BF/ll.-2 .ctl~itil.) ".n hat""'e.t.el b.t1re'll~ ~ ,

" ta 72 Iao.rs. Thi. 'crue' c.ltue sllpenaat&At BF/IL-2 "a. (llt.reel

Oll a .22 !III .11l1pore ... braD' fil t.r allAi stored • t .'C.

Mitol.1l sllalllat.d n.-2 ".s obt.uaed froa altollA .etl .... t.d cllltur.

,uperlaatut. of baaaa PBL. or toaall cIll •. Cells ".re ,tua_l.t.d "lÛ

1 III PHA/l Jt 106 c.lla/.l ,)

ud 0.-2 " •• r.co .... r.d followlAI • 48 houri

lIacùa t.1 ail. na PBA C'A bl r_OY.d by Ixt.aah', ab.orptioll of ûe

s.perlla tut. "1 Ua fr •• h claicUIl. sla •• p. or h_ .. DCa. n.l" ".r. AO

la •• of IL-2 aoti..,ltl •• foilowlili IBC .baorptioll. a .... c.lla "Ire .1.0

Iti.llla uel to pr04uce IL-2 Il.illl Prot.ia A p.rt! ied frGa S. Aare ••

(Phu •• cta) .

a Crue. c.lture .Ilperaetaat "hiola co~t.ill' BF/IL-l " •• Il.ed .s it " ••

hat""'.ated "ltbOllt prior coacelltratioll or purifieatioll of prot'llls. Full

blolol~cal aot1 ... 1tl •• "er •• %pr .... d ia tlL... c.ltlU'e •• peruta.t •.

BF/o.-2' 1 blo1ol1cal .Ctl ... 1 tu. .er. al sa cOILC •• trat.el by Alucoll

w •• sar. ftl tr.tlO. Oll a muo .... bru. fil ter. n. coa.celltrat'el cru.

cllItllre IIlperuatut "as tlLaa .ppl iad OlltO a S.pbaellx 67'. or Biole1 P60

preparath. colaaa "ltb a becl 't'o11:llUl of fi .... liters. T.1l al fr.ctiolls

"ar. coll.ct,d .Ilel ."sayad sep.rately for proteia coat.At (O."'. 210ua)

.Ad BF/n.-2 bl01ol1cal .cti~1tus. BF/IL-2 .cti .... fractl0A' ".re pool.cl

...

.. 4 coac •• trated alai. by Aaico. pr ••• ~. filtratio •• lIo.t •• naa

prot.i.. ai,rat.d •• parat.ly fr~ th. fractio.. .hieh ooatai. IL-2

biololieal aetiTiti... Tb.a G75 BP/IL-l r.f.rs to ooac.atrat.d BF/IL-l

.hi,ch had b ...... ip~ifi.4 by P60 or G15 ,.1 fil traUo ••

IF/n.-l ••• re roatiuly prodDe.d i ..... q ... Utie. by IUl., PBL.

obtaiud froa lte4 Cro •• 4o.or blooG. Ooea.io .. ll, a11ol •• stiaalat.4

h .. a. to •• il IL-l • •• r. al.o prodac.d. Althoaah .aeh crad. BF/IL-2 .a.

a •• ay.d iD4iTichaally for blolo.ical aetiTitie •• oft._ ...... ra! IF/IL-2

acti .... preparatio •••• re pooled a.d coace.trated prior to ,.1 filtratlo.

o. G75 or P60 colaaa •.

li) Mari .. IL-l prodactio.

ht a.4 aariu IL-l ••• r. prodae.d by aito,.. (CoU or PIlA) or

alloa.til ••• ti.alatio. of .ph.ie lyapàoeyt .. (114.11j). n. u~c1s.d

rat or 80... spi... ••• perf ... d .ith 10 ta 10 al of .ter il. aaak.

(8S'S) or IBPIII 16.0 asi., a 13 ,aa, .... dh. n •• ple.ic lyaphocyt ..

coll.ct.d •• n caltar.d at 5 to 10 s 106 c.lls/.1 iJa , .. FCS-IlPIII.

IUto.e.1 n.d •• 'n 2.' to , t'I CoDA/al or 1 .. PlIA/al. n ... perutaats

.Auch co.tu. IL-2 actiTiti.. ..r. ha" •• t.d alter 24 to 4' hoar.

110 .... allo_.tl,e •• tiaalated n-~ prodDctio •• a •• iallar to tll •

•• tllod. cle.crib.d b1 Fitch 1.1.I.L.. 110 •••• ph_tc lyaplaocytu froa t.o

hiatoiacoapatible strai ••• ar. aixed at al: 1 ratio at a f iul cell

coac._tratio. of 5 x 106 cel1/al. IL-2 acti .... caitar. saperut .. t, .ara

.. " •• t.d baty ••• 36 to 60 hoarl folloy!a, i.iti&tio. of caItar.s.

17-

1 HU IL-2 produt ..... u U ..... u. UCD 1« ~lj U ••

n. G1hbo. ori,ia UCD 1« c.ll li .. pr04.c •• D.-l co •• Ut.the1,..

Cal tu. ..,.naataat. ..ra ooU.ct.4 .u .... r Ü. c.ll1 ,r.. to

coafl .... cy. Suc. tla. UCD 1 .... cells 40 aot .tUb. U.-l. tU _o .. t of

a-2. urY.st.d fra. th. C1l1 tu. '1lperu tut •••• dir.ct!,. proportio .. l

to Ü ...... 1' of .i.ble ,rowi., c.ll.. Tke UCD 1 .... c.ll li ••••• ,row.

,a la.. FCS-1PIll alUl req.ired to be t.d .ith frull .. di_ e.ery two to

tlar •• d.,. •.

B. A ••• ,.. fol' IT8Phot~ •• acti.1ti.s

1) IF •••• ,..

a) 2- ~ c.ll prolil.r.tio.

BP/IL-2 ••• r01lti .. 1,. .... ,..d h,. it. .bility to tri"er c.ll

pr011'.ratio. of 10 to 1" da,.. old 2- MLR c.11s (221).

BlIaU ...... '1" •• t.bl ialled •• iA, do.or PBL. iD .Ilicll tll •• tia.la tOI'

cella .'1" i&&cti.at.d b,. ,.... irradi.tl0. (Cobalt 60. 1800 l'ad.

irradiatio.) 01' aita.ycin C treataeDt (2' ~, a1ta.ye1A CIal at 2 to 10 s

10' c.Us/al. laacthatioD of c.Us .a. perfora.d at 37-C " COl for 30

ai •• te •. ) ne i .... cti.ated .tia1l1ators aAd the r •• poad.r cella .'1" co-c1llt1lr.d st a 1:1 r.tio at a tillAI cell COBeeDtratioD of 1 s 106

Occ •• 10 .... U,. aacroplla,.s •• d plla,oc,.tic cella .'1" depleted

b,. carboDyl iro. a.d "Illet trea~'At. (Cell. .8re iacubated .itll

c.rhoayl lrOD for 30 ai .. t .. at 37-C followin, .Ilicll th. c.rboayl troD

ial.sted ceU. .ere del'leted b,. aüerellce to a .troa, aa,Dat.

-fl-

No'-'-.,ocyUc c.U. ".re ooU.ct.cl ia da. ..peraatut de,h tecl of \

"'-----. c.rbo.yl lros. B.t.r .... t.lala, k.cl .kowa tki ... tkocl to b. affecti ••

Ooo •• io .. l1y 1. MLa c.l1 ,rollf.ratios ••••••• y.cl oa clay. , or 7.

Cau prouf.nUo •• a. el.Umi_el by Ua •• ,tab of 1 IICi of 13 tla,..ieliu

• ,eoUio acU.t ty of 10 C""). Ceu .t.bUity " •• dau~ia.cl by dy • t

.zelalo ... iD, 0.0.'- trypta blu. S.oo".r,. ... c.ll •• '1" ••• el batw •••

dayl 10 to 17 to .... 1' loI' BP/IL-l acti.ity. • A ... y. for BF/IL-l .othUi .. "1" roaUuly perfor.ecl la " .. 11

.terot! tn plaUI. ODe ku4recl ",lof BP (.1 U.r c.racl.. 915 or P60

001 ... fraotio .. or coac •• trat.cl IF) "ar. t •• tael o. 1 s 105 2. MUt 0.11.

par 100 ",1/".11. C.l1 ,rol1t.r.tio. la r •• po ... to 8F ••• 4tt.mLs.d by

th. apt.b of 1 )lCi B3T i •• 5 u.r pal •• 1.ballia, dur • 41 to 72

hoar iaoah.tio. at 3J·C "~. Cell •• '1" karT •• t.cl OD • lAS. II Cell

Bar. •• t.r o.to ,la •• flbr. filtar .trip.. I&clloacti.it7 .a. el.t.~i •• cl

by aeleli., 2.0 al of Oaal.o •• t-tol ..... ci.ti11.tio. flaiel aad c:o .. t.el i.

a Packard B.ta co .. t.r.

cdh (224). lhariu 1· MLb ".re •• t a, iaiU.lly at a 1:1 IUa1L1ator

to r .. po .... r ratio ho. .ph.io l)"apkooyt .. of tla. <:$7.1/6 &Ad th. D8AI~

Itrai •• of .ic.. n. Itia.latOI' calla .'1" trraeliaUcl "tU 1100 reel.

a.cl co-c.lt .... el "ltla th. r .. po .... r. iD 1011 FCS ta Dulb.oco aoclifi.el

..... ti.l .. dia. •• 'pl .... t.el .itla l~ , • .-Str.pt. ..cl 2 s 10-'.

2-"l'o.,toa thuol. n. 2· JLJl c811. .'1" ... el OA clay 10. St-U.r

.. i ..

oo .. lUo.. for t •• ti., BP/D.-l acth'iU... ...". • .. d.

OOG.aioully 8U'i .. 2- ... cel 11 ... ". lalt.U.d .. itJa IIlT fo" 12 to 18

IIov.. Opt.b of UT iato û. oell', DNA il .. 1 .. 10. tio. of &la., oell',

" •• po •• i...... to .,/IL-l.

b) 1- MLI oytolytl0 .otl.1t1.,

BP/IL-l .. ill Itt.al.t. ,,,~d cytolytio r ly.pkooyt •• (CTL.) lA balk

1. It', .bil1ty to t"l"." CIL •• di.t.d oytoly,l, of t."I.t c.ll,.

".tiaal.tio. of 1- 1LIIcn.. .... pedoraed la balk c'&1 t1lZ'U

ooata1.i .. ~ BP/IL-l (.1.) .t 1 x 106 0.11/al. Aa a ~.{tl •• coAtrol,

0.11 la • 1:1 ntl0 ... o.UU'.d .t 1 x 10' o.lla/al ( .. ,.t1 •• oo.troll

.... y •• -o. clay. l &ad 3.

r.",.t e.l1a fol' CIL .... y ••• " •• Ula.1' S to 7 .aY8 01' 0'&1 t1lZ'.d

atillal.tor l,...lIooyt ••• • • .nh.t.d IJ'IIPJaob 1.,t a. 0"

t"aa.f~. 8 c.ll IJ'8Piaoltl •• told 0.11 14 .... Tu,.t 0.11, •• ".

l.bell .... i~ c.zoai .. 51 for JO ai •• t., .t 37-C, .t 10 x 10' o.11a/0.2

to 0.3 al Cr51 (.,."oxLaat.' 1 .Cl). To .. 01' •••• b.ok,ro ... Do •• peoifie

bl"lD, of ra4loâOthUy. lab.ll •• ta" •• t cel la •• n iDo".t •• At .. -C

fo" 30 ai .. t.. pdo" to tke l .. t " •• Ia. Qro.l .. 51 label hd tal'l. t

o.lla ".". aise. "i~ .otl.,t •• 2- ... c.l1, .t •• "io •• "ati08 ( .... 11y

.t 100:1 aad 50:1 CIL to t.",.t oall ",tio.). r.. c.ll1 y.". iac"at.d

,t

•

-70-

" to 6 Jaou. at 37·C. , .. ~. FollowiA. iacllbaUoa. the aiorotitre

plate. "U', oeatrifu.eel at 1000 rpI for 5 aiallt •• to pell.t the intaot

oeUa. 100 ill of tlle oeil tr.. superu taat "a. ooU.cuel to el. te na iIl.

tll. _o_t of CrS1 relea.eel frOli ly.eel tal'.e,t o.Ua. llazi .. rel ....

" .. clet.naiJleCl by "at.r ly.is of th. .q1lÏ..,al.llt "IlUllb.r of labelieel

tu.et 0.11.. Cytosi. " ••• %pr •••• el as th. perc.llta •• of lysis "hich

"u eleUnaiaed by

(.,;perlaeatal rel .... (opa) - ailla ... rell ... (cpa» .. ly.ia • 100%

( .. zia ... rel.... (opa) - ailliaua rele ••• (cpa»

Sùailu oollClitiolls ".re u.eel for auia. CJIL " .. ys. nell the PS15

aurlae lyapkobla.toiel 0.11 lia. ".s us.el for tar •• t cella, the killer to

tar.et c.ll r.Uo "a. incr.n.el to 200:1. To iapro..,e CIIL acthitie.,

pl'ior to iAcllbaUoll. tll •• iorot! tl" plat •• "er. o'lltrifu.ed at 800 rpa

for 5 .isat •• to ~ro..,. kill.r oeIl to tar,et ce1l cOlltact ••

li) Jluia co-stia.lator auay

ne co-stiaatator a ••• y ••• perfoxaed aooordiai to the procedur ..

• 'Ubl hhecl by Pae tuu .ll .IL. (128). Tlayaocyt.." ill r .. poll:" to lcnr

ooueatratio •• Il (CoDA) ollty in th. pr,s.llce of ezo.ellou. IL-2

• ddi t i41l.

na,... ... frca B.tb/o aioe "ere r .. o.ed .Ild ainc.el

"ith .ch.or, OT.r a .t.rU •• ie.e. n. aiace4 ti •• u. "'1" " •• k,d "ith

IISS aad Ua. aillil. caU I •• pe.doa collected. le la... UsUd

tlaJ'llOcyt.. froa u.eral .trai.. of aa1aala. C57Bl/6. DBA/1. hi ..

0.tbre4 CD. Bioad olltbreel hiaJa aatibociy r .. pouerl &!ld (C57Bl/6 z "

-11-

Th. Balb/c thy.ocyt.. ha.e ,i •• n .or. consist.nt

r"pon... in th. co-.ti.ulator •••• y.

Ta tattiate a co.U.1l1ator .... y in .icroU trI .... l1s. 1 x 105

th,.,cyt.. w.re cul tured wi th 0 • .5 Ji, CanA and .5Q1i IL .... ;2 (v/v) iD 200

Jil/w.ll.~-Cultur •• w.r. incubat.d for 3 day •• t 37·C, ,~ C02 and pulsed

with 1 JiCt H3T for th. final 12 hours. A po.itiv. responM was seen by

H3T uptakt -as • ruul t of cel1 prol iferation in respons. tq CanA and

IL-2. Th. ne'ativ. control was perform.d with thymocyte. incub.ted ... ith

ConA in the .bsenc. of IL-2.

IUto,tn înduc.d rat IL-2, mouse IL-l and hum an BF/IL-2 ",re all

.ffectiv. in stiaulatinl thymocyte proliferation in costi.ulator ass.ys.

-AlI batches of FCS used in 'the coati.ul.tor ASsay w.r. prescr.ened for

the abaence of tadol.noua aitolenic .ctivity for aous. thymocyt.s.

iii) IL-2 dependent, IL-2 expand.d c.ll line.

Th.re Ar. nuaerou. established .ous. and human cell line .... hich are

d.pend.nt on IL-2 for prolifer.tion and sUrYi.,al (9). Sinc. the •• cells

wUl die in th. absence of IL-2 for 24 hours. th.y "rT' .. a r.liablo

c.ll to .... y for th. pr ••• nc. of D.-2.

F.or .011. •• IL-2 d.pend.nt c.ll lin ••• 1 x 104 c.lls .... r. cùltured

... i th 50-. IL-l (in 200 Jil final vol ua.) for 2" hour. • t 37·C S'II C02.

Cell prolif.r.tion in ruponse to IL-2 ..... deUntined by the upt.kt of

H3T alt.r • , hour pul... Cell viability wa. d.t.r.ain.d by trypan bIne

dye .xc 1 us ion.

Ou .ou •• D.-2 cl.pendent cell lin. w .. est.bU.hed by Dr. H. Rode

usinl

-72-

a C57Bl/ ~ re .ponder and irradia ted 1

1

(CS7Bl/6 x C3H)Fl stiaulator.

Thr •• days / followinl lI'?

resti.ul a tioD. the respoDdiDg cells "ere')

tran.f.ned into cul tuo. "hich contained SOlI rat ConA sti.ul a ted IL-2. ~

This partioular IL-2 depeDdeDt oeIl line had beeD maintainod in 3011 IL-2

for over three .onths at ~he ti.e t4ey "ere used for our a.says. This

cell Une "al unresponsive to ConAt but it did respond to rat IL-2,

.ou.e IL-2 and hum an BF/IL-2. ,These ceUs "ore Iroyn in the abseDce of

a filler or feader coll layer.

Huaan IL-2 dopendont coll linos are extr .. ely diffieult to maint.ln

due to the irreversible 'criais period' leadinl to eell death.

Therefore "e have been "orkinl "ith IL-2 and aaitoleD oxpanded huaaJl'

cells. Tne.e eells are dependent on huaan IL-2 for cell proliferatioD,

however they are Dot totally unrespon.ive to the aito,eDs CoDA, PRA and

Protein A. Therefore cell prol iferation. ..en are a co.biDStioJl of

re.pondV.Dess to IL-2 ud aaitoleDs "hich are pre.eDt iD the IL-2

preparatioDs.

Huaan IL-2 o%panded colIs, 1 x 104 to S z 104 celll "ere iDcQbated

"i th Sm. IL-2 in 200 ,,1 final voh_e. Cell prol iferatioD aDd cel 1

viability ".r. detenained at 24 and -48 hours.

iv) IL-l a.say

Th" preseDce of IL-l "a. testod OD PRA sti.u1ated aouse thyaocytes.

Sinee ,

th. thJ'1luS lacts aatur. IL-l prod.cinl .acropha,,,s, thyaoeyus

"ill Dot re.pond to .it0leD. &lOD •. Up(Jn the additioa'of IL-l (or D..-2)

and aitolens, thyaocytes "ill respoDd to the aitol.nie .ti.ulus. Unlit.

-73-

the costi.ulator assay which was perforaed at low concentrations of

aito,ens and low cel1 nuabers. the IL-1 assay was performed with 2 x 106

th,..ocytea. 50'- IL-l and 1 .. PRA in 200 Jil vohme. CelI proliferation

was deterained by lJ3T uptake followilll three da,.s of incubation at 37·C

S, C~.

-7<4-

Ch.ptor III

, I.aUDiz.tions .nd Hybridaa. Fusious

w. h ..... att .. pt.d to us. s ..... ra! ~1UliJ,a tion protoco 1 s .s wo1l as

dilferent straius of aie. to obt.iu ï..aunized B lywphocytes .. hi ch will

• produce antibodlOS 1I'ith lpocitic;::i ty diroct.d taward. the hua.n rL"""2.

Bl.stoloule Factor.

w. ha .... a1so lacorpo~ated tho hy~ridoa. fusiou tec;::hniquos ta obt.ia

a aODOclonal hybrida •• whieh s.cretes a aonoclo~l product.

'0 This chaptor dlscusses the varlOUS i .. UOlz.tion proc.dures, and the

>

~esults of the hybridoa. seroenlnlS. Bonco, 'Inti-8F' refers ta

hybrido.. c.lIs .. hich secrete an antibody wlth spocaficity dii.ctlld " .

towarda BF/n.-2 as eshiblt.d by the inhibitiou of BF/IL-.2 stiaulated 2'

.c; eell proliferation. ,

IlAl'EJUALS AND JŒDODS

l') Th t' ~ o an lions wor.:

BSA (100 l'I/al), GA (100 l'I/al) and paeked (SJlBC) (501'0 woro

injeetod IP.

A BF/n.-2 prep.ration, conceutr.ted ta the equivalent of 0.8 llter

of cru410 BF/IL-2/a! 1I'.S UNd for t-UDü.tioDS. This prep.ratioD of BF

retaiued i ts or llinal biol 0lieal·..,..act hi ty throulho~t the course of

v.rious ~1UllzatioDs and .... u.ed in aIl t.8UD1.t.hou pl"otocols. This

(

"

t -75- 1

BF/IL-2 preparation was not mitogenic for unstimu1ated hum an PBLs. It

,ave a BF stlmn1ated 2° MLR cell proliferation of 12113 ~ 114 (cpm) with

aD. ansti.n1ated back.ground of 270 + 99 (cpm) as determined by ... H3T

uptak.e.

il) In vivo ilÏiJDunizations (Balb/e, (C57B1/6 x C3H)F1

To 1maanize animal s to BSA, DA and SRBC, each animal received an

initial Ulaanlzation IP followed by three weeUy booster immunizations

IP. The ani .. 1s were saerificed It four we~k.s and immnnized Iymphoc~es o.

obtained from thelr spleens.

To obtain BF/IL-2 immunized eeIls, a two months old Balb/e male

mouse received an initial Uumnnization of 1.0 ml/G7S BF by IV injection

throup the tai1 vein. This was fo1lowed by three monthly boosts of the

eq ui val e.n t .. oun t of BF IP. Twenty four hours after the final boost.

1yaRhocytes fro. the ilm.unized spleen were fused to the Balb/c P3I.20

a.yeloaa cells. Each animal received the equivalent of 3 ~ ter! of

crude BF in four iDuaunizations. The (C57B1/6 x C3H)Fl mouse received

the ... e imaunization protocol.

ili) In vltro iJlllllnnlzation (Ba1b/c)

Lnben and Muellet: (230.231) hfave shown the effcctiveness of ln in

vitro iaœunÎ%ation procedure t~ generate antibody producing cells ta 4

soluble antiaens of reputcd low antigenicity. Wc attcmped to immnnizc

Balb/c lymphocytes to BF/IL-2, BSA. and OA using the in vitro

l .. unization aethod.

•

,

,

-76-

Thymie conditloned lIledum! was III a de Y1th 04 yee~s old Balb,c

thymocytes ln wh~ch 5 % 106 th,..ocy-tes/al yere used to condItIon Ea.le' s

.odified eSlontial aedia. suppleaented Ylth 2~ FCS and Incubated for .8

to 72 hours. The eell fr~o superuataDt harYested COQtalDed the thy1uc

couditipned aedla. whlCh wa. kept at .·C for thre. weeLs.

PreliJainary in vitro i_UDlzatl01l was perforaed w1th 5 % 106 •

sploDocyte. 5Q1\ thym1e eonditl.oned .edilla. S~ fresh aedl.tDI (2are FCS ln

~) and ei th.e,r 50 J'l sec. or 100 J.l,Jal OA. Th~ f iDA 1 c Dl t ur 0,

contaiDed 100 % 106 spl.nocytes in 20 aL Cultures wore incubated at

37°C S~ C02 and cells were harvested at day 4. \

The ce Il vub 111 tle s. \

the number of lymphocytes produeing the speclfle antibodtes (PFCI) and

cells se~retins mouse illllllunogiobuilns (l, socretins plaques) were

de termined.

For the in vitro iDmIunization ta 8F, 5 % 106 8alb!c spleen

lymphocytes wero incubatod with S~ 8alb/e thymie conditionfd medlum and

S~ of a concentrated G75 BF whieh had been extensively d~alyzed against

fresh EMEM. This was the equ.ivalent of 5.0 l1tors of erude 8F. The

-~ concentrated equivalent of t1l0 liters of erude CanA stimu1ated Balb/e

\IL-2 ns a1so added ta the dialysis. This Balb/e IL-2 was biologlcally

active and gave a posi tive costimulator resp?ns~. Therefore in the

final LœmUDization mixture. thora wero 100 x 106 8alb/e spleen cells, 10

ml of fresh 'medlllDl whieh also contained the dialysed concentrated

equivalent of 5.0 lit,ers of erude 8F and 2.0 liters of erude CanA

stiaulated 8alb/c IL-2 and 10 als of thymie conditioncd mediWD, Cells

recoTered after 4 days incubation were fnsed to the P3X20 myelomas. The

/

-77-

ln "'ltro l_unlzatlon eell free culture supernat&JI.t. harTested .fter ..

daYI ... re •••• y.d to detenl1.ne the .. oant of BF/IL-2 and .ur1ne ll..-2

blOlollC.l actl ... ltle. rea.lninl.

1 ... ) In ... 1 ... 0 ~1Uuz.tlon (B10ZZl. .1ee)

The Biozzl. hl.JÀ .ntlbody re.pollder .Ou.e 1. fra. • stralD f1f

outbred an~ls orillnally •• lected for ~elr hllh tltre ant1body

respou. ta SUC (232.233). Sub.equlltly ~ey h .... e been shown ta

re.poDd .. lth a hl.lh antibody tl.tre ta a hait of sollLble anti,en •• Our .6' source of Bl.OZZl _lce .. as suppll.ed by Dr. G Bio%zi.

The in ... l.VO l_anl.z.tl.on of B10ZZl. .iee follow the protocols of

Boua.elI li.!l. (23"). On the fint day. an ei,hr,~ets old Biou.1 lIIale

... s liven 1.0 .1 of G7S cOD.ce.ntr.ted BF IV thrqtilh J

the tall veine

Sixteen hours later an 'equl.valent boosting dose of BF .. as gl.ven IP.

Four d.ys later the l.JDIIIunized spleen lymphocytes .. ere fused ta the

Balb/c P3X20 myclom. cells.

/' \ -

v) Adoptive transfer of immunized cells (Biozzl. mice)

An adoptive transfer of BF immunized lymphocytes .. as attcmptcd

into an irradiated Biozzi mouse.

An cight .. ecks old Bio%zi male mouse .. as given an irradiation of ""~, " ..

920 rads from a;amma radiation Cobalt sourcc.

hours, the anima .. as given 30 x 106 BF LDmunizcd

After rest1ng for six

splecn cells in 0.5 ml

of G75 BF through the tail veine Four days later the LDmunhed spleen

cells .. cre fused t~ the Balh/e myelo.a P3X20. , ' ,

-78-

"1) AAtlbody •• cretinl cell as •• y.

a) Plaqu a.say. for ~UJlo,lobulin •• cretlu, cella

n. ass.y for Il s.cret1ll' colls "as Pttrfo~.d accordinl to the

•• thod describ.d by GrollOWlC% ~~. (23'). The prlnclpl. of thlS assay

_as bas.d 011 th. blndin. of UlalUl'O,lobulln. fro. Il socretin, cells ta

Prot.in A coupled SiBCs.

ODe al of Protein A (0.5 .1 Ph.raacia Protein A/al) "as alxed wlth

10 als CrCl (2.5 x 10-411 pH L5) and ou .1 of "uhed .. nd p.ckcd SRBC.

This aixta.re "as incubated ln • 30·C _ater bath for 60 .inutes,

followin, "hlCh tho secs' were extensively _ashed with O.~ NaCl to

remove free Proteln A. Protein A coupled secs were stored for three

days ol11y.

To ini tiate the plaque usay, 20 )11 of • 301J1t Protein A-SRBC

solution. lÔO 111 of Ig socreting lymphocytes, SO 111 mediam. 20 111 of

1:20 diluted rabbit anti-mouse Ig}I antiserlDD. 20 III of 1:4 diluted

guinoa opii, complement (Cedariane) and 400 111 of' O.S" agarose prewarmed

ta 42°C were mixod 'in a tost tube. This mixture was immediately spread

thinly onto a 9 cm diameter petri dish and incubated for 6 hours at 37°C

S" C02 humidified atmosphere. The plaquos were counted macroscopically.

Random plaquos were selected to determine the presence of a lymphocyte

in the centre of each plaque.

AlI antisera and complements used were previously tested ta

de termine an optimal dilution. 'As thi. plaque as say employs a

monospecifie aftiser1llll (rabbit anti-Illonse IgM), applications .ere made

-79-

ta visu.alhe I,G secretin, plaques. or total l, lecretin, celll by the

chOlce of different .ntlsera.

h) AAti,.h specifie plaqua foraill' celI .... y '(PFC)

( Anti,en specifie PFC .... y ".s perforaed usina specifie anti,en

coupled SiBC .nd anti,ell t..unized lyaphocyte.

Ta initiate the .... y 0.1 al of a 20111 SOC solution (with,ulti,en

coupled ta the sec cell surface) w.s aixed "ith 0.1 ml of imauniud

lywaphocytes. and 1.2 al of 0.5" 42'C prewanaed a,arose iD i.PJlI-1640.

This aixture was poured over a b.se plate of 4 ml 1 .. a,arose in a 5 CIII

di .. e ter pe tr i dish. This was incuhated at 37°C for 90 ainutes. To

develop the direct PFC (IgM secretin,) plaques. 1.0 al of a 1:15 diluted

luines pi, cOlllpleaent "as added ta the plates directly and further

incub.ted for 30 ainutes at 37'C. After relllovai of the cOlllpl elllent , the

plaques were developed overni,ht at 4°C before enumeratiOD.

To de""elop the indirect plaque s (IIG secreting plaque s) folIo" ing

the ini tial incuba tian. 1.0 ml of al: 300 dilllted rabbi t anti-mouse Ig

o antiserUIII was added and incuba1:ed for 30 minutes at 37'C, after which

the rabbit antiserUIII was removed and.replaced with 1.0 ml of guinea pig

complement fot another 30 minutes incubation at 37°C. Pl a tes were

stored at 4'C overn,ight to deve10p the plaques.

Severa! methods :'l'ere used to <(louple soluble protein antigens ta

SRBCs. BSA .nd DA "ere coupled to tannic acid treated SRBC. !WO ml of

"&shed and paeked SRBC was lDixed with 20 JDl of tannie aeid (0.1 m,/ml)

"'. and incubated for 15 minutes at 37'C. This ,w.~/~ollo"ed by w •• hinl ODee

-80-

with Ca+-+- Il.'' fr .. PBS and diluted to • lOI sac 1011ltl0n. Twentyal

of lOI tallA1C acid treated sac wa. aixod with 20 al of anti,oll., BSA or

GA at a coacentratioll of • a,/al ill Ca++ M,++ fre. PBS. Incubatioll was

lor 30 ailll1te. at 37·C followed by thre. wa.he. with Ca++ M," ir •• PBS

and two wa.he. with RPKl 16<40. hti,ell coupled taAllic acid troated

cell. wor •• tored for 7 day. at .·C. BSA alld OA were alla cOl1pl.d ta SRBC by chroaiua chloride. Into a

reactioll ai%turo wal addod 0.1 al packed alld wa.hed SRBC. 1.1 al .aline

and 0.25 al BSA or OA at a cOllcentration al 10 al/al. OIle .. l of CrCl2

(2.5 % 10-.11 pH ~ .• ) wa. addèd dropwi •• and thi. ai%tÛr. wa. al1o.od to

lit at roc. teaperaturo for 5 ainl1tes. ne reactioll wa. teminatad by

tha addition of 1.5 ml PBS. The anti,en eoupled SiBC. ware washed three

ti •• s in .aline and RPKI 1640 and stored at ~·C for 7 days.

c) Ouchtorlony type ùmDunodiffusion

An Oucht.rlony type ol ~UIlodiffusion was perforaed to detect tho

presence of mou.e immunollobulin in solution. Ten ml of 1.. aSlrose

solution in Ca++ MS++ fru PBS was pound into a 9 cm diameter petri

dish to form a base p~ol1s of 2 mm dieoter wero punctured at

appropriate spacings. The source of developinl antibodies were affinity

colamn purified goat anti-mouse Ig of 'various subelasses (Meloy's).

Kouse iJDDlUlloglobulins, hybrido.a supernatants, and the developinl loat

antibodies wore placed in the ap'propriate wells. The plates wero

a110wed to dev.lop overnight at roOIII temperature. aL preclpi ta tion

band. were observed at 24 and 48 hours.

, -

-81-

?ii) Hybrida •• F .. ioa aGd Cloaia,

a) F .. loa procedar ••

th. f.sloa procedare wa. ~ri?d froa th. EMBO cour •• oa hybridoaa

faaioas (136). th. 1IY.100a w. cllo.. to mN .a. the Bal1»/c

Dr. P. BaRd ••• a. the P3120 .,.108a 40 •• Dot •• cr.te À chaia. althou,h .. it i. ~OWD wh.ther i~ .. cr.t.s a & or 1 liallt chaia. Our choie. of

tlle P3120 .,81oaa .a. due ta it. reported abiU.ty to produce stable

aati,ea specifie l, s.cretin, hybrido ••••

ODe huadred .illioa u..uai%ed .plooa lyaphocytes w.re fused ta 2' %

106 P3120 ayeloaa. Tho fusiD, a,ent was "2" polyethyleae ,lyco1 (PHG

l5~0. Baker), 15" DIISO (Fisher) iD RPIlI 1.6"0 (G1bco). 'Iwo.1 of this

solution wu added to , eell pellet coaliltin, of 100 % 106 1aa1Ulized ., lywaphocytos and 25 % 106 1IY0008al. The cells .ere resuspended for 30

secoads in PEG u.iD, a pipette. The reacUon was termina ted by the .,

dropwhe addition of 5.0 ml !lAT lIedium (hypounthine 1.361 lIallll (1 %

10-4~). &IIinopterin 22 ~g/lli (10-8~) and thymidine 0.388 IIgllll (1.6 %

10-S~) in l~ FCS-RPMl) ovor a period of 90 seconds. This yas folloyod

by another 5.0 III HAT 1I0ditllll to obtain a final volume of 12~.0 111. The

cella yore pelletod by gentle centrifugation (900 rpm. S minutes) and

aIl the supernatant relloved. Fivo III of complete HAT lIeditllll was added

ta the pellet very carefmlly ta avoid o%cess saitation. The pel lot yas

re.uspended in tll. 5 ml BAT aediua carefully·. Euo .. agitaUon at this

point can r.~ult in dissociation of fus.d cells.

(

-12-

. ne fu .. d cella .en added to a Iphell fted.r layer of 1 J: 106

Balb/c Iple.1l ceill/~/.eli. Filty pl of the hybrida.a f ••• d celll •• r.

added ta .ach •• 11 (th •• q~i.al.at of 1 J: 106 hybridoaa c.ll./ •• ll). 1

n. BAX .. di.. .a. pr.par.d froa • 100I cOIle.atrat.d Itock

loliltioll. Thi •• a. dil.t.d 100I iato la. FCS ia RPMI. AlI batch •• of

FCS (Glbco or FLow) .er, pr •• cr •••• d to .apport P3IlO .rowth .ad cloDia,

by liaitJ,a. dil.tioa.. Th. cOIlplet. lAT •• di ...... of tell .upple •• at.d

• ith Pea-Stnpt • .oeli .. bicarboll& t.,

l-..~capto.thaaol. ayco.tatia alld faa,izoa. .ere •• ldo. if ... r ••• d.

TWo day. alt.r th. illit1al f •• ioa, aIl •• 11 ••• ~ ••• ppl ••• at.d .ith

O.!I al of BAT •• di_. alld tl!-ia .a. np.ated oa day 7. If c.ll lro,rth "

.a. opti.al (a. d.t.rain.d by c.ll aorphololY. and chan, •• iD the pB of

th. ,ro.th •• di .. ), th. individllAl •• lls •• r. slowly •• an.d off the RAI

•• di .. into KT •• di ... startinl on day 14. Slow Irowin, .ells .ere of tell

proaoted ta ,ra. by supple •• ntin, th.ir •• diUII .i th 30 ta !101ft speat

cul ture snperna tant fram the P3X20 lIIYeloma. By four .eeks a11 wells

.ere trusferred to lOI FCS-RPHI supplemented with L-a1utamine only.

AlI su~sequent cell IrOyth was maintained in 1~ FCS-RPMI.

b) Cloniua

All clonius was porformed by limiting dilutions onto a Balb/c or

(Balb/c ~ Bio%%i)Fl spleon feeder layer. The Bio%%i mous. is an outbred

animal, therefore ta avoid aD allo,eneie response of th. feeder celfs ., ',/. .~

.,ainst 'the hybrida.a ce11., .. 0 chos. to us, a (Balb/e J: Biozzi)Fl

f •• d.r layer. The Btozzi fath.r for th. Fl &Di .. l ..... th ..... 'Ai.al

"

, (

) -13-

Cloaia. OD a Balb/e f •• d.r layer a10a. la.. 1/10th th. oloDi ••

effioi •• oy of a (Ba1b/o x Blo&xi)Fl f.ed.r layer.

tato each .icrotitre .ell .aa added 1 z 105 fre.h splee. cella iD a

filaI vol .. e of 100 ~l. lbe hYbridoaa~ oeIl popalatioD .aa dil.ted to

obtaiJl 5 celhlal .. d 100 III (o~ 0.5 ceUs/IOO "l/.eU) .u added to

.ach .icrotitre •• 11. Startia, OD day 3 •• el1 ••• r. oheckad .lsual1y to

cleUm.i:M' that oa,1y 0 .. cIo .. cl.ve1oped trOll .. ch •• 11. '.ll •• i th .ore

th .. 0118 010118 "er. eliacarel.el or reclo_d.

CIODlal .e11. "ore feel e.ery two elay_ aDcl tho po.itl.o cloaes .ero

i ... dlately expaadeel lato •• ltiplo .011, .poD coDflueDoy ot cell Irowth.

A,ai. -.11s can be auppl ••• ated .ith 501 P3120 .po.t cultare ,.pernatant

to Ùlprove oeIl ,rowth. AH cloDla, •• ore perforaect iA l~ FCS-JlPIU

1640.

viii) Sereeainl of hybrido.a supernatants

a) Sereoni.I criteria for .011s/elonos positive for

immunOllobulin proeluction anel 'anti-RF' activity

Followin, coll fnaiOIr "0 initially obsorveel for hybrido.a coll' \ ...

growth in oach of the '96 ori,ina! fusion "ells. The myeloma ceUs are

HGPH.T (-) all,el thus lac1:. the enzyme neeessary for the' salva,e pathway of

DNA synthesis. Thorofore, iD the preseace of aminoptorin, • dru, .hich "

blocks the synthesh of DNA procuraors. aIl HGPB.T (-) cella .. ill elie

(i ••• unfuseel ayeloaa colIs, or ayeloaa:-roloaa cell fu.ioaa). Bowever

if a ayeloaa coll "a. fuaeel to a noroaal c.ll, the nqrmal ce11 supplieel

1 -1U-

th. 'IU,... BGPKT , th.refor. all faMd hybrido •• , will 'V"f'i.,. iD tll. ,.

pr •••• c. of _laopt.ria. Nor.. 1 c.lla woald 81'0 .a"",i.,. ia the

.. IIOraal Cilla do aot po..... th.

'iaaort.lity' of th • .,.eloa. or hybridoa. c.ll.. na.rdor •• ay cell

whioh .urTi.,.a b.yoad two •• eks la • BAr a.1.otio. a.di .. i •• oat litaly

a hybridoaa c.ll r .. al tia, frGa th. fusioa of 8 .,..108a IUld. aor..l

.phu 0.11.

Fol1owia. ob .. "", .• Uoa _ of hybrido •• è.ll prol if.r.Uoa, ...... y.d

th. c1Ütur •• uperutaat for aou •• t.a1lll0,lobalia .,crIUoD. n. P3120

i. a DOD-l. ch.ia prodac.r, th.r.for. whaU.,er aoa ••

iaa1l.DOllobuliD prodactioD we ob •• rve "ould be th. product of a hybrido ••

~ oeIl. l'1l1'th.J:Ilor., thl .,.cifio~ ty of thl aatibody or aoQ."

i"1l.Do,lobulia reco.,.red woald b. d.t.raiald by the lea.s 'Dcoded by the

iaa1laizod B lyaphocyt ••• W. lait1ally a •• ay.d for iaaUlloJlobuliD

productioa by an O1Icht.r1';lDY type i_1lDodiffudoD. The crudo cutture

sup.r .. tant harv.st.d tram .ach Will was testod for I,GI, I,G21 liA and

IgK Iler.tioD without further cODceDtratioD or purific~tioD of prot.in~r

W.lls whieh "01'0 positivo for lDouse imIIl'linollobul iDs in ho eona.c~tt~e / assay. would then proe.ed ta the sereeDin, for biolo,ical aetivities.

All wells "oro .crooDed ".ekly for the contin1lOus seeretioD of

i.mmlUloSlobul in ••

e) 'Anti-BP' biololieal scr.eDial as.ays

SiDce BF/IL-2 Un inclue. 2' MLlt oeIl proliferaUoa (221), then

th.or.Uc.Ily "'th', pre.,ac. of &JI aaUbody •• ai.at BP O.Jl 1ahibit BI'

\

..

\.

-85-

Itia1Ûateci 2· IILA c.ll proUleratioll. l'herdor. w. a .. ayed the ability

of the hybridoaa luperutUl~ibit BJi' .tia1Llat.d 2· JLll cel1 ~

proliferaUoA.

81010,lea1 aetiYity lereeAinl1 of bybridoaa .uperaataAts wer.

2· ... oells iA a fbal yolaae of 200 p.1. lAollbatioD "a. for 48 to 72

hours followed by a , heur B3T pul •• with 1.0 p.Ci B3T. AlI w.l1s "er.

 pod the cODtro 1 was

,edonecl wiU e.lh .tiJIulate,d with BF iA th. absence of hybriclo.a

.uper .. tut • Occadoaa1ly pod the control s ".r. perfora.d "ith

• uperut&Jlt of aA I,G1 produeiAI hybrido •• with s,.cificity directed

a,aiAst SIBC. or the P3%20 81elo.& luperutant. N"ative cOlltrols were

cells cul tured in th. ab •• nce of HF and - hybrido.a .uper .. t .. t.

Bybrido.a superna tant "hieh contaiD hypo%anthine and thy.idiAe had DO o

adverse .itolenie nor inhibitory .fhct on BF sti.u1ated 2' MLR cell

proliferation. Bawever

DF ,ti.ulated respouses

complete BAT .e~iua can inhibit 25

non- spe ci f iCI ll~.

to 50.. of the

Murine 2° KLRs "ere tested in th. sam •• anner as the human 2° MLRs

l'n .hich bo·th hmnn BF/IL-2 and murine .mitolen indueed IL-2 were ·~sed as

the stimulus. Hurine 2° MLR cells wero pulsod with a3T for the last 12

hour~. Ali murine cultures "ere supplemented ~th 5~ FCS (224).

DISCUSSION OF RESULTS

1) In vitro i_lLIlizaUon to .pecifie anU,.ns. BSA alld OA

~'-

-' -

1

(

.!

•

-86-

(prel iainaries)

to uti •• ns b.ad been Ihown or i.iully by Dut ton li li.. &Ad BenlartAer

(237.138.239). Lüen and Muller d •• erib.d a .odifleation of th. in .~

yi tro ~1IIli~ation _Iliell resv.l hd lA th. eüuu:ed reGoyery of spec i f le

ant~body pro41loinl celll directed towardl lolubl. antil.n~ of low

utI, •• ieUy. Luben a.d ... 11.1'. UlAI the lA yitro UallllhaUoa •• thod

iD the pre ••• ce of ,0. tllJllie eoui tioaed .ed'1118 "1' •. a/>1e to sU.ul a t.

&ntibo4y p1'o4lleln, ce1ls _ith speelficity direct.ci towarcis th.

1 ywaphokin. osteocyte aetiyation factor (OAF) • Furth.ra~. in

i i th i i ' -' ,'-------.6 th co.pa l' son w n v y 0 ua.1lA1.:& a t 1 an. • ia vitro i_llDization •• thod

i •• Ilcc ••• fui at a lower concentratio. of aDti ,ea and ~t caA ,enerate &

lar,.1' allab.r of sonoclaD&1 antibody producial' hybrida_.. _ith

.pecilicity diroetod tawards OAF.

w. used an adaptatioa of the in vitro i_unization protocol ta .>

sti.ulat~ specifie antibady prodncinl cells with specificity directod

tawards the human lymphokine BF/IL-2.

PreliJainary u:porimoats. yoro performed to tost the ef!cctivoness

and v~,J:j.di ty of the nso of sa .. thymoeyte condi t,ioned lIIedi mil. 'e chose

ta nse tho soluble anUgens ovalbumin and bovine serlllD album in. Ta

determine the snccess of the immunization protoco!, we used t"o assays

to de termine tho presence of antibody prodncing colIs, 1) Tho

Itiaulation of the antisell specifie antibody produ~inB clones as

de.onstrated by the recoverios of antiaea specifie plaque formins cel1s.

2) The stiaulatioll of any IgJI prodll.cina colls. us in, the reversed "

,

-87-

" h .. olytic plaque •••• y •• de.cr1beë by Gronowicz ~~. (23~).

a) &ati,.D apeoifie plaqu. fora iD, cell as •• y

A. ahowD in Tabl. I. GDstiaulated sple.n cells incuhated for. daya t.

<1 in 2'" FCS-EIElI aloue ,a"o tew CA specifie PFCs. A tew OA .p.cific PFCs

were •• ell which a.y poadbly be due to th. anti,eDie cross reaetiviJy

with a aou •••• r. protein. or it a.y be due to Dou-spetJ.ific aitoleDie

f.c:tors fOllAd in the FCS. Cell •• tiaulaud with 100 ~l/al of 0'Y • .J.ba.a1n

la"e aipific&ntly acre .Dti.eD specific PFCs. Bowe"er. apl.eD eell.

atiaul.ted with OA (100 Joli/al) in the pre.eDce of th,..ocyte

conditioud aedia lave • two fold increa •• in anti,eu specifie PFC.

recovered. Addi tion of S~ thyaocyte condi tioDed lIIedina appe.red ta

iaprove the erp.nsioD of the OA stiaulated clone ••

1r. also studied the leDoration of BSA specifie PFCa where the BSA

w.a supplied by tho 2~ FCS present in the culture medium. Agaiu. 1.6

fold increaso in BSA specifie PFC was seen when cells~were stilllul.ted in

the presence of ~~ thyaoeyte conditioned lIledium. Sinee the se we re

indirect PFCs which ~epresent bath IgM and IgG seereting clones, we werc

therefore looking .t the total number of antigon specifie (OA or BSA)

antibody produeing colIs stilllulatod.

Experiments performod wi th nnconj uga ted RBC as indicator cells

gave in.ignifieant numbers of plaques (data not shown).

~ . b) IgK secretinl colIs lenorated following in vitro illllllunizatioD

In vitro tmmunization involves • short imauni%atioD period (4

•

-11-

~ars) . n.oreticallr .e .ou1d selectiyely recover aore 1&1' soerotin,

clou. than ItG •• cretin, clan •. n.refore .e looted for the total

1l1lllber of 1'" •• creUnl cella followiD, an in yitro t-lUliution.

III p.raIl_l cul tu.. in .hich "e .t"t9ied th. Inti-BU PFC., ••

foud tA.t c1Ll tu.. .tiaulated in th. pre.ence of '()II. thyJlOcyte

coAditiobad •• di_ ,a"e a t .. o fold incr.... in total IaJl .ecretinl

cetla. "The't~tore it appears that addition of 5()1l thyaocyte condition.d

•• di_ did e&hance the clonaI .xpansion of &lIti,.n specifie IJM

prodllcinl cells tOllaYin, in vitro t..lllDlz.tion.

ii) Balb/e in vitro UDallDlzation of BF/IL-2 -Lub.n and Mueller had shO'Wn the advanta,es of Ilsinl the in vitro

i_llDiz.tion ._ thod ta Iti.llla te antibody secretin, cells .i th

spc eif ici ty direc ted toyards a humail lyaphokine-OAF. le have con! inlled ..

the enhanced clonaI expan.ion of nsponding 'colIs sti~u1ated in the

prosence of thymocyte conditioned medium. We then adapted this same

method to immunizo mouse colIs to the human lymphokino BF/IL-2.

To avoid a1logene ic response s from shed Hl antigons, "e chose ta

use syngeneic Ba1b/e as a source of thymocytes to gep.erate TC]( and

Ba1b/c immunocompetent spleen colIs for in vitro immunization.

Another problem whieh .lIIay interforo with in vitro immunization to

BF/IL-l was due to the potenthl of human IL-2 (BF) ta stimula te murine

T lymphocytes. TheoroUeaUy our anUgon, BF/IL-2, can be used by the

murine T colIs as a T coll secondary sianal rather than be presented to

the B cells as a foreign antiaenic stimulus. To ovorco.e th!. problelll

, -89-

we hoped ta saturate the murine T cell IL-2 b1nding sites by the

addition of excess murine IL-2 (CanA stimulated Balb/c splenoeyte

produced IL-2) to the in vi tro iJllJlluni:ution cul tures. Al though there

ha. been no dir8.)lt evidence to suglest 50, it is possible that mouse T ., cells would have 'a prcf~rential bindi~g for murino IL-2 rather th&l1

hu.an IL-2. Therefore by saturaUng the- IL-2 binding sites on the

murino T colIs, ye hoped that yhatevor human BF/IL-2 which wc supply (as

the antilenic stimulus for in vitro itmDuni:z:ation) yould not be u.ed by

tie mouse T cells but rather it would be available

th .. UaDunocompetent mouse B cells • . '",

to be presented ta ... The culture supernatant wh1ch yas harvested following the fBur days

in vitro immuni:z:ation was assayed for the presence of BF/IL-2 aetivities

(Table II). If biolog~cally active BF/I~-2 yas recovered, we can assumo ~ .

that not all the BF/IL-2 had beon Iltilized by the T cells.

Thorefore, theore tica11y, there should be sufficien t BF IIL-2 "remaining

to stimulate the immunocompetent mouso B~~ ! .

/

•

As s~own by Table II, the spon t ,cul turc supe rna tant re tainod 44 ta /'

54) of the original BF/IL-2 biologieal activity as determined in tyO

consecutive assays on two preparations of 2° MLR colis. Since .fur ine

. IL-2 cannat stimulate human 2° MLR cells, "wc arc confident that the

stimulation seen yas a reflection of the amount of remainin, BF/IL-2 ~

activitles". Therefore cxcess BF/IL-2 was available ta be ~ti1ize as an

antigenic stimulus.

UnfortunUely, YO obtained a very poor - f~on ef.ficien~y aad

hybridom. gro.th from this' particular fusion. Df the 96 ori,~n.l fusion \.

''''

-90-

"ells. 18' (17 "e11s) ,ave evidenee of hybriclo.a cell Irowth in a HAT

.election .ediua. This low fusion efficieney "a. attribnted to the peor

viability (les. thaJl Sm. viable) of the t..auAiud celh ued in the

fu.ion protoeol. Therefore it wa. pos.ible that we obtained a lar,e

pereenta.e of .yela.a and dead cell fusions. Furtheraore. of the 17

wells which exhibited hybriclo.a eell ,rowth. only 5 "ell, exhibited

a01ue 1.aUllo.lobuliA production &Ad very peor inhibition in Ue

biololieal .ereeninl usay for uti-HF activity (leu the 4011t

inhibition of BF/n.-2 sti.1l1ated ceU proliferation). bdeed it is

questioDable "hether the ilLhibitioD.s we .a" were due to the hybrido.a

products (.OUH i.aalUlollobulins) or rather due to the .. iAopterin

pre.ent in the BAT .ediua.

lit.hiD ODe ag.Ath of th. ori,inal fu.aion. aIl fiv. "ells had ceased

1. productions. althoulh the hybride.a ce1ls continued to ,row.

iii) Adoptive transfers of 1aaunized cells (Biozzi aice)

Adoptive trusfer of ~lUlized cells iAto aA irradiated host. alonl

with a concurrent restianlation iA vivo in the adoptive hast "ith the

d •• ired antilen had been shown to euhance the clonaI expansion of the

speeifie anUlenic reactive clones (142). We chose to perfora adoptive

trusters of BF/ll.-2 w.uuizod cells fro. an iA viTo i_UIlized Biozzi

aale .ollse into an trradiated Biozzi .ale.

Al thoulh we were trusferrrinl potentially alloreactive alloleneie

cel Il froa oue Biozzi .ouse into aD. t.8UDosuppressed irradiated Biozzi

hosto it was uulikely a suffieiently stroll, ,raft versus host reaction

..

..

-91-

wa. elicit.d whieh would illterfere "ith our .tt_pt. to obtaill an

anti-BF/IL-2 reactive B cell ClOD'.

lIow ... er. followiD, the fusion of .pleell celh froa the adopti"'-e

tranaf.rr.d hoat to th. P3X20 ~elaaa cells, we wer. uaabl. to r.cov.r

any ~o.lobul in prodllcinl hybridoau frOli this fusioD • No "ella

• ecreted a hybrido.a product which eould b. r,coJJlized by a ,oat

antibody with specificity directed taward •• ouse 1&61. I,G2. l,K. or I&A

b.ea~ chain. After a 6 "e.ks period. ". abandoud thi. fusion due to

th. lack of evideDe. of an ï..auno,lobulin produciDI hybrido.a c.ll or

Iv) In vivo i .. unizatioD. Balb/c and (C57B1/6 x C3U)Fl .icl

Our initial choiel of th. (C57Bl/6 % C3B)Fl .ou.. for iD vi ... o

t.aunization to BF/IL-2 was ba •• d on pr.ltainary result. "hlch SUI,est,d

the presence of BF/IL-2 inhibitory aetivity reeovered fro. a BF/IL-2

Waunhed aouse s.rua ... pl.. At that ti.. this was a aipificant

findinl sinee previous atteapts to Umauaize JUine a pi,s.' chiekens.

loat.. rabbits and other strain. of .ie. did Dot yield a ser1lll s .. ple

whieh oould inhibit BF/IL-2 sti.utatad eell proliferation. Dowever. in

" lipt of recent findillis by Hardt .!1 ..!.l. (2-40), we .ay have been

ori,inal1y aislead. Hardt ll..!l. have ShawD the presellce of an TI.-2

ser,. inhibitory co.ponant found ollly in Iloraal .ouse ser1lll. but ab.ent

in .thyaie nu/nu aouse ·ser,.. Therafore th, inhibitory co.pollent '"c

found in the ser,. of the BF/IL-l u..unized (C57B1/6 x C3B)Fl .ou.e .ay

be duc to the inhibitory factor deseribed by Hardt .!.1 u .. rath.r than

-91-

4u to a .pecific anti-HF &Atib04,. a ••• ha4 b.li ••• d.

FusioD of th. (C57Bl/6 z C3B)Fl WaUAiz.d .pl .. D. c.lla Yl"Û th.

Balb/c P3%20 .,..lo.a 414 D.ot yi.14 any wells .hich uJdbited

~Ollob.liD. production. nia fusion wa. al.o aban.doud as Yith th.

adoptiv. tran.ferred i..maizatioD. with th. Bioz%1 .0 ....

l'lle B.lb/c fusioD. In. e ... i .. D.c. of 99." (9'196 "ells) hybrido.a

c.ll Irowth (Table III). Of th. 9' w.ll. which exhibited hybrida •• c.ll 1

proliferatioD... 29196 (30ft) .howed ... i40D.c. of .ou ••

produc:tloD.. 27/29 (93trt) •• creted a .ous. IJG!, aael wo of th. 29 •• 11 •

•• cr.ted a .ou •• IaJI (BB4 &Ad CDS). n ••• 29 wells w.re COD.d.teDt ia

th.ir iaaUAollobul ia product tOD. for tho iD.i tial thr.. aollt.hs pr 1or, ta

clonial.

Alt.r four cons.cuti •• as.a,.. with 4iff.r.at huaaD. 2- MLI colis aD.d

4iffereD.t BF/IL-2 preparation. (bath G75 coacentrate4 aad crudo BF w.ro

tost.d), w. fOUDd that 11/9' (11."") .ell. eÜibit.d Ireater than SO'-

iallibitioD. of HF .ti.lllate4 2- IILH. oeIl proliforaUoD.. All 11 ".11 •

•• orotod a .ou.. IJGl. Althoulh tho 4.lre. of iD.hibition varied ho.

50'- to 8011, w. ha ••• rbitrarily tabn 50tt iahibitioD. as the cut off

point for positi.e or Ilel.ti.e &AU-HF UlUbod,. procluciAl .eU •• ~

An alteru.ative sereeD.lD.1 as.ay whioh .e used wa. to atteapt ta look

at the direct billdin, of hybrido.a antiboclie. to h1lllu 2- lIUt cells

.hich .xpre •• IL-2 OD. their oeIl .uri.oe.. We h.d previously .haWA that

alloantl.en pri_d but n.tial 2- MI.Jl coll •• xpre ••• roceptor for

BF/IL-2 (Chaptor II and 221). Our Protein A biD.4in, .... y took

.4.snta •• of th. ability to sb.orb BF/IL-2 onto 2- KLR cell .~f.oes. A

, \

-93-

hybrido •• Ultiboely direoted towards BP'/U,-l "o~ld billd the cd 1-- surfac.

bouael HF/IL-l. lb •••• loeli .. 1Z5 lab.lleel Protein A •••• eldeel. ". "o.lel

••• biAdiA, of 1115 Protein A onto c.11 •• kich ha •• hybric10aa aatiboeli ••

bouact ta th.ir c.ll .uriace.

GodiA, (2.1) bd .hown that Prot.in A d.ri •• el fra. St.ph. Aar •••

caA elfectiv.ly biAd ta tho h.avy chain of iaa1Ulo,lob.Un. fra. .. ny

.,.oi... a2..ver th.r. "ore pr.ferential binelinls of differ.nt .,-ci ••

of laauollobuU... 110 ... laGl "ill biAd Protein A poorly. To iapro ••

the 1115 prot.in A bindiA, .... y. • •• s.d a .andwich t.ch.a.iq.. iA "hich /

a r.bbit Ulti-ao ••• i_uao,lob.lin .a. add.el to th. c.l1-Bll-hybric1oa.

aatibody co.plex. Addi tian of Protein A "U ~ reco .. 1u th. r.bbit

aatibody 801" effiei.ntly. How •• er t~ Prot.in A bindi.'1 C.A .i •• U.

fal •• poli ti •• re.pon .••• liAce • hybric1o •• aatiboely cUr.cted a,aiA.t a

cell .urface co.po.a.ent·"ould b. reco~iz.d ••• 'po.iti •• r •• pen .. '.

f'roa the 29 1aa1Ulo,lob.UA .ecreUn. ".11.. 10 ".1h provid.cl . "

.videnc. of th. pr .... c. of an antibocly .hich '~01l1cl bind to BF/D.-l

ab.orbed o. 2· KL2 cel1s. This howov.r does DOt a."oat that hybrido ••

a)ltibody biJUtill, is via BF/IL-l ablorb.d onto the 1· JILJt. Wells "er. , !

co •• ider.d Pratein A poaiti •• il th.y show.d • two 101d i.cr •••• i. Il15

Prot.in A bindin •• bov. th. b.ck,roucl 1 ••• 1s. Co.tral '%poria •• ts •• re

perf01'1l.d "ith 1· uasti.datecl culture ceUs. or ze. JILJt c.U. "i'thollt

.b.orbecl BF/D.-l onto their c.ll surf.ee.. Al! tell 'positive' "ell.

".r. De,ati •• for Ill' Protein A bindi •• s on the cOAtrol cells.

Uafortua.te1y AODe of th. 10 •• 11. po.itive for Prot.in'A bi.ctiAI'

oorr •• poAdect ta ".lh "hich .xJlibit biolo.ical iahibitio, of BP/IL-l

,. - ----- - ------ - '

('" 1

) •

-M-

.till1llat.d c.ll prollferatioa. Du to our 1Ulo.rtaiatill oOILe.mla, th.

112' Prot.ia A biDdiA, ••• ay. •• dicl aot pat too .uch .. phaai. oa the

ProtetA A bindiD, re.d t.. Thereforl" cbo.. ta 010D. tbr.. ".111

"bioh .üiblt.ct th. ItrODI •• t biolo,ioal iDhibiUoa of IIF/D.-2

atill1ll.t.d c.11 proliferatloD ••• ".11 a. the two "e11. "bich •• 0ret.cI •

ao... 1 ail. Th. "ellI oloa.ct .er. A.U. B83. CD1. 884. &Act CD5. 192 .

olouial ".11, •• re .'tablisb,cl froa ,ach orillA.l y,ll (Tabl. IV). Th.

CD1 •• U did AOt procl!l~e &Dy c10ul cell ,rowth. Th. r.aiDiA, ".111

,a". 2' to 50. 010u1 c.11 ,rowth (!Al 52/192. ~ 101/192. BB4 104/192.

ud CD' 91/192). AU oloall ".re .... ,..cl for .0111. t.a1mo,lOb1l1iA

proclllctio .... ,11 a. th. iabibitlo. of BP/IL-l .tia1l1.tecl 2- JILa 0.11

pro1ifer.tio.. 23/960 cio... ..re cha... for furtla..r .xpaDdo. &Jlcl

oharaet.rizatio.,. The ba.i, for oboo.iD, th... cIo... ya. th.ir

repe.Ucl ability to iùibit BP/D..-2 .tialllated oel1 prolileraUo. lA

four co ••• outi"e •••• ,.. uaia, diff.re.t BP/IL-2 preparatio •• &ad 2- MLI

oell. (T.ble IV .how. • repr .... t.U". iahibltioa of 2- JILI c.l1 (

proliier.tio. re.ult.).

1. cho.. to coaceatrat, our .ffort. o. tilt cloM' "hich .zh~bi t.cl

th •• troa,lIt biolo,ioal iJIlibitioa.. n.,. y.re W-II-All. A.A1-I-A12.

A.U-I-G'. A.U-I-B6. BB3-I-G12 .. cl "'-II-Pl. Al! tilt 010 ... procluo.cI a ....

ao... IIGl at tbat Ua.. Table V .boya • repre ••• tati". biolo,ioal

iùibitio. re.lllt b,. the •• 11% clo.... The AA1-II-Al1 cIo ••• üibitecl

72Y1 iahibitioa at • 1: l cUlatioa of th. orud. hybricloaa 'llperaatut. Ât

a 1:16 clUutioa. "e .e •• 2 .... iahibitioa of 2- IILI oeU prolif.r.tioa.

For th. oUer oloa... th.,. ubibU.d approltiaat.l,. ",. iaJaibiUoa .t th.

--. ----- - -----_._------~----.

-95-

10., •• t el11atio. U .. :2.) to a "or. ID1l1bitio •• t tJa. 1:1' 411acl0.. ft.

4ep'.. of iaJllbi Uo. ••• IlOt .,.ry .tro.,_ &JUl tla. iAJalbitory .fhot of

008Pl.c. 10 •• of biolo,loal iDIliblCory actl.,rti •••

w. att_pt.4 to coac •• trU. tla. I.penuacut hybricloa. aDtib04ie. by ~

laIIOai_ .alfat. pr.cipit.Uo. a.la, SOft .atuaC.4 lIIIIOai_ .alf.t. ad

to pa ••• ,. th. llybr140aa •• 8alb/e a.cU ... T.. fo14 cOIaG •• CraC.4

-.oai_ .ulfat. pr.cipiU.u4 cuJt..:. '.apenaatoc , • .,. • oh ... r

pr.par.Uo. of lIOaooloul .. tib04i ••• 1l1011 •• 1'. clep1et.4 0' tla. ujority

of PCS prot.i ••• Al thoap .itla ••• clo... •• ob •• rY.4 .üaao.cI

iülbiUo •. (Glol. Atol) ott •• •• 4i4 80t obCaia .. laor •••• la 1ûlb,ttory

\

aoti~iti •• (Tabl. VI).

Pl~. cio ..... 1'. ..cabliall.d a. Balb/o •• 01t... tla. AlI. All. 05.

G1l &ad Pl. ne G.S aD4 Pl, • .,. poor •• oit •• ,rowth. Approz~ta1y O.S

to 0.1 al of a.oit •• naicla •• 1'. ooU.ot.d ••• tl,.. boa th ••• two elo •••

• ur.a. th. AlI. Al2. aacl Pl 010... oa. pr041lC. 11"".1'48 of " al of

.' •• oit •• tlaida ''''1'7 .1 to 3 d.y~.

A.oit.. fluiel. coU.oteel •• 1'. clepl.te4 of c.11. &ad clebria by

e •• crifq. UO •• Pool.el . a.cit.. nuicla Ir_ tJl. .... 010... ..r •

... lp~ifl.el by ..-oai ... alf.t. 9r.olpitatl0 ... d r.oo •• titat.4 to tla. 1

ori,iaal .,01_.. A. •••• 1»7 Table VII. •• ob .. rfl4 .. iaprO'f.eat of ,

oti-JIll bio1.~~ical 1ù.ibltioa ~iJl' tJa. ..oiC.. fiai'.' -.0010 .. 1

.. tlbo4ie.. Por tJL. es, a.oU •• fiai •• at • 1:10 elUatloa, •• ob •• rY.d ... 1. iDIlibitl0. of mr .ciaal.t • .s c.11 proUferatio.. 1 ..... at • 1:10 ",

411atloa •••• a •• SOi iDIllbitio •• , coap&re. to SOI ~lbltio. at • 1:1

...

..

411.Uoa 01 10:1 aOQ'lltrab4 ov.!tur. 'llperaatuca (Tabl. VI). 1Iow, •• r

IlOt .all pr,p.~.tioal 01 .. oU.. l1aid. ,zJalbit.4 th, •• 1 ",r .. of

laprn,cl iâi'bitloll. na. BB4-II-P2 414 aot .zJaibit lûaao.d ltl010,io.l

tahibitioll 11'0. ~. 0011.ot.4 .,oit.a Ilaid. (T.bl, VIll). Partk'Eaor ••

tl1Ücl, ". "Irt IlOt abl. to ahow ~ •••• dearl' 01 !aprOY.d biolo,ioa1

ialalbitioll (ooapar. G5 ill T.ble VIlA ucl VIm). A.... clid Ilot lin. •

NadU.. • ... ,. to clat.mill' tot.l ItGl. oOlltlllt la .. oJa •• oit ••

p~ap.l'.tioll. .1 ".rt Ilot abl. to ooap.r. th. utibod,. titI" .. itll tIl •

. uar •• of ialaibitioa.

App~osiaaul,. fi.. .ath. .'t,r the illiU.l tualoD. ..cl ~r •• "

aGath. .Iter olGala •• · .. e ,." a pro.rlad.e 'cladi .. iA thl le.lta ,01'

t..ao.10bv.UII proclutioll by .. oh 010... ni .... s •••• botll. ill the,

tf ..... cal tVI .1Z'0WIl h7bridoaa •• peraatut, .d th. ..oit.,. tl~dd. By ,.

GotoNr 17. 1911. lb aOllth. alt.r th. illltl.l , •• 1011. .U sis clo ...

.. ".zo. DOt procluial .ay .011 .. ~0.10b1l111l.

!kt f.l1ar. of th. B.lb/o IIybricioa. 010... ta pr041&O'

~.lob1llilla zoeader.cl tlae.e laybricloaaa ... l .. a to... Bowe.11' .... ':

"a~e ooal iclta't .. of tl&." •• 00. ,. of ou iai ti.l att.pt to procllloe ,.

Thi. .... ..pport.d b,. the

ObHI'Y'tioaa that 1) oaly o.~ture •• pel'utaat.. Ot •• olt.. ft.icla

uriY.d 11'0. a U IU.-l \

WalUlb,tioll '.d cuill f.aioll .a... bl010,ioal

~bitiOIl of BP atiav.1aud 2- JIIJl prollleraUoll. A aoatl'ol cv.! tar.

;. ... l' .. t .. t (1.1. uti-SltBC, or the P3nO .,..l.a) did Ilot 'Z!Libit • .,.

ÙIJli'bitioll of BP .tia1l1at,d oeU prollleraHo. (T,ble IV), 1). •• l1ao

" l' ~

'\

\

..

t " ,

,\ l'

-n-" " i\

ob .. " •• that hybrldolla .a~+-t .. t Ir_ .. 'uU-D/IL-%' 010 .. oa. (

laki.ât l' ILR oeil prolif.ratioa aa4 dl4 aot a".ar to !&kib!t • B 0.11

4 aito, •• atillalatact o.U proUferatios. It ia "aU eatablisJa •• tIlat l'

lLa .tt-alat.d oeil proll'.r.tioa la dept"'.t oa IL-l. ,,"r ••• B 0.11

alto,.. .till1l1.tiou Ar. n.-l i ... ,. .... t (Table VIII. tJah "u~ b.

di ...... ct i. ~r. cletaU 1& CJlapt.r V) •

• ) 11011:.1 110... la TlYo I.aa.t.aUoa to ./IL-l

s~ .... ... r. prod1Utla, a Jaybrt .... 0.11 popalatio. Ir. tIl ••• 11

.. ftaioa of .. oatbr •• Bio •• i s Balb/e or~ ..... pr.par.~, la adYa.o •• th •

... ~opri.t. (Biozai a .alll/c)"1 .traia of uiaal" for t.tu, ..... . n.

If/lL-1- .... i •• 4 Bioz.l aal. .... la U. ,0.U '.doa tg th. 'P31.20 , (1

.,.10..'''., ori,i .. ll,. .. t.d .. iU' .. ~.r.l B.lb/o labr.41 .. al,. prior to

b.i., .aoriflo.d for •• 11 '-.i.... na 13 FI olf.~ia.. of th •••

.. tia •• prOYided th •• P1Wopri.tt .pl •• a c.ll I •• d.r ).,..1' for cloda,

&ad to c.rry th. cloud hybddolla ... acU •••

Iltllia 10.day. followia. o.U l.aio ••• 5 of th. ori,i .. l 9' ".U.

,skiblt.. c.ll .rowth lÀ a BAX •• 1.oti08 .. 41... Pollawi.. fi ••

oo ... o.ti.... .. •• y. of hybl'l4oa. ..pal'ut .. t - laJaililtio. of BP/.D.-l

.thnlat.d 1- na odl prolifu.tioa, _16LJ3_~t1l. !.r. oho ••• tOI' '"

farCh.l' .tuetl... AlI 16 ".11. prod.c.d a a~u •• IaGl.

'1'0. th. 16 "eUI, lhe ... U. ".r. oloud b,. liaitiAI dUutions

(ACt, DIW, DD2. DO, .. cl BC4. Table. IX a.d X) oato a (Bioazi z

B.111/0)Pl Iplaea , •• 4er l.yer. A total of 1632 cloDia, ".111 ".ra

utahlhhad. of .. hich 27.. (441/1632) ,zhibit.d pOlitiv. clonai cel 1 ' ...

1

..

..

(

"

, - . ,~.C(fr cJ&. :p~ ~ - -

" V.Ua CIl ... ti-" 111010,10.1 t •• tla, ot da. ,uolo.4 t .. io. ".lls •

". foacl 010 ... "lIioli iûibit.d tla. JIll .t~at.4 r •• po... (tu (-)

oloa.. o. T • .,,, X)', •••• li •• • atplfio .. t -"r of 010... ,,111011

_aao.4, o~ • .tillaiat •• ' • BP .tiasl.t..4 0.1,1 p~olif.ratl •• r •• po ...

(ûe (++) 010 ... oa T.b .. X). nia ... &&O .... t. o •• 11 ........ 0" to

two, fo14 laer .... la o.U ,roUt.r.Uo.. 1. ti •• oo.~o.U ...... y ••••

laIlibitory la .......... r •••• y.. n.r ••• r. DO oorr.latio •• b.tw •••

(T.bl. Il. XII). f>

Alao.t ail 010 •• pr0411H4 • 80." IaGt •• at..Dll .. d

w. ou •• to ooao •• trat. ~ .ffort. o. 24/441 010 ••• 111011. •• r.

clao ... b.o.... tlaey Jaa«l .Il.. ' ..... o .... t·. or iala!b! tio. (b1 'Ofa) of

CIl. JIIl/IL-2 .U,aal.t.4 oeil ,roUler.tio.. Of tla. 24 do.... • •••• .

010.. .Ûlbited .ü .. o.d BI .tiaâl.tJ..oII iJa •••• r.l olQ_ t •• till' ••

21/2<4 010... ..r. IaGl pr04.c.r.. uul 2/2 .. •• r. • ••••• t.4 to b. 1'­

a prod ... r laiti.llT. bat .t l.t.r .... y ••• r. 'to'" to b. IaG1 prodao.~. ,

at.o. n. AÇ4-V-PU " .... IaG2 pr04~.r (T.bl •• X. n. III) •

.u ••• tala. 4 to 1III.d.r.t .. d Ji .. hybridoa. aaperu t .. t. O.D 1114ao. BP

.~"O'.'Dt.. tir.t " •• ~alled to r~'. o.t tJl. pouibUiU •• tlaat tilla

'.ab .. o •••• t' " ••• 1I08-.,.oifio .T.llt d .. to .1to, •• io factors deri •• 4

tJaat tla. laybrido.. 1."1'1&&t .. t iahibitio.

-proliferatioB " .. Dot du to 1; Doa.,.oifio 1.I.t .Idi.ted by cytotosic: ,

, <1

I-------.-~---~--- ~ -- -------,-----~-------~------

L

(

(

1

to Al. nt th. po .. iltiliU.a of J'CS iaflu.o.a. a,bridolaa .u,er1latut

oolleot ....... 1' "l'11li fr.. 0.11 p-""th aJa •• d • 1-5 fol4 laon... ip

IaSt "CO •• 1'7 C.a .ûibit.. by 11:1'0.'.1' pr.aipi tut baad. la

t __ 041ffuiou>. TJùa, wou14 b. np.ot.. aiac. arowtla of a.lla i •

.. rwa fre. GOadiUo .. iJwol •• el a t •• folel lAGr •••• la o.U ....,.1'. pel'

.01 •• of 0.11 oaltu. C ••• CJaapt.r IV). n. i_bUity tô obtaia a 10

fo14 iAcnu. ta IaGt titI' ... ,. lt. partially .tuibtt.el ta tJa. alIort

iJunab.tio. tiJM Cl-4 Jao1&l'1) ... el i. tll. proel.atioa of I.r. 11' ••

Ilyttri.to.a nperaa t .. t •• rl1&a th. 72 JaoUl ill01lb.tioll U.. .I.el to

plfodu. Jaybri4oa& .aperaat .. t ill tll. pr •••• o. of 101 IfCS-IPIII.

A. .howa b1' T.bl. XII. hybrlclo11. a.pena tut obt.i~4 froll ''l'WI

fr •• 0.11 p'owtll ou _.ff.oti •• l,. ilIIaibit tll. HP atia1l1.t.d 0.11 r"poll"

~ SOfa. 'ut •• raor. 010 ... wllioh w.re pr .... io .. ly cJa.r.cteriz.. •• 'Ill

.üuola.' b.c ... IF lüibitory wh •• th.ir •• peraatuu w.re prod.o.4 1 •

.. ~ tr.. c~lUliUo.. (i... olo.e. DOl-II-AlO. AC4-III-ü. ..d

~

AC4-III-Dl). W ••• re IlOt .bl. ta fiA. tll. 'JIIf .ah .. o .... t' pIl •• _ •• o •

.. i.. "1'. fr.. produc.d hybri40aa Iqleraatsat.. na.r.fore it would

appar th.t· tll. 'BP .ü .. o .... t' ph •• _ •• o. wa. partiaU,. ... ta ail

artefaot fOGBd III th. PCS. B"" ..... r w. oaJl1lOt 1'1l1. o.t Ue po a lib il i ty

Claat 'BI' .ab.uo· •••• U'. ..,. he 4.. to a .0IlOola .. l utibody whioll. o ••

oro •• , liat oeil lurf.oe •• tl..... prot.i •• , 01' rec.ptorl a.d tll.1

tru_it 1 atia.latory sipal bto th. o.U. It il al.o poa~lbh that

th. aoDOaloll&l anUbo.y a.d BPln.-2 ca. fora •• t.a .. & ooap1ez whioll iS

.. t -100-

atia1l1àtory for 2- .... ceU ••

1. are coafideat Clast th. Jlybridoas 'lIperat .. t iDhibitioA of

BP/IL-2 .UlI1Ilat*d oeil prolifentioll ia IlOt .edi.cad by a DOlllpaoific

PCS Ga.po.at. This la 'b ... d GII th. obaenolUoa U.t iûi'bitioll of HP

of lA PCS-RPIII. A1.0 1 slIper_tu.t dari .... eI "frO* .. IiGl proelllcilll

hybri~ cio ... itla .pecifloity elir.cted tawsrel. sISe. elid Ilot illhibit

s mr ,tialLla.teel r •• poa .. (T.bl. IIII).

Of partiolLlar illter .. t •• , th. AC4-II-B5 c10_ yhiclL .zJaibiteel 9~

iaJaibltioll III .e .... ra! .... ,.. (r.pr ... llt.ti .... r.'111 ta of no ... ,.. .r.

üowa III Table nI). Thar.for. y. dacicl.cl to CODe.atrate aIl our 'v

. . effort. oa the ohar.cterbatioll of the AC4-II-"" clou.

J

na. AC4-II-'" clou lla. b •• 11 r.a1oJleel , ..... er.l ti.... ne cloda,

effiGi •• oy •• a ua .. 11,. 29'4 iA wllich 8. to 98 .. of the clou, eûiblt.cl

.. ti-IIP lDhibi tiOIl lA tJae b101011e&1 •••• ,... Ye clid aot pur.ue the

.üc1oaecl _t.rial furtla.r beyoad th. c1etemiJlatioll of ce11 arcnrth.

~aao.lobIl1ia proeluctioa IIld tll •• bility ta illhibit BF stialllat.cl ee11

prol if.ratioA. P.riodieall,. the AC4-II-B5 cl o Il. ... 1'8e10Ile«1 to

clet.Dliu ita .0IlOc~è:JDality. A11 hrtla., .%periaellt. to be clisoUI •• eI

are ICllCU .. of th. AC4-II-B5 e101le.

,

-,- -- - ---- -._-- ----._._----------

o

-1-01-

Table 1

In vttro Imnunization ta Ovalbumin (OA) and Bovine Serum Albunin (BSA).

Experirœnt 1

Culture Conditions

unstimulated

DA stimulat;on~)

OA stim. + 50% TC~(4) .

Experiment II

Culture Conditions

BSA st~7~

BSA s tim. + 50% TCM i

-----_ ... ---~

OA specifi c indi rect

A B

5 16 _

21 32

44 67 ,.

BSA 5 peci fi Ci ) indirect PF01 /106 cells

..

10

16

PFC.(l) /106 ce" s

dl)

0

21

48

Total ~{JM ~ecreting plaque }lD celTs

'.

,

-102- \

Tabl. 1

1) Indioator oells for anti,en specifie PFC assays are OA or BSA coated

SKBC. A rabbit anti-aouse la antibody preparation (1:300 dilution) was

uaed to cleve10p the indirect PFCs.

2) Three representative expeiraonts. Values are expressed as the

nuaber of plaques seon per 106 ÏlUIunized ~pleen cells reeovered after 4

days in vitro ûmBunization.

3) Spleen cells sUmulated win 100 '~,/DÜ OA for 4 days at 5 x 106

spleeÏl 0011 sIal..

1

4) TCM- Th~ooyte conditioned medium - 2~ FCS-EXEM conditionod by S x

106 Ba1b/c thymocytes/DÜ for 48-72 hours.

5) Indicator oells for IgM (socretin, plaques are Protein A coated SRBC.

A aonospecifio rabbit anti_ouse I&JI (1:20) antibody preparation-~

used to develop the plaques.

6) Values are expressed as the number of plaques recoverod from 107

~unized spleen cells.

( 7) Spleen oells are stimulated with 201J1 FCS in EMEK to obtalin BSA •

ilImlunizod ce 11s.

-103-

Table II

.-R.esidul BF /IL-2 activ i ty recovered from cul ture .uperna tant follow in,

in vitro iJlllllunizatio.n.

Source of BF/IL-2 2· Ja..R BF/IL-2 responder cells (1)

A (0103x) B (9900x)

cpm(2} .,.(3) cpm .. 1

nil (med) (4) 276 + 16 0 270 + 99 0

control BF/IL-2(S) 7662 + 115 100 12113 + 114 100

spon t supe rua tan t (6) 3429 + 291 44.7 6524 + 71 • 53.8

1) 1 x 105 2° MLR cells are cultured "ith S~ BF/IL-2 for 72 hours.

Coll proliferations "ere determined by the uptake of 1 ~C H3T/10S cells.

Two e:z:periments are shoyn.

2) Values are expressod as cpm + sn of uptake of B3T into activated

colls' DNA.

3) 'Tt ~/IL-2 activity recovered.

\

4) Unstilllulated 2° MLR cells cultured ,..ith medium alone (negative

control) •

5) Fresh BF/IL-2 "hich had nover been in contact ,,!th murine/htmlan

" ....

..

- -- -- ----- -----------

f -1().4-

cells (positive control).

6) The spent cul ture superna tant fro. the in vitro u.nnization ot

Balb/c spleen cells to BF/IL-2.

.. \

\

\

,.

-105-r-

Table rrr .. Ba l b/ c in vivo ;nmunizatian

Hybr; dama Screen;"g Resul ts

Fusianwells r9 subclas~l) 20 MlR inhibit;o~2) Surface blndinJ3)

A Al Gl +

2 ~ 6 .. +

84

~ C2 +

4 eii +

B A2 ~

+

82 +

3 Gl +

4 M +

5 G1 6

~ Cl

C Al G1 3 G1

+

BI Gl +

CS G1 6 G1

01 G1 +

2 G1 +

3 Gl +

5 M + 6 G1

0 A3 G1 +

5 G1 +

6 G1 +

Cl G1 +

2 G1 3 G1

- )

-106-

T.ble III

1) Kou. l, .ubelas ••• cretioll d.t.~ill8d." by an Ouchtorlony type of

~1LIlocliffusioll uailll erud.. hybrido •• sllperllAtant and ,o.t allti-.ou ••

l,.

2) Th. ability of hybridoa. luperutants to inhibit BF/n.-2 sti.ul.Ud

2· IILR cel1 prolifera'tioll' (100 III BF/IL-2. 100 Jll hybriclowaa

.upe rna tant. 105 2· lII.Jt cells).

(+) .... idellce of >501t inhibition of 2· JILJl pr'oli~eratioll in "

cOIl .. euti .... as.ays.

(-) <2q1 inhibitioll of 2· MLR proliferatioll ill ~ cOIl.ecutive ass.ys.

3) Th. ability of the hybrido •• proclllct to billcl to a (2· ML2 + HF) cell

surf.c. co.ponellt. and th. subs.quellt biDdill1 of a 1125 lab_Iled Protein •

. A.

(+). poai Uve 1125 Protein • biAclilll in 3 COllsecutiv ••••• y ••

\ , (-) Iack of 1125 Protein A bindiAls.

'v Suaaary of Balb/e ill vivo i.auaiz.tioll .cre.nilll result ••

, IaGl producè r s

IaJ( producers

inhibi t iOIl of 2· JILll

27/29

2129

11/29

10/29

. ..

Table IV

Balb/c in vivo immunization

Clàning Results

Fus ion wells cl oned ~""',

AA1 BB3 B84 CD5

-107-•

Wells expressing clonal growth %

52/l9-}J:} 27.0 101/192 52.6 104/192 54.1

91/192 47.3

Representative 20 MlR inhibition results

Clones

AAI l G5 II 812

COS 1 Al 812

AA1 (I) A12 84

BB3 (I I) BI AAI (II) AIl COS (II) D6 B84 (II) G1 BB3 (1) 012 Ml (I) H6

'C6 8B3 (1) G12 8B4 (1 I) 012

G12 AAl (I I) GS BB3 (II) CS BB4 (II) F2 ~

CIl BB3 (II) A10 884 (II) E2 AAl (I) G12 (3) P3X20 mye 1 orna (4)

'":~ (S)

A

4786 + 12512 ) 5090 +" 516 7205 + 379 5978 +" 1691 5153 +" 1430 4724 +" 811 5512 + 1231 3767 +" 561 6470 +" 420 4536 '+ 285 5803 + 122 5115 + 355 4541 +" 820 5244 +" 186 5123 '+ 321 4077 '+ 751 5233 +" 508 5040 +" 459 4821 +" 85 5482 +" 177 5819 +" 454 5608 +" 442 4457 +" 463

17524 +" 1056 794 +" 10

19361 +" 1304

B

13831 + 441 16418 +" 1371 17448 "+ 360 15457 "+ 2030 11599 +" 1630 11501 + 547 12336 +" 393 10441 + 1496 15649 "+ 830 10995 +" '511 11953 +" 806 12823 +" 2378 14674 +" 231 14144 +" 1631 12644 "+ 1373 10384 +" 351 10728 + 2210 10982 +' 660 10062 +' 858 9821 + 455

11094 +" 1665 10733 .. 897 9287 +" 927

24374 + 1962 846 +" 297

19803 +" 2218

(

-101-

T.bl. IV

1) tta.ber of •• 11 •• spres.ia. cloll&l c.ll arowtJJ. per th. total Il1IIIHr of

•• u. olo .. el.

1) TIro' repr •••• t.ti ........ ,... Valu •• r •• %pr •••• 4 •• the opa ± SB of

B3T apt.t, b7,105 h .... le XLI c.l1s ati.ul.tael .ith 100 pl BP/IL-l la

th. pr •••• c. of 5011 cruel. h,.bridoa. supenaatant. Celh •• r. cultur.4

for 3 4.,. ••

3) 501 .peat culture superutut y ••• 4484 to th •••• al1s aa a coatrol

to àJlcnr tJa.t P3IlO 1I1e1c:.. superutut 40.. Ilot po..... illhibitory

.oU.lty for ,,-n.-z .. Un.ad h_~ 2' JILIl •• 110.

~/; .. . "-le KLI c.11s unati.u1.te4 allel culture4 for 3 d.y ••

5) Po.itive co.trol. hua •• 2a KLR cells stiaul.ted with 100 ~l BF/IL-2

vé-&10 .. i. the .b •• llca of hybrido •• or lI1elOli. superll~t.D.tls •

• ."

J

--~. "---_._-~----- -~

i : .' " ~ ';

• , g ., " ~

,1 ~ '~ ,~ J j ~

.;J -

(

~'O9-

Table V

Inhibition of 2° MLR cell proliferations

Representative Results fram 6 clones.

Hybridoma supernatant dilutions

Clones 1:2 1 :4

Al II Al! 14724 + 2371) 17689,+ 213

1 A12

G5

H6

83 1 G12

B4 II F2

16048 + 469

20839 + 1437

20608 + 538

23433 .:!:. 211 -

21031 + 196

Controls: unstimulated~ - BF/IL2 stim-. ---

18006 + 184

20328 + 491

26197 + 586

26897 .:!:. 954

23798 + 156

705 + 11 52107 + 2129

' 1:8

22841 .:!:. 1600

24539 + 270

29136 .:!:. 872

285~1 .:!:. 236

28459 !. 136

26106 + 120

1:16

39805 .!. 1741

25894 .!. 136

29182 + 890

28780 .!. 194 ,

28845 + 833

26004 .!. 390

1) Values are expressed' as crin :t SE of H3T uptake by 105 2° ~1LR' cell s - -

stimulated with 100 ul BF/IL2 for 3 days. Cells were stimulated in the

pre,~ence of various hybridoma supernatants.

2) 20 MLR cells cultured for 3 days unstimulated

3) 20 MLR cells stimulated with 100 u1 BF/IL2 for 3 days in the absence

of any hybridoma supernatants or other 'inhibitors' .

•

.+--:-t-~ __ -.,.. ____________________ _ , -,....~" .....

• ... '

'.

\

..

-110-

Table VI

Inhibition of 20 ~LR cell proliferations by lOX

ammonium sùlfate precipitated culture supernatants l ) ~

Clones G5 G12 All F2

Dilutions

1098 + 6592 ) e

1:2 108 !. 36 2876' + 269 2684 !. 198

1:4 2977 + 520 1687 + 214 6855 + 238 4583 + 965

I l :8 7969 + 868 1-

4939 !. 785 4805 + 65 6745 + 161

1:16 6055 !. 176 7634 + 275 10742 + 188 9517 + ,67 - .

AI2

114 !: 48

355 + 159

165 + 65

268 + 90 -1:32 9667 !. 1963 9366 !. 1106 11031 .:!: 364 11439 !. 1112 1152 .:!: 440

0

1:64 1837 + 750 11788 !. 112 12010 + 760 11199 + 440 2684 + 545 . . -Controls: med (unstimulated) 301 + 37

BF/IL2 stim. 11336 ~ 1532

1) Spent culture supernatant from each crone was concentrated by af1lOOnium .

sulfate precipitation (50% v/v). The concentrated material was extensively " , dialys~d, and found to be {gG! positive.

2) Values are expressed as cpm + SE of H3r"uptake by 105 huma" 20 MLR

cells stimulated wi):h 100 l1l BF/IL2 in the presence/absence of 10x .... '. 1

aJmIOni um sul fate precipi tated hybridoma supernatants. Cells were ,cul tured

for 3 days and H3T pulsed for the last 5 hours .

(

.'

-111-

Table VII

Inhi'bition of 20 MLR cell pral iferations by ascites fluidJl) 1

. dilutions of Ascites "fluids

A) Clones 1:10 , 1:20

AlI 4480 + 39·~{'!) 7540 + 18

A12 3048 + 260 4' >

5392 + 630

G5 1648 + 244 4335 . .:. 1386

. G12 2494 + 365 7895 + 909

F2 5227 + 196 7934 + 812

Controls: med (unstim.) 231 + 21 Bf/IL2 stim., f 13144!, 176

1:40

11578 +0 709

8341 + 104

5310 .:. ~90

. 7758 + 168

9828 + 263

dilutions,of Ascites flufds

B}'Clon~s 1:3 . 1 :6 1:12

. AlI 26063 + 541) 28334 ! 506 36975 !:. 460 "

A12 14807 + 1462 30425 + 229 39224 + 565-

G5 24223 .:. 1031 293<15 .:!:. 3008 35509 .:!:. 325

G12 13519 .:!:. 185 23393 + 303 38673 + 899 --.. F2 16220 + 324 31249 + 2258 28669 :!:. 321

Controls: med (unstim.) 4~~~; * ~~6 r , '

BF/IlZ stim. .--"-::/

.~

1:80

9723 + 127

8736 + 789

6523 .:!: 228

6991 .:!: 798

12732 .:!: 1093

" '

1 :24.

36692 .:t 2279

41576 + 532

44248 + 745

-. 42492 .:!: 681

37214 .:!: 471

.')

1

-112-

Table VII

. 1) Alcites fl~id y.s collected fro. several tappin,s, and precipitated

wi'th ... oni1Dll sulfcate (SOI v/v). It .. as reoonstit~ted to its orilinal .'

• voluae and 'extensively dialysed against PDS.

2) Tyo .representaUve assays. Values are .%pressed as H3T uptake by

105 2' MLlt oel1s sUmu1ated "ith 100 p.l BF/IL-2 in the presence of

a.cit •• fluide ~o preparations of ascite. fluid yore assayed agalnst l'

~o preparations of 2' XLi oe1ls •

..

•

.'

(

ta li

1

1

\ 1 __ , - ,

Ta~le VU!. Effect of hybr1doma ~upe!na_~ants_~!l_)o MLRs _~~~_B'_cell mitogen stimul_~_tio~s~ __

PWM st1mulatipns dilutions of hybridoma supernatants

clones

Âl Il Al!

1 Al2

65

H6 -

'B3 1 612

B4 Il FI2

1:2

67120 + 1154(1)

~62390 + '3385

63676 + 3188 ,

57592 + 2864

65783 + 3742.

65489'+ 6557

Control': -PWM 4541 + 62 +PWM 58643 + 341

1 :4

71132 + 2595

76803 + 243{) - - '-

790~3 + 3870 -~

76524 + 3588 .

71704 + 6300

76938 + 2413 -

1:8 , 1:16

74463 + 38,70 76745 + 2459 ,

75485 + 1992 70859 + 1423

79636 + 5693 66443 + 9608

75515 +'9318 74268 + 3468 - -74962 + 6505 67463 + 2588 -

- 79695 + 7957 75847 + 8119 - ----------8

) ~ (J

-.--,

1 ~

~

~

t -114-

Tabla VIII

1) Fre.h Huaa. PBL'. (105 cel1./wel1) ware .tiaulated with l~ PWK for 3

day. in thepre.ence of 10% ~oncentr.ted hybrido.a .upe rna tant.. Valu..

are a%pre •• ad as cpa ~ SE of B3T uptaka by 105 cell ••

2) alllian fresh PBL', wen set up in a l' JILlt by staulatioll with a 1:1

ratio of responder:sti.ulator cella. CaU. wen cul tuecl for 6 days in

the 'pre.ence of 10% cOllcentrated cul tue superna tants. Value. are

u:pre .. ed as opal ± SE of a3T uptaka by 1 % 10' respollder ceUs in a. 5

hours B3T pulse.

(

---.--- ~--,---.-._-.... _---

" , ,

't

• -115-

TableIX

8iozzi in vivo il11l1unization

Screening Resu 1 ts

Fusion wells i nh ib itian of20Ml~) Ig secretecP ) cloneJ3)

A plate C4 + G1 yes

B plate C4 + G1 yes 02 + G1 no 06 + G1 no

C plate Cl + G1 no C2 + G1 no 03 + G1 no

0 plate 82 + G1 no B4 ' + G1 yes B6 + G1 no C2 + G1 yes C4 + G1 no 02 + G1 yes 04 + G1 no 05 + G1 no 06 + G1 no

1) These wells exhibited inhibition of BF/IL2 stimulated 20 MLR cell

proliferations in ~ consecutive assays.

105 human 20 MLR cells were cultured with 100 ~l 8F in the presence of

100 ~1 spent culture supernatants. Wells exhibiting 50% inhibition were

considered positive.

2) Immunoglobulin secretion determined by immunodiffusion of crude

hybridoma spent culture'supernatants:

3) Wells cloned by limiting dilutions onto a (Balb/c x Biozzi)F1 spleen

feeder layer. Clonin'g efficiency = 27% (441/1632).

--- ---~ _ ... -~-_ .. _-_.

-116-

.. Table X ~ ..

Bïozz; ;n vivo ;rrrnun;zat;on . -Clon;ng Results .

Clones 19 secretion l ) Effect on 20 MLR cel,2)

AC4 1 Dl GI E3 GI II AB GI B5 GI III A6 G1 ++

Ag G1 ++

• 02 GI ++ 1-

F2 GI IV A4 GI AB GI B4 GI 04 GI E9 G1

++ G2 GI

V F12 G2 H2 G1 H3 G1 Oe2 II 03 GI F4 GI G4 GI 002 II A4 GI ++

A7 G ++ AIO m_~S,3) ++ GIO m-~G 1

(

-117-

Table 1

1) Secretion of mouse immunoglobul in is de termined by immunodi ffusion

Assay on the c~ude culture supernatants.

2) Effect of the crude culture supernatant on BF/IL-2 stimulated 2° MLR

cell proliferations

( - ) inh i bit ion

(++) euhancement

3) These AO clones showed a mouse IgM secretion originally but became

Ig01 secreter in s~b5equent aS5ays.

" #

-..

Table XI Inhlbition of BF/IL2 stimulated ~o MLR cell proliferations (crude cult!re supernatants).

Clones 2° = 6165*1) 6566x 3840x 4244x

DC2 1 1 E3 4540 + 144~2) 5020 + 425 17724 + 2899 9241 + 1262 F4 4582 + 458 37130 + 888 23223 -+ 3054 13742 + 1500 G4 5505 + 187 5568 "+ 1058 30969 "+ 1571 12460 "+ 2796

002 1 1 A4 4024 "+ 283 3236 + 380 23072 + 112 7595 +" 365 A7 4231 "+ 377 3443 "+ 842 29608 +" 2757 15110 +" 1739 AlO 6070 + 385 7918 + 1517 33335 +" 5216 13833 +' 1065

III GlO 6013 +" 1435 7115 ~ 1048 35065 +" 3977 14937 +" 1018 AC4 1 Dl 6441 +" 367 5116 + 220 31333 +" 1558 10263 +" 8340

E3 5141'+ 756 7222 "+ 535 23733 + 5371 . 9315 +" 533 .rI 1 A8 4773 +" 60 5385 +" 1106 20932 +" 972 11468 +" 3655 1 .... B5 5099 +" 1665 6361 +" 716 , 24260 +" 747 ('] 13195 +" 2116 ....

00 III A6 4165 ~ 224 1 4048 + 1209 22E09 + 1225 8946 "+ 985 1

A9 4133 + 1040 1 4576 "+ 977 37925 "+ 9732 14182 +" 719 02 6577 "+ 658 4664 +" 329 30075 +" 3449 12216 '+ 1443 F2 5881 +" 1750 5373 + 113 27354 +" 4351 11888 "+ 609 IV A4 3486 +" 1127 5279 + 377 23720 + 9663 8480 "+ 1316 A8 4377 "+ 918 5234 +" 1684 18868 "+ 6 14537 "+ 2820 84 4708 "+ 133 7084 +" 1128 28426 +" 2988 16766 "+ 2319 C4 3993 + 642 4956 + 1722 11169 + 2641 10215 "+ 1720 E9 3072 "+ 510 4534 +" 1812 4805 "+ 483 11547 +" 3967 G2 4949 "+ 825 7224 "+ 288 32296 "+ 2612 13437 "+ 3148

V F12 i S67 I952 5226 "+ 633 32747 "+ 3566 13128 +" 4755 H2 061 + 313 5079 + 940 25796 + 1481 11715 +" 1828

- P3X20 3) H3 4160 "+ 460 4801 +" 1983 32786 "+ 579 12117 '+ 4817

7156 + 1643 5929 +" 2017 28748 "+ 1306 13082 '+ 278 -4 ) 912 + 295 616 +" 115 986 + 107 1899 + 601

,/ med 5) BF 78Q2 +" 608 6266 + 598 23382 + 1245 14423 "+ 2996

-119-

Table XI

l} FolU' representatiTe experiments ~itl1 • diffe~ent 2- IILR cell

preparations. The .ame BF/IL-2 preparation _as used.

2} Re.ul ts are expressed as cp ! 'SE of U3T uptake 1nto BF/IL-2

stiaulated 2- )([.R colls. 1 li: 105 2- IILJt cel1s. 100 J'!. BF/IL-2 and 100

J'lof tl10 hybrido.a supernatants (1:2 dilution), 3 da,.. incubation. 5

hours labellin, _ith 1 J'Ci U3T.

3) P3X20 - spent cul ture· luperna tant frOID the P31.20 lIYeloaa. poli tivo

control s •

• ) Cella _ere Dot stiaula~ed _ith BF/D.-2 - nelaUve controls •

.. 5} Cella stiaulated _1th 100 ,,1 BF/D.-2 in the absence of any hybrido.a

lupernatants. or other inhibitors - positive controls.

-120-

TabLe XII

proLlferatlon by serum free hybrldama supernatant

Hybr l dama Expected experlment A experlment B cLone actlvlty 1

1

P) 2420 + 145~) 'l,

OD2II A10 3515 + 193

~Ctt- l E3 2105 + 203 4290 + 393

\ II A8 2599 + 455 4687 + 422

II 85 239 + 152 480 + 26

III Ag E 2744 + 346 3625 + 85

III 02 E 2370 + 249 3797 + 783

IV C4 2601 + 338 3637 + 204

Controls5) med 92 + 18 104 + 14

BF IIL2 4781 + 827 6061 + 119

-121-

Table III

1) ho rf/pr .. entaUve asaays with no preparations of 2° MLR colls.

The .... BF 1D..-2 was l1sed.

2) RU11115 are e%pressed as cplt .:t SE of H3T nu.~ako by 1 % 105 2° MLR

cells stiml1lated with 100 ~l BF/IL-2 in tho presenco of 100 ~l hybridoma

Sl1pernatant. 3 days incubation. S brs H3T pulse 1 ~Ci/well.

3) E - enhancomont, 1 - inhibitory. Provions tosting of the s.rua

containinl hybridollla sl1pernatants from these clonos had shown either

enhancement (i.o. e%coss stimulatory response) or inhibition of the

BF/~-2 stimulatod response, soe Table II.

4) S.rum free snpernatants are produced by incubatinl 10 % 106

hybrido.a colls/al tor 24 hours in RPM 1640 (no serua).

5) Controls aed ~ no BF/IL-2 or hybridoma sup.rnatant was added.

BF/IL-2 - 2' KLR cells sti.ulated with 100 ~l BF/IL-2

in the abunce of hybrido.a I11perna tants •

•

,

~

Table UlI,

AC4 Il 85 clone

20 t~LR proliferation inhibition by various hybridoma preparations

dilutions: (4 )

20x concentfajed culture suP.}

1:4 }:8 1:16 1:64 1:128 1:256 no BS -- ----~---- ---A 1951+661~) 11538+2927 23125+2598 26928+4819 27618+3413 32510~3025 35924+3

B 2098+1536 9942+1559 23426+2723 28054+2690 25800+7913 28765+547 30749+5 --------------' . - -- - . ------ - -- ---

dilutions:

Ascites fluid'2) 1:20

(8lozzi x Balb/c)F1 184+14~

Balb/c nu/nu

Control supernatants:

• 20x co~c~l) 'P3X20

5623+342

dilutions:

1:2

40915+446

20x conc. anti SRBCr23992+296

1 :40

779+82

1 :80

1159+461

------ -- - ----- -

1:160 1:320 1:640

4730+491 9199+748 8431+121' 12410+150

5681+2353 16660+3073 22015+1507 20640+3798 18552+1849 35938+8 ---- -- ---_. - _._-

-l.

N N 1

,. -123-

\ \ Tabl. XIII

1) ACf-U-B5 hybriclotla. super_tu.t ".. COllc.Dtratecl 20x by IIIIIOU_

lulf.te precipit.Uoa. Lib"h. th. P3120 lpellt cul tur. luperutaat,

au the aODOoloul aati-SJlBC hybricloll. Illperutaat "ere ...,ai_ sul fau

cOllc.lltrlt.d 10 lolcl. ') 2) Asoites fluid. "'1 collect.d fra. (Biozsi % B.lb/e )1"1 or Bal ble

UU/IlU aie.. _olli... sul fat. pr.cipitate" .Ilcl recollstltute" to iu

orili .. l vol .....

3) ae.ultl ar •• xpr .... " as cp. : SE of S3T upt.t.. by 1· MLl cells

It!alll.ted by 100 III BP/IL-2 la th. pre.ellc. of differellt clilutioa. of

B5. Four cliff.r.llt .... ys are show ... ith Hs eorr •• polldiAI po.iti .....

co.troll •

• ) Po.iti •• co.trol., 2· XLI ce11 •• ttaal.t.d by BF/IL-2 ia th. ab ••• c.

of ..,. B5 proc11lCtll.

t -124-

. Cla.pt.r IV

n. 4Ct-II-Q5 Clop •• Purification of th. Hybridoma v

FollowiJl.I rep.aud sere.ninls and eloninls, w. chose to

conc.atrat. our efforts on th. AC4-II-B5 clon.. This is an IIG1

prodllc:inl clon. whic:h ~ad .%.hibi t.d • ilnif icant inhibi tion of cell

proliferation inducecl by BF or marin. IL-2.

S.T.r.l •• thod. yer. us.d in .ttempts to obtain a large

CODsistent quaJl.ti ty of parified and hi,h titre monoclonal AC4-II-B5

antibodi.s. Thi. chapter discusses tho various mODoclonal antibody

productions and purifieatioDs we have us.d. A. weil. the subel.ss of

.ou.. ~1lDo,lobulin produced by AC4-II-B5 WIS charaet.riz.cl by

affinity chro~.to,r.phy. ion exeh.n,. chroma to gr aphy and

1.0.l.atricfoc:usiD,;

IIA'lDTALS AND JIE'DIODS

i) "i,t.alpe. of th. AÇ4-II-B5 clop ••

- -Th. Ac.-II-BS cloaé (BS) wa. ,roya in 5-1()1l PCS in &PJU 16~0

without IUPl'1ea.ntiJl.' th •• edilla yith L-'lutlain.. 2-JŒ, BEPES. or

uUbiot1cl. 411 PCS yas prtler •• ned to support th. ori,iaal P3X20

-.ye1oaa ,rowth ancl cloDin,_ Th. B5 clone Irows poorly----at a coll

cl.aaiey of le •• thu 0.1 x 106 e.lli/ai .nd wUl n.ah •• xia. coll

,rcnrth at 1.5 to 2 x 106 celh/al. n, a.ll doublinl Ua. wa.

approxiaat.ly 16 to 20 houri. AlI B5 cnltur •• w.r. fed .very 48 hours 1

>.

•

(

.. -12'~

to lIa!ntain the eells in losarithaie Iro"th iD order to .axiaiz.

imàunoglobulln pr04uction. Forty-ei,ht.hours old culture .uperu.atants ,: - ,

contain t~. B5 c~,ll product. the monoclonal liG1.

Tho AC4-1I-B cells woro frozon in a -700C Roveo uain, 2~ DlSO

in Sa.. FCS. at coll concentratioll of 20 x 106 colls/al. Th.

effieieDoy of viable cell recovory froll freezinl yas 9S~ aftor a 6-8

lIonth periode AlI BS ClODOS frozon were recoverable and eontinued to

socrete 19G1 aftor an initial la, poriod of 7 to 10 days.

ii) Ammonium 'sulfate precipitation.

Crudo cul turo superna tant yas concentrated by Aaicon pressure

filtration and ammonium sulfate precipitation of JIlouse ilDlDUDOllobuliD "

yaa porfor.ed by the drop-wise addition of .qual volu.os of saturated

amaoni'\JID sulfato. (This was perform.d ovor a t'Wo-hour period.) Tho

prec~pitates wore allowed to fona at room .témperature for 6 hours and

at 40C for 12 hours. The precipitates 1I'ore thon spun d01l'n at 20,000

l'pa for 30 lIinutos at 40C. They 1I'ere then resuspondod in PBS pB 7.4

and ~xtensively dialyzed against four ehanses of PBS. and tyO changes

of il'ill. Routinoly 201 concentrated supernatau.t was producod by

... oniua s~lfate precipitation. AlI preparations of 20X concontratod r

snp.rna tant 1rere testod for 1&61 content by' imaunodiffusion and

bioloaical aotivity to .stablish an antibody titre in ordor to cOIIPar~ \

vadous preparations of conc.ntrated superutant. IgGl lovols 1I'ore

al.o dotormin04 by a seDsitiv. radiotlDaunoassay.

iii) In$roduction of as hybridoll. Ipto a.ci, •• Iro"th.

,'. ,

t

(

-126-

Saco th. AC4-II-~ "a. dorived froa u outbred Bioui x Batb/ c

fu.ion, "e "er. re.triet.d in 'tera. of "hieh an~ll ta carry tho BS

asoita.. le have tried uAin. (Biozzi lt Batb/c)F1 aice. I10raal uel

irradiatod Balb/e, and hoaoZYIOU. nu/nu Balb/e aico.

Ta initiato alcite. ,rowth, anlaal. bet.oen tho alo of 6 "oek. ta

6 IIOnth. "01'0 priaed, io~ a ainla_ of 7 days "ith 1.0 al Pristane

injected IP. B5 hybridoaa eells in dilutions of 0.01 x 106 ta 1.0 lt

106 colls/O.SaI "ore introdllcod IP iuto Pri.tano priud aniaal ••

CoUoction of asc Hes fluid "u by the p1lDC ture of tho abdo.on

as.p~ical1y "ith an 18 .au.e needl. and the peritoneal fluid draina.e

colloctod. The cells "ero pelletod by contrifulation at 3.000 rpD for

10 ainutos at 40C and the .upe~natant colloctad. A.cite'. fluid can be

concontratod by SOli (v/v) saturatod IIIaOni_ sulfate. AlI eolllcuel

ascites fluid "as to.t.d individual1y for Il content and biololica1

activity.

iv) Sor_ (r.e proparatioD' of AÇ4-II-B5.

ne aaaoni_ .ulfat. 20I concontrated hybrido.a superllatant

contain the equivalent of 2I cODcantrated FCS prot.in.. In arder ta

rulo out the pouibiHty of .eru. proteb. inhibition of coll

proliferation, and ta obtain a chan proparation of B5 for

purification purpo.... ". att.ptod ta harvest hybrido.a lupernatant )

froa AC4-II-B5 Irown 1lDder .e1'1l. free candi tion.. ("'

B5 eells did, Dot arow ill co.plete .erua freo condition.

Thorefor., 3 daYI olel- 10larithaical1y Irowinl B5 cells "'1'0 harYe.tad

Alld cODClntrated 10 fold ta 10 lt 106 c.lls/al in 1er ... fieo !PMI.

1

(

\

-127-,

Cel1, "ero incubated for 14 hours at 370C 5 .. C02. Tho seru. froo

luperaatant harYostod "as as.ayod for IrG1 titre and biololical

actiTity. Furtherwaoro tho sera, free hybricloaa superJl&t~ts "ere

ooncentrated bl" ABicon pressure filtration.

le have also atteaptod to Irow BS in serum froo .edia.

luppleaented "i th insu! in. transforr in and seleni 1lIII accordinl to tho

.ethod doscribod bl" Jlurakaai.u..!.L. (243). Coaaercially available I1'S

•• dia "as dilutod 1:100 in RPMI 1640 to obtain a final concontration

of 5 ~I/al insulin. 35 ~8/.1 transferrin and 2.5 mM selonina.

" Occasionally cultures "ore supplolDonted with 20 uJ( o,thanolmine or

1.5" FCS to iaprovo Il prOduction. Coll proliferation. cell viabilitl"

and Il production wore lDonitored daily bl" a3T uptake. trypan blue dl"e

exolusion and i .. uaodiffusion. respectively.

,>

T) H3 loucipo biol.~ellipl of B5 lcil.

Tritiua labolled BS I1G1 wu prep.red hl" ,rowinl cells in th.

presence of a3 leucine.

Lo,aritbicaill" Irowin, BS cells "ere cul tured at 106 colis/al ia, .......

leuciu freo JlPI(I (Gibco) I1lpploaentod "ith s.. dialyz.ed FCS. (FCS"u

diall"ud a,ainst four chu,es of PBS). ,.. co.phte RPJlI 1640. 1 ..

L-a1utaaine an~ SO ~Ci H3 leucino pel' al (NEN a3 leucin. lot nuaber

1618-087). Colls wore cultured for 24 hours at 370C '" C02. ne

colIs colhctod "ero assayod for a3 leucine taken up into tho ceUs ..

Allo th. supernatant "as procipit.tod by ~niua sulfate (SOI v/v) to

d.teraine the .. ouat of radioactivitl" associated "ith th. precipitable

proteiDa .(b.eludiAI 116'.) aJld non-precipitable aoleeulu {i.e. fr ..

I------------·----~---------- .----------------\

( ,

-128-

H3 !euc ille ) •

AlI superna tant coUected froa H3 leucine biol abellinl wu

precipitatod by ",oDilDl sullate at SOli v/v and oxtonsively dialyzed

a,ainlt four chaD ,es of PBS • The precipitated and concentrated

• upernatant .... a ... yod for i ta IaGl titre _nd total radioactivi ty

incorpor at od.

vi) Physicoch •• ical analysis of AÇ4-I1-15 products. DEA! ion exchanle

chro.atolraphy.

DEAE ,ols (Phana_cia) (2<45) were n,tellsively pre-washed at .. OC ,

with th. elution buffer of 10 aM Tris at pB 8.0. A proparaUve 'colU1Ul

.i th a 100 .1 bed vol mae .... packed at "OC and the colman wuhed with

10 al( Tris pB 8.0 by ,rarl{~tion elu~ion.

Sixty .1 ol a 20X cODcontrated BS superna tant was extensive1y

dialyzed a,linst 10 ail Tria pB 8.0 and applied to the DEAE colman.

T.ll 81 fractions woro col1ectod and each fraction assayed for IJGi by

~UJlodi f fus! on.

Mouse IgGl'S are Dot retainod on a DEAE iOD exch&D,e colaan at pH

8.0 • Dy coUectinl th. colaan ru throup which contain the B' IiGl.

.. hope to pur if y BS 1&61 fro. the .ajority of FCS proteins which are '1

retained on tho DEAE COllDlD at pH 8.0. The coluan boud .ateria1 was ~--------.

eluted with _, salt ,radient of 0.0 JI NaCl in 10 mM Tris pB 8.0 to 0.5

X NaCI in 10 .. Tris pB 8.0.

AU fractions iro. th. cO 111lln , 1'1l1l throulh and the NaCl Iradi.nts

woro assayèd for content and protoiD content by

spectrophotoaetric absorption at 280 Jœ. IaGl. positive fractions fro.

(

t

::..... .... ·1

-129-

the col_n ruA throllp .ore pooled. concontrated by .baicon pressuro

fil tration alld dialyzod alainst PBS. Tho 1,G1 nogativo. but

proteiuaceous fractions from the salt ,radient eluted fractions '01'0

ala6 pooled. cOllcentrated and dialyud. Thia IIGl nogaUve fraction

aerved as a ne,aUve control for the DEAE-IgGl fractions in subsequent

biololical assays.

Tii) Physiochemical analys!s of AC4-II-B5 products. rabbit anti-mouse

1.G Umaunoabsorbent columns.

(a) Preparation of affinity col UDn by ,1 utaraldehyde activated

,els.

AB Sepharose (Pharaacia) .a$ extensively pre-yashed in 40 C PBS pH

.7_4. The .ashed ,el yas added slowly to 2.5'4 glutaraldehydc to obtain

a final 1oncentraUon of 1.0 ml AH Sepharose gel/lO. ml 2.5'4

,lutaraldehyde. This mixture was gently lIIixed on a lIIagnctic stirrer

for 15 .inute" to activate the gels. The reaction was tcrminated by

yashin, the gel $ rapidly .1th 100 vol umes of 4 0 C PBS pH 7.4. The

aUvated gel yas addedfl ta tlio protein solution (rabbit anti-mouse

1,Gl. or id 1&(2) yhich was to be coupled onto the gels. Optimum

conditions yoro 2 1111 of actlvate,r gels/20 1111 PBS which contain 30 mg

of imM IgGI or RaK 19G2. This mixture yas kept overnight at 40C on a

rotatinl platfonD. The UlOunt of protein coup1ed to the gel yas

deterlllined by the decrease in optical densHy of the protein solution

al101' 24 ho ur s. The efficiency of protein coupl ing YU 80 to 9&Ja

binding to the gels.

To saturate the unbound but ,lntaraldehyde activated sites, gels

._~-._. . -_ ..... _. __ ... _---------,------------

(

-180-

\, wero washed in O.l'M NaHC03, pH 8.1, rosuspendod in 0.1 M ethanoamine,

0.1 M sodium bicarbonate and O.S M NaCI at pH 8.1, and kept overnight

at 40C on a rotating platform. The gel s were f inally washed

altorn.toly with 0.1 M sodium acetate, 0.5 M NaCl. pH 4.0 and 0.1 M

sodium bicarbonate, 0.5 NaCI in pH 8.1. Gels were stored at 40C in

PBS pH 7.3 with sodium a~ido.

(b) Cyanogon bromide activation of gels.

Sepharose 4B (Pharmacia) was washed extensively in cold distilled

water and 10 mg of gel was resnsponded in 30 ml of cold water. This \,

mi.x~ure was kept stirring gontly on a magne tic stirrer while 1 gm of

cyanogen bromide per 10 ml gel was allowed ta dissolve slewly. As the

CNBr dissolves, tho solution bôcomes very acidic. Thoroforo the pH

. was maiwtainod betwocn pH 10.8 to 11.2 by the continuons addition of

SN NaOa. Gel activation was terminated by washing witl!· 10 volumu of

cold 'lrater, followed by washing with 0.1 M sodium bicarbonate with 0.5

M NaCI pH 8.1. Activated gols ure added to the protein solution in

0.1 M sodium bicarbonat~ pH 8.1. Optimal conditions wore 2 mg protein

(RaM 1961 or RoM IgG2) per one ml of activated gel (at 10 gm gel/30 ml 1

volume) • Protein coupl ing was performed overnight on a rotating

platform at 40C. Saturation of binding sites and washing of thè gllis

wore the same as for glntaraldehyde activated AH Sepharose gel. AU

gels were packed into a disposable syringe. Tho elution buffer WIJS

PDS pH 7.3 or 0.5 M NaC~ in PDS pH 7.3.

~ Various preparations of hybridoma samples wore al1plied to the

affinity columns. Following the entry of hybridoma product into the

--_._-_ ..... _-_ ........ --- ~--_ ... ~--..,.. __ ...

t -131-

affinity c01tmln. the flo" was stopped for 30 minutes incubation to

encourage binding of the hybr idoma product to the col umn bound

antibodies. , Various means were attempted to e1ut'e the co1umn bound 19G1.

Fractions eluted with 0.5 M NaC! in 0.2 M glycine HCI pH 2.4 were

immediately neutralized with 3N NaDH to prevent dissociation and

inactivation of mouse 19G1 at 10" pH. Elutlon of column bound 19G1

was also attempted with 0.05 M diethyluunc. 3M ISCN. 0.5'11

deoxycholate and 6 M guanldinc HCI pH 6.1. AlI clutcd fractions were

, concentrated by Amicon pressure fll tratton or millipore cone

fil tration and dialyzed against PBS prior to Assay for biologicai

• activity. Dccasionally the H3 leucine labelled B5 hybridoma Ig61 was

used as a marker to determine the success of each affinity column to

retaln mouse 1961 and to monitor the success of the various elution

methods. Each fraction was ass.aycd ,for the presence of Ig61.

viii) 1soelectric focusins.

, To obtain purified B5 19G1., we chose to purify 1961 ~y

isoelectric focusing. Seven pOlnt five grams of Biogel P60

(Pharmacia) ·~.-s washed in distl.!1cd water and resuspended ln IIJlre

sucrose (RNase' froc) in dl.stllied water. This gel was poured into a

glass trough and allowed to tlry partially. A mixture of 2.5 ml pH 5-8 , ampholine. and 1.5 ml pH 8-9.5 ampholine was inJected intol the

( partia!ly dried gel.

Flve ml of B5 1961 (from 20X concentrated culture supernat,ant

"hich had been semipurified on • DEAE ion exchange collDlln) was lIlixt!ld

'.

1 -132-

"ith 200 111 of 83 !eucule BS 19G1 as a lIIarker. This lIIixture was

dialzyed overnight against 1" glyc lne "i th 1.. ampho1 ines. (An equa1

mixture of pH 5-8 Lnd pH 8-9.5 mapho1lne.) )

The dialyzed BS IgGl "as loadod in the centor \of the gel and

electrofocused lenlth"ise using.2' ethanolullne as catholyte and loS ..

1

phosphoric aCld as anolyte. E1ectrofocusing' was carrled out for 19

hours at 5 m-amp or 1055 using a LKB Bromma Mu1tiphore 2117.

Subsoquently the gol "as 51 icod lnto 1 cm str1pS and proteins

el ut e d "i th S ml sa llno. Tho protein content (optical denslty) and

the pH was deteqll.ned for each fractlon. Alter extetnsive dlalysis.

oach fraction was assayed for the presence of IlG1 and the ablll ty to

inhibit BF/IL-2 stimulated cell proliferation.

lx) Radloimmunoassay.

A RIA wu de 5 igned to quanti tatively de termine the amount of

_ouse in e.c~ preparation of concentrated BS hybridoma

superna tant. The principle' behlDd the 1UA was based on the

co_petition between unlabelled BS IgGl and 83 1abelled BS IgG1 binding ,

to a rabbl t anti-mouse 19 antibody. lncreaslng .. ounts of unlabe lIed

19G1 will result in decreased blndinl of a3 labelled 19G1 to a

prede termined R,aH 19G1 dll ution.

Optuul condItions "ere pre-determined uSlna

available standard mouse 1&61 (Bloneties). Rabbit anti-mouse IIGl

(Batch Nuaber 12) "a5 used at a 1:4 dilution. a3 labelled BS 19G1 was

use d a t al: 20 d il ut ion. A standard curve was prepared daily using

standard aouse IIGl in the range of 0 to 15 '" 1,G1/al.

1 -133-

Into a test tube wu added 100 111 of the unknown hybridoma

superutant. (or standard Ig61). 100 111 of a 1:20 diluted H3 labelled

BS ~gGl. 100 111 of a 1:4 diluted rabbit anti-mouse IgGI and 300 111 of

o.u. BSA in PDS pH 7.3. (All IgGls were diluted in 0.1411 BSA-PDS pH

7.3. hbbit antibodies ure diluted in lQ1i normal rabbit serlDll in

PDS. ) , This mixture lns incubated for tyO hours at room tempe rature

following which 100 111 of an undiluted goat anti-rabbit antiserum wu

added ta precipitate the rabbi t antibodies. To facilitate

precipitation of the rabbit antibodies. the reaction mixture w.s

incubated for 30 minutes at 370C folloyed by an overn~ght incubation

a t 40C. The precipi ta tes were extensively washed wi th 0.1411 BSA-PDS

three t ime s. Ta dissolve the precipitates. 1.0Ilft SDS yas added and

heated at S60C for several hours. The dissolved precipitates were

c011l1ted in 3.0 IDI Blofluor in a Packard p C011l1ter. 1&61 quanti ties

are expressed relative to the daily standard curve. \

RESULTS. PRESENTATION AND DISCUSSION

Our lnitial source of BS IgGl had been the crude culture

superutant. or cul turo supe rna tan t J

froe serlJa free ce Il gro ... tll.

However. quantitatively 1itt1e biologically active BS 1161 was

obtained froID the crude culture supernatant. Approxi.lll&tely 50 ua/ml

of BS 1&61 was obtained from the crude cul ture superua tant "hereas

ser,. free produced BS 18Gl often represented a tyo-fold increase in

1,61 recovery. ,

In our atteapts to obtain a larler volume of consistent BS I&Gl

1 we decided ta try various purifieat~o~- àÀd concentrations of the B5

.~ ~ \ produet aAd ta r .. ove the .. jority 6f conta.inatinl FCS prot.ina.

~niua sulfate preeipitated. 201 coneentrated culture

Sllpernatant •

~oni1Dl sul fate will precipi tata IROus. ï..aunoalobul in wh.rus

the .ajority of the FCS prot.lns remain in solution (i.e. BSA). The

LBaunollobulin cont.nt of the 20X concentratèd culture supernatant was

approxi .. tely 1 IIIg/llÙ. as de tenained by RIA (Table XIV). The 201

concentrated culture supernatant showed an inerease ÏD\ th. inhibition

of BF/IL-Z sUaulat.d eeU proliferation (Table nII). At a 1:4

dilution. Iruter than 9a.. inhibition of cell proliferation wu s.en.

/ This wa. an illlpro-.elll.nt over th. S()II. inlu:bition se.n at 1:2 dilution

of th. crude (UAcone.ntated) cul tur. superna tant.

1re do 110t belJ.eve that the increased inhibition s.en with the 201 ~

concentrated supernatant WA5 a non-specific illhibition due to th.

concentra ted proteins. TYo control supern.atant •• P31.20 And an I,GI

secr.tinl anti-SRBC. were also conc.ntrated 20X by the s.a ••• thods as

B5. Th.so control supernatants did not u.hibit inhibition of BF/IL-l

stiaulation (Table nII). L1k.wise the crud. culture supernatantl

froa the P3X20 and th. anti-SRBC did Dot inhibit th. BF/IL~l

st iaul. te~ ~:sspon5'.

H} S'rua fre. and rrs aediua Irowsh of AÇ4-II-IS. To facil i tate

purification of B5 IaG1, 1re att_pt.d to ,ro1f the 85 hybrido •• iD

ser~ fre •• ediua. thus el1.lliutinl th. aajority of containatiDI

t -135-

ser,.- proteins.

BS hybrido.a celIs elic! Dot proliferate, nor produc:e IIGI when \

Irown at low cell conceDtrations in co.ploU ser_ free colLCiition.

Dowevor lo,ar i thaically Irowinl BS cells c&n be CODcentrateel to 10 %

106 colIs/al &Dd cul t1l.1'ocl in serua fru .ediua for 24 hciurs. 1

As shown

by Table ri, snch sor,. frae supe rna tant showecl Ireater BF/IL-2

inhibitioD than the correspondinl FCS cODtainini cruele cul turo

superna tant. Rowever in repeatecl attempts to ... oni1lll sulfate

concentrate the as IIGI in serlDD free supernatant, we were un&ble to

recover biololically active B5 IIG1 froa the concentrateel .aterial.

We attributeel th!s f.i1ure ta the low protein cODtent in the serma 1

free superna tants which de na tures uneler ... 0 ni 1111 sulfate

precipitation. AdditioD of a carrier protein woulel defeat the purpo ••

of producinl BS ISG1 1I.Ilder sorlDD freo conditions.

Atteapts were aade to Irow the BS clono in serua free rrs aediu..

It has boen shOYD by Murakali .tl .!L.. that hybridaaa colis can bo

iuuced to ,row and secrete iaaunollobulin if IrGYn in serua free ITS

aodi1lll suppleaented with 20 pM ethanol .. iDe. The AC4-II-B5 clone elid

not proliterato in coaplete serua free ITS .oeliua. rrs .. eliua

suppleaented w i th 20 pJI .thaJlol .. aiDo. or in 2.'" FCS in rrs aodi1lll.

Althoulh we elid not oba.rve ai,niflcant cell eleath. "e alao did Dot

ob •• rve cell proliforations, nor secretiOn of tho AC4-II-BS proeluct

into the supernatant. ITS ae~i1lll's spent culture supernaUnt (whethor

crue, or ... oniua sulfate conc'ntrateel) eliel not contain aDY aGus,

i-uAo1lobulin. nor did it ehihit any illhibitioll of HF stiauhted

cell proliferation. Since AC4-II-BS colIs CUlJlot be .. lntained i.

1 -136-

coaplete serua free or rrs aediua. we had to find an alternative "aDS

"to obtain ' pur if bd' BS 1&61.

iii) Ascites Itoyth of AC4-II-BS. Introduction of hybrido.a. into

a.cHic Irowth in aiee has been shown to clr_aUcally iDcre .. e the

antlbocly titre produced by th. asc i tes. lIowever, ye were restrieted

in tenas of the choiee of animals ta carry the AC4-II-BS ascites. 1re

att .. pted to srow the BS ascites iD the (Biozzi x Balb/c}Fl. Balb/e

nu/nu, aAd lrradiated Balb/e aice.

Irradiated Balb/c: .ic:e wen i.maediately kUled (3 ta 7 days) by

the coabined offocts of the irradiation and the Irowth of B$

hybridoaa. If non-!rradiated Balb/c aice yere used, at low cell

nuabers (loss th.n 0.5 % 106 cells/_l), no ascites Iro.th yas evident. o

1rith laraer n1Ulber of cells (,reater thaD 1 % 106 eells) the ani .. l

ditd .ithin 7 ciays due to th. invasion of hybrido.a c:e11s into the

thoracie cavity, no ascite. fluid wa. eollec~od.

We aU .. pted to .,stablish the BS hybrido.a in th. Balb/c nu/nu "

aiee. Very low nuab_ra of eells (0.01 to 0.05 % 106 co1h) yere'

introduced into pristau prbled Balb/c nu/nu .iee. In the .. aiee, it

took up to 14 to 21 days befon ascites ,rowth was evident. lIowever

the ani_l'a usually clieci .!thin 3 to 4 days followin, the initial \

collection o( peritoneal draina,e. Very litt1_ ascit.. f1uid .as

" collect.d froa the nu/nu .ice.

We have b.en .ueelssfu! in th. ,rowth of th. AC4-II-BS c1ô •• la

th. (Biozzi x- Balb/e)Fl aiel. A pristan. priaed aAi .. 1 'i .... n aD

inoculua of 0.5 % 106 BS coll. IP~ill .xhibit a.cit •• Irowth by clays

"

-137-

7 to 10. Asoit .. !llLicl "a. eolleet.cl .very 24 to 48 hou ... ith an

avera,. collection of 2.0 al. Th •• e ani .. l. .urviv.d for

approxiaately 20 to 30 days after the initiation of a.cite. ,ro-.th.

CeU. collected iro. the .. eit'I fluici clraina,e, or IÏn,h cell

••• pension .ade fra. the exc ilecl t1lll0U .. sael ean be reinj eet.cl into

aaoth.r ani .. l to initiat. ascit.s ,ro-.th. or b. ro-introcluc.d into la

vitro th,'ue cul tues. l'h.s. hybriclo.a eells ... hen 1'0- introducecl into

..iJl vitro ,ro"th, continued to proliferate ancl .ecrete 1161 iu.to the

cul tare IUp. rna tallt. l'h. npernatuts cOlltinued to inhibit BF

Iti.ulated 20 KLR ce11 proliferation. There dicl Ilot .ppea~ to be Lny

phellotypic chan, .. of the B5 hybriclo.a cells "hich had be'll palu,ed

..iJl xi!2 fo11owecl by r.-adaptatioll into la ~ tillue cultur. ,1'0-. th.

AlI ale i tu fI uicl. "'1'1 poo le cl aeccordiA, to th. 5train of

ani .. 15 froa "hieh th.y w.r. coll.ct.cl. Approxi .. tely 11 a, of 1&61

.. al raeover.d per al of ... oD1aa sulfate precipitatecl •• cit •• f1uid

(Table IIV), wh1ch repr •• eDt. 10 foid aor. 1,01 thall th. 201

CODe.lltrat.cl cultur. 5uperJ1&taai. -The &lcit .. f1uicl. a,lthou,h clilf1cult to obtaill,

iDhibi tiOD of BP/IL-2 Itiaulated proliferatioD a't a clU UtiOD , b. t .... n

1:160 ancl 1:320, a. coaparecl to the lOI cOllc'Dtratecl culture

•• peru tant .h1ch ,av. 5a-. iJÛlibi tion be na.n a clU ut10n of 1: 16 and

1:32. nere clicl Ilot appear to be any cliff.reDc.I b.n.en ascU ••

flaicl obtain.d fro. th. (B10zd x Batb/e )Pl or th. Batb/e Illl/llU aie.

(Table nII).

iv) PIA' 10. 'Ieh,... colppp. •• h,.. pravio •• l,. d.t.~in.d by

lU .. • a J 1 •

(

(

-138-

WaUDodiffuaiou that BS produce. a .011..0 i.ma1Ulollobulin of the IlG1

subela ... -\

Furtho1'1llore. in imaunoelectrophoresis of the B5

coacentrated ~ulture supernatant. the B5 product traTels as One .ajor

protein band in the region correspondinl to the aouse I,G1.

In our subsequent att_pts ta pur if y 85 hG1, w. chas. to use the

\ DEAS ion .x~.ng. cohan. As shown by Table IVI and Figure J • when

B5 .a. applied to a DEAE colaan, the flo. through app.ared between

-fractions 8 to 25. 'hen wO applied the salt gradient bo,innin'g at

fraction 30. the salt ,radient eluted protoins appoared at fractions

35 ta 45. Similar results wore seen .hen FCS was applled ta the same

DEAE col UlDn (F lpre" ) • Ouly tho flow through froD the BS-DEA!

colu.n coutain signifieant amount of mouse IIGI. No I,G2. IgM or lIA

_a. found in any fraeti.-ons. Lib_he, no aousé immunoglobulins _ere

recoverod from azay fractions of FCS which had beoza throll,h the DEA!

col UIIln.

nen .e tested these pooled and concentrlted frictions in the

Inti-BF biological JSSlyS, we found that only fractions which contaln . ...

l'IG1 exhibited inhibi tian of BF stiaulated cell proliferation (i~ o.

tractions 8 to 20 from the B5-DEAE col_n). Nei thor of the salt

,radient el uted BS-DEAE cohan fract'ions, nar Any fractions fro. the

FCS-DEAE 'coloan were capable of iDhibitlnl, BF sUaulated eell

proliferation (Table IVI).

The B5 prodllct which iDhibits BF .Uanlated cell proliferation

co_i,rates with the rgGl fractions ollly. It _appears to have the same

.0Iecul.r charae charlcteristic of a .011se IIGl. The inhibition of BF

sti.ulation by as I,Gl don not appear ta be a non-specÜie serua

(

/

,

-139-

proteill iJlhibitory co.poaellt dAC. the .... fraction. fro. a control

colaan (FCS-DEAE) did not appe.r to po ••••• the abi1it1 to inhibit HF

,tiaulated c.ll prolifer.tioll. Furtheraore, the •• jority of proteills

iD, the 20X, coneentr.ted cul ture .upernat'Dt appeàred in the saI t 4

,radiont eluted fraction' (I.e. the DEAB colmaD bound .aterial).

The.o ,aIt ,radient oluted fraction. did not exhlbit .l,nific.nt

inhibition of BF/IL-2 stiaulated cell prolifer.tion.

By p •••• ,. of the 20X coneentrated BS culture supernat.nt throu,h

a DEAE Ion e~han,. colaan. we have' removod tho majorlty of

coat .. Ina tin, FCS protein whieh Adheres to the collllDn. and recoyered

.... nUaUy aU of th. IIG1 in th. colaan flow throu,h. This ellables

u. to recover inhibitory activiUes in th. I,G1 polltiy. frae.tioll

wh.r.a, aIl oth.r prot.inac.ous non-IaGl fraction. did Ilot inhibit th.

BP/IL-2. re.pon.e.

v) I.bbit .,t1=,0,., l, iPRUpoab.orb.,t co1pa's. In or de l' to

definitively .. saoeiate th. inhibition of BF .U.alation with the BS u

IrGI'S .nd to parify BS 1&61. wo docidod to study B5's char.eterl.tic

on a rabbit anti-aous. la i .. unoab.orb.,t co 1 aaa.

Two .ethods of coupli" rabbit antiboclies. onto Sephad.x bead.

wore usod. AB Sepharose activated by ,lutarald.hyde or Sopharose 4.B

aetiyatod with cyaDO'.1l broaide. Both Ilethod. of '01 activation ,ave

a protein couplin, .fficloncy of ,reater than 9S~ as deter,ained by th.

los. of protein. in the superaatant fol.lowill' tho overnipt couplin, '.

r.aetion.

Ca.aercially availab1. rabbit antibodie. (Moloy'.) and 'hoa .. ade'

... _--'--.......,-........ ---~

t -140-

l.dJI I,Gl or 1 aG1 .e re ule cl. AlI rabbit antibocliel .ere conai4erecl

.onolpacific base cl on purificaUons on IDOUS. or IgG2

~uaoab.orbant colaans, or by UmaUDoeloctrophorosi ••

Wo ·.ore interestocl in shawin,. that BS'. inhibition of HF

.ti.utat.d cell ,proliferation .as reaovocl on the RaN I,Gl coluan. ancl

DOt on a control RoM 1&62 co1œan. Also.e .ould like to shaw that BS

laauao,lobulins recoverecl iro. tho Rail l,Gl colaur can inhibit BF

stiautated coll proliforation# .heroa ••• torial rocovorocl frolll tho RaN

I,G2 colaan cloo. not shaw any inhibitions.

As shawn by T.blo %VIlA, .e appli.cl DEAE colaan purified B5 laGl

Cohan flaw throll,hs •• re

coUectecl fro. the two COlUlUlI ancl assay.d for inhibition of BF

ati.utatod coll proliferation. In two cons.cutivo assays. colaan flaw

throupi froa th. RoM laGl cohan did not oxhibit ilÛlibitory 1'ea111 tl,

.horo.s tho colaan flow throu,h fra. the RaM laG2 col aaD oxhibitocl

full biolo,ical inhibition of BF sti.ulation. Thorefore th. B5

biolo,ical inhibitor,. co.ponent .as retaia04 on the l.aN l,Gl co11lll.ll

and no~ on th. RaM IaG2 col Dan. Anal,.si. of th. colaan flow throllah

.howocl the ab •• ace of laGl fra. tho RaM 1&61 co 1 aan. but tho pro.ence ,~

Vsrioui .ethocll "ore att.ptecl to oIute the co11llUl bound l,G1.

In prel iainary exporiaentl, .e appliecl a D3 leucine labeHecl B5 l,Gl

( ta 'luto off the coluan bouad raclioactivity.

As shown by Table %VID. if 112.000 cpas' of radioactive B3

labellecl 85 IIGI ".a .ppliecl onto th. l.aJ( IaGl ucl aall IaGl coluana.

~ ---------- -~-~- --------;----~-----

"

(

/ -141-

9~ of th. ra4ioactivity appeared in the colaan flaw though oL the KaM

IaG2 collllUl. E .. ontiaUy no radioactive B5 IIG1 "u bOUlld to the RaM

IsG2 col1l8n. Wheroa. 65.5'" of the radioacU vi ty wu ro tained on the

RaIl laG1 coluan. Elution of tho collllUl bound radioactivity with 0.2 M

,lycin. Bel at pB 2.4 or pB 8.0 4id not e1ute off Any radioactivity

froa tho Itd IIG1 co I1111n. Diothyllaino e1uted approximately 3 .1~ of

th. ra4ioactivity. The best olution was "ith 3 K ESeN in which 26.7'

of tho radioactivity was recovored. Bowever this represented less

than 50'- recovery of the cohall bounci laGl. Elutions "oro d50

att .. pteci with dooxycholato anci luanidine Bel "ithout succoss.

W. next atteaptod to use 3 M ISCN to olute unlabolleci BS 1161 ,

bOQllci to the RoM I,G1 coluan to study the recovery of B5 II61 mediatod

biolo,ical inhibition activity. As shawn by Table XVIIe, we could not

rocovor any anti-BIl/IL-2 biolo,ieal iahibitoty activity troll tho ESCN

olutod ,aateria!. Bowovu tho ISCN e1llted fractions woro "eaUy IJG1

positive whon assayed in ÙDaGDocii} fus ion. Further olutions "ith ESCN

We have shown that BS laGl will bind to • Rd laG1 co11lllJl.

lib"he the r .. oval of anti-OF biolo,ica1 inhibition activity on a

RaIl 1 aG1 e 01 1I8n. W. ha.. b •• n unabl. to neov.r auti-HF/lU

biolo,ica! inhibition acUvity froa the cohan bound I,G1 fractions.

Perhapa the biolp,ica1 active site on the t..uao,lobulin (B5) was

inactivatod ciurin, the el ut ion prousa. No g, I,G1 or bioloaieal 1

iabibitory activity .as rotain.d on a IoN IaG2 coluan.

vi) I.oel.etrie focu.ipl. In order to definitively associate th.

J

-142-

anti-BF biologieal inhibition activity with the BS 19G1. wc must

produce a homogenous preparation of BS IgG1. Since wc "ere unable to

recover signifieant amounts of IgG1 from the RaM 19G1 column. we

decided to use isoelectric focusing to purify BS 19G1.

A 83 leucine labelled B,5 IgG1 was used as a Dlarker. The elution -fi

profile is shown by Figure 5 in whieh the peak 83 labelled BS migrated

in fractions 10 to 15 corresponding to a pH of 6.8 to 7.8. Wc decided

to pool the fractions containing tho peak 83 leucine labelled BS 19G1

and the fractions before and after the 83 19G1 peak. Each pooled

fraction was assayed fo,r IgG1 content by immunodi ffusion. and the

ability to inhiblt BF stimulated cell proliferation (Table XVIII).

The peak which contain radioactivity (i.e. H3 leucine labclled BS

corrcspondod to the only fraction which contain IgGl.

Furthermore the only fraction which possesses the ab il it y to inhibit

BF st imula ted ce Il' 'pral iferation corresponded to the IgG1 pas i tive

fraction. and t~e fraction which contains the peak 83 BS IgG1.

Bascd on the above da ta, we are conf ident tha t the mOnOc 1 onal

IgG1 produced by BS indeed possess the ability to inhibit BF

stimulatod cell proliferation in a specifie Dlanner. It is also '"

encouraging to sec that there was no signifieant non-specifie

inhibition of BF stimulated eell proliferation in the non-IgG1 but

~ proteinaceous fractiQDs. Therefore. we can rule out the possibility

of nOQ-specific excess protein inhibition ofe

the BF stimulated

proliferative response.

vU) 03 labe 11 iua of BS' s h21. We have boen able to biolo-aically

\

-143-

label BS IgG1 internally with H3 leucine. Logar ithmically groY ing

cells yere cultured rn ser11Jll free c.onditions in leucine leficient

lledilDl supplemented yith HS leuc ine.

At different times fol101t' ing the initiation of B5 ce 11 groyth in

leucine-deficient medium. we took aliquots to usay for the amount of

radioactivity incorporated into the cell. the precipitable proteins.

and the non-precipitable molecules. As sboyn by Table XIX. there was }'

/

a linear increase of H3 leucine incorporation into the cells. and the

~onium sulfate precipitable proteins. Likeyise a linear decrease of

radioactivity in the non-precipitable molecules was seen.

After eight hours, the radioactive ammonium sulfate precipitablc

material was found ta be IgG1 positive in immunodiffusion. The

ammonium sulfate non-precipitable fraction remained IgG1 negative.

After 24 hours, 38.4' of the radioactivity ya, incorportted into

the ammonium sulfate precipitable IgG1 positive fraction. This

ropresentcd • radioactiv ity of 341,504 cpm/100 "lof the pree ipi ta ted

proteins.

AlI cul ture supe rna t an ts af ter 24 hour s of BS ce Il groyth in HS

leucine medium yere precipitated by SOI saturated amllonium sulfate. (

The H3 labelled BS had b~en used in the radioimmunoassay to deter.mineo

the total IgG1 content, and as a marker in various column

chromatography, isoelectric focusing. and binding studios.

viii) Rad~oimauno.ssay for II~ content. With the aid of the HS

leucine lab.lled BS I,G1 ye developed a RIA to quantitatively

determine the .. ount of IgG1 in each preparation of BS IgG1'

(

-144-

As shown by Table XIV, and Figure 2 standard curves yi th

increas ing amount of st andard mou se immunoglobul in y ill compe te yi th

a3BS 19G1 b,inding to the rabblt antibodies. This yas reflected in the

lower amonnt of radioactive B5 IgG1 precipitated by the rabbit

antibodies.

Our 20X concentra ted cul ture superna tant contains approxima te ly

1.1 ta 1.S mg IgG1 per ml. The two fold concentrated ascites fluid

contain approximately 11.2 to 14.4 mg 19G1/ml.

As y111 be dlsocussed ln Chapter V, 'II'e have detennined that ln

most anti-BF biologieal inhibition assays, we require approxl.mately

3.12 ta 6.25 Ilg of BS IgG1 to glve 5~ inhibition of 100 ~l of G7S

BF/IL-2. Thu would suggest that BS 19G1 has a very 10'11'. affinity for

BF/IL-2 since a large amount of 19G1 was required in arder to give 5~

l.nhibitl.on of cclI proll.feration.

Alternatively, in Any biological systems, there are competitions

betlfeen BF/IL-2 binding to the monoclonal antibodies, or the specifie

IL-2 reeeptors. Perhaps the IL-2 reeeptors have a stronger binding

affinity for IL-2 than the BS IgG1 monoclonal antibody.

\.

•

\

\.

-Tab 1 e fr 1(.

AC4 Il 85 clone

20 MLR prollferation lnhibltlon by various hybrldoma prepardtlons

20x concentfa~ed culture sup.l

fi

B

-------

- -- - ---- -

Ascites fluidFl

(Riozz; x Balb/c)F 1

Balb/c nu/nu

COlltnll 5uperna tan ts

dIlutIons

1.1

19'11+66P)

2098+1536

dl lu t Ion S : - - ------

1 2n

184+145

5623 + 342

di lut 10IlS

1 2

20x conc~l) P3X20 140;15+446

?Ox (Onl dnt 1 )!WCI23992+-296

1: 8 1 16 '} 1 64 1 128 (~ }

1 256 no 85" -...i

11538+-2927 23125+259B 26928+4A19 27610+3413 32510+3025 35924+3 -

9942+1559 23426+2723 2fl054 +-2690 25800+-7913 28765+547 30749+5 ----"-- -

,~

1.40 1 80 160 1 320 640

779+82 1159+461 4730+-491 9199+748 8431+121 12410+150

5681+2353 16660+3073 22015+1507 20640+3798 18552+1849 35938+8

., 1 . ~

1

.t-­\.J1 Cl! 1

1 -145b-

Tabl e IIII

1) ACB-II-B5 hybrldo.a supe rna tant w.s concentr.ted 20% by UIIIOUlm

sul fa te precipl taUon. Lib"ise the P3I20 spent culture supernatant.

and the .onoeloual anti-SJmC hybrlda •• supern.ataat were aaaonilla sulfate

concentra ted 20 foId.

2) Aseltos flui.u .... collected fro. (Biozzi x Balb/c)FI or Balb/e

nu/nu lIIice, Glaoni .. sulfate preclpltated and reconstltuted to its

orlglnal voluae.

3) i.esults are erpressed as cp + SE. of H3T uptak.a by 2- lILJt cells

sU.ulated by 100 ,,1 BF/IL-2 lD. the presence of diffeunt dilutions of

B5. Four dlfferellt ••• ays 'ro shawll with it. correspoDdill' positivo

controls.

4) Po,itive controls. 2- XLi colIs stiaulated by BF/IL-2 ill tho absence

of any BS products.

Sldndanhlï )

Ill] 19c1ml

A 100

/')

'ln

2')

12.1'")

6.25

8 7'1

ri. ')

lA.75

9. 17

4.hA

"--~------

,

, ,

'l'dl>le XIV.

( ) ppl. qJn qrn/ppl. -fn..>C C1lll

~--- ----- --1-- ----)')22 O.IOIH

') H5 O. j 152

hflH4 O. ')f)fj J n u') ') 7~5 ~ O. Il BH H 20X Slip

7962 O.7Ij2'1

HOH4 0.028j

7]hh 0.74 U 1 B'lb

9601 1.2h

1 ~Clte"; 9')(J2 1. Hi

l()I)02 1./2

I1070 ) • HO

R_ll! 1 ( ) 1 IlIll\UlOdS!:icly t O[ 1 t1; l ('olll eflt .

1 q lln/ppl .(2 ) ~n

B') dlluLllvl ppl. qt tree ClIIl

-------- -------

1: ~ 41,2 .22 J6

1: 10 4')Îb .2H-'O

1 : 20 fi} ()l) .4/82

1 ; 40 Il ') 1 .114':>4

1 : HO 107 i • 7B 3h

1; IhO -"02 Il.HO

1; 320 (lU) 3 I.H

1: h40 10!1)() 1. 52

--"------ ---- -- - - -- ~------------------ - ----

...

~

1'1 l'onlen J4)

lIt) Iml - ---

660

1160

} ')611

1640

1120 1 ~

f'

'" QI

1

1120n

11200

14400

-------

-146b-

< ==

~

<! '" ,

.' .. 1

~

<T

<J

~ 1 / 1

/ ~, 0

-='/8

~

1

........

-j 1

1

J

o CD

o "7 .....

0 ru .....

0 0 ~

0 CO

0 CD

0 "'7

o ru

o

E ro L Cl a L tJ ..... E

-146-"

Table XIV

1 Standard curves are sot up with Moloy's affinity cola_n puifi.d

aou.o laOl usod' at th. concentrations indicatod.

2 cp/ppt. - the radioactivity recoverod in the pr.cipitate fonaod 'by

the 1161. rabbit &Ilt! IIG1 and ,oat Inti-rabbit antibodie ••

3 ppte cp./fr •• cpa. The cp in the ppte are as dotemined by (2).

\ Th. free cp are th. total radioactive 85 IIG,. added to ,the reac:tion

ai%ture. The standard curv.s are plots of I,G1 content (~I/.J) versus

the ppte cpa/fre. 'cpa values.

4 The IIG1 content can be derived froa the studard cun •• usU., the

ppte cpa/fre. cpm v.lue •• nd multiplyinl that by the dilution factor.

i.e. ppte cp/fre. cpa - 0.4786 • 78 ~1/8l

78 ~1/81 % 20 - 1560 ~I/.l (1.56 8,/al) '.

5 The lIA p.rfona.d on 1 repr .... nt.tiv .... ple of B5 201 cOAc.Dtrated

culture supernatut.

6 The RIA perfonaed on the (8iozzi x 8alb/c)F1 ascites .fluid.

-147-

Table xv)

8102:71 ln ~1.() lIDmunu.atlon-Inhilutlon of ~(J MLit cell

prollfprutlon hy !>erum free hyltrldoma superu<ltnnt

lIyltrldomn clone

DD2 II A 10

ACLt 1 E 3

II A 8

II B 5

III A 9

III D 2

IV C •

Controh (5)

lIlod

BF/IL-2

E(3) •

1

1

1

E

E

1

[xperlment H

2.20 + 145(2) 3515 :!:. 193

210S + 203 (

-4290 :!:. 393

2599 ±. 45S .. 687 :!:. 422

239 + 152 480 !. 26

27 .... + 3 .. 6 3625 :!:. 85

2370 + 249 3797 :!:. 783

26010 ±. 338 3637 :!:. 20 ..

92 + 18 104 :!:. 14

4781 + 827 6061 :!:. 119

t

(

T.ble xv

1 Two r.pr •••• t.U., ••••• y. -ith two pre .r.Uo •• of 2,0 ... c.ll ••

Th. • ... IF n.-2 ••• a •• cl.

2 ".al ta al.'. .spr •••• cl •• ct- ~ SB of B3T .pt.b by 1 z 10' c.Us

ataal.t.cl .ith 100 III 8F/n.-2 la tho pr •• oace 'of 100 III hybriclo ••

saperaataat. 3 cl.y. iacabatio~. , hrs a3T pal,. 1 Iloi/ •• ll.

3 E - .ahaac ... nt. 1 - iDhibitory. Pr • .,ioa. t.stial of th. ..rua

coatainin. hybricloaa supernatants trOll th ••• clon •• h.cl shown .ith.r

eah.nc ••• nt (i... .zc."Ü sti.ulatory nlpon •• ) or inllibi tio~ of the

BF/IL-~ sti.ulated re.pon.e. S •• T.bl. Il.

• Ser~- ire. lupernatutl are 'prodaced by incabatlni 10 z 106

hybrido., c.ll./al for 2. houri in 1DM1 16.0 (no I.rua).

'Controls. JI.cl - no IF/IL-2 or hybriclo.a .aperutant .as .clcle..ct;)

~ BP/IL-2 - 20 MLR cel1 •• ti.alatecl .ith 100 III 8F/n.-2 " .

in the .b.ence of hybriclo.a saperaatantl.

_. '. ~---.. - --- "-- "~' - - - - ---------

! :

/,.

,-

B5 -- DEAE

FCS -- IEAE

B5 -- DEAE

1 Table XVI. DFAE Ion-Exchange Chranatography

". --- .. - •• - ~ ••• __ • __ o ••• __ ••• _______ , '----------- .. -Fractions positive for: IgGl

8 - 201)

rn1e 1-------------_.-

Fracti90s 1:XX>1ed and concentrated:

7 - 24

IgG2' M, A

o ~---

111 1: 1 , 1

36 - 5&2)

-FΠ- DFAE 1 5 - 25 35 - 50

fx

B5 G1

(G2

MA)

ffê. G1

(?2MA)

r~ i \

1

. --~---_ ... - - ~ '--"-" ----- "- .- -2° ~q inhibition results . .

1:2 1 1:4 1:8 1:16 Control

4914 ± 163Jl) 5679 ± 1494 8130 .t 1235 12082 ± 967 -BF 760 .t 364

9426 ± 562 10307 t 1773 13863 t 1071 12635 t 1077 +BF 11813 ± 868

11280 ± 860 13265 t 335 13042 ± 1038 , 16744 t 274 -BF 2138 t 588

,1)643 ± ~ll 12640 t 969 13929 ± 3591 ' 15039 ± 1610 +BF 10778 ± 353

,

•

1 ....&

~ II/ 1

{

II \ '

/ /

/

/

~ / "'" qj ...

1 =' / cc' 1" ..-4 t:..

( / .. 0 -2

/

. '" '

-149b-

\.

\

1 -/

\>-

5 ·1 a .! S·O o '0

·0 ·0

------~-----

a tn

a (11

a N

a ....

0

fi)

c 0 ... "., u 0 1..

t+-

-149c-

\ 1

f

( 0'2 o "1: S'O

"g."O

l~

l 1

r 1

r 1

t ~ r !

1

o "0

c ('T')

a ru

a ....

a

en c o ... .l

'U a L

<-1-

(

-l~o-

Table XVI

1 The fraction she .aa 10 all fnetioa. Oaly fractioD 8-20 froa the

B5-DEAE colllaD ez.b.lbitecl atroD, IiG1 content as cie·temin.cl by

w.1Ulocli f fuai oa.

2 This fractioa. after coaceat.tioa/purif le& tioa of Ixo,eDous , ,

proteins, elthibitecl sliaht I,G1 positl.e.

3 Valuea are expre.a.d as 01'8 1: SE of the upt.ke of a3T by BF/n-2

atiaulaUd 20 IILa celh. 1 x 105 IILR celh •• re stiautatect _ith 8F

D.-l (100 ",1) in tbe pre'seace of 100 ",1.85 I,G1. or other ' I,G1 '

fractions.

1

t -151-

Table XVII

Fractio~tioa of B~ OD Rabbit Auti-Moua. IIG1. I,G2

L..aaoaffiaity Coluans

'-- .. A. Coluaa fla. throu,h iahibitioD resulta

Exp.

1

2.

No B~ I&61 (l)

1637" :!:. 871(,,)

11104 ±. 593

10536 !. 785

92.~5 !. 59%

aa ti - 1 &62. (3 )

153" !. 632

659 !. 22

B. Elutio~ of H3 Labell.d B5 Fro. r..uaoaffiDity Colaaas

Liu îïGl AuU ~----

Cp. , Cp. , ! ,

: B3B5 appUed 222000(5) 100 222000 100 1

colaaa fla. throulh

t,:: 3".5 206400 92.0

DΠe1utioa(6) \ 26.7 7200 3.1

di.thyl_iue e lut iOIl(7) 6960 3.1 1~60 0.70

- - --- ---- ------

7

1 -152-

c. Da. El uU ou- IAh i b i ti OD Re sult s

No B~ DCN elution 1

1

1

R a If IaGl 16374:t 871(8) 18496 + 613 t

\.Il a JI 1

IaG2 16374 :t 871 18996 + 2014

•

--"

1

•

-153-

Table XVII

1 Inhibition Assay was performed by lncubating 1 x lOS homan 20 MLR

100 J'l BF/IL-2, 100 J1I B5 IgG1, or fraction samples. Cells "ero

incubatod for 3 days, 370 C, 5~ C02 and labeIIed "ith 1 J1Ci a3T for the

last 5 hours.

2 The colnmn flo" through from the rabblt anti-mouse IgGl colomn.

3 The column flo" through from the rabbit anti-mouse IgG2 colomn.

4 Resui ts are cxpressed as cpm ± SE of the aptale of a3T by 1 Jo: 105

human 2 0 MLR cells stimulated with 100 J'l BF for 3 days.

5 H3 labelled B5 IgG1 was used as a

expressed ~s the cpm (of 83 labelled B5

a ~ of the 10~ response arc expressed.

6 3 M ISCN eluted fractions.

7 0.5 M diethylamine fractions

tracer, thtrefore resal ts are

19G1) in jCh fraction. AIso,

/

8 Results arc oxpressed as cpm + SE (see #4) of the 3 M KSCN oluted

fractions •

o

•

\

<J • < •

--:-

Jf ~

< L/ -------------

()

<

•

•

;

< <

..

sos ,

1 -154b-

Table XVIII. Isoelectrlc Focuslng Inhlbltion Resu1ts

\

"--..

~ fractions 1-8 9-15 16-24 \

Ig6i !content (1) +++

Inllibition da ta (2) 15486 + S~ 6043 :: 424 16094 + 657 - -

" 1

cttrol S lIIed 933 + 285

3006,8 BF + 2398 -

(B5 + BF) 898 + 359 -------

1 II61 posItlve fractlou3 " .ere the Ouc:hterlooy

l.IIIIIIUllodlffuSlon mct.hod.

.. 2 Inhlbition data are expressed as the ablllty of as 1161 to inhlblt

BF stlmnlatcd 20 MLR cell prollfe~atlons.

-155-

,

Table Ill.. BlolabelllDI of pS's 1,61

IncorporatioD of 83 leucine lnto.(l)

Tae (hours)

0

4

8

12

16

20

24

Ce 11 pe 11 e t (1)

cpa(2) ,

125361 20.8

271371 35.5

328S26 39.6

398116 48.0

381452 46.8

396911 47.4

414S57 46.6

SOlI. UIaO D 1 ua

sulfate preclpltable Cp. ,

74429 12.3

930:'6 11.1

200154 24.1

2lZ-S61 25.6

265765 32.6

300155 35.8

341S04t 38.4

Don-prec lp 1 tab 111 cp. ,

401!10 66.i

399S23 52.3

300082 36.2

117799 26.2

167094 20.5

139655 16.6

131824 14 ,8

1 Cul tures were separated lUtO three fract 1 ons follow Ill' the

appropriate IDcubatloD tl.es· (1) lDcorporatloD of 83 leucHle ,1l1tO

cellular structural protelDs, (1) the culture supernat&Dts .. ore

preClpltated .1U 5011. (v/v) satural.ed maoDlaa sulfate aDd separated

~~~~Dto preClpltable aDd DOD-preOlpltable lIacra.olec:ules.

2 le.ul ta are expresaed as cpa. the .. ount of 83 llulclDe label

1.l1corporated lDto each of the t.hree fractloDs. 1. e. DNA, cells.

a.aQDlaa sulfate preclpltable aDd Doa-preclpltalc aacra.olecules. The

reluIts are .also expressed a. t.he' of Ua. total radlo.ctlVlty

1 -156-

recovered at •• ch ti •• point.

r

1 -157-

Chapter V

AC4-II-B"s Biololioll Activities

BF/IL-2 is ,aA oblilatory soluble aediator involved iA lymphocyte

.. responses to alloaAtil8n5 and various T cell aitolens (176). le

therefore studied the abill ty of AC4-II-BS' s I,G1 ta inhibi t various

~lUlolo.ieal response s in whieh IL-2 had been directly iapl ica ted as

an obli.atory sl,nal.

M,terials apd Methods

1) AC4-II-BS lphibitiop of BE ftinhted 20 ML& cell proliferatiop.

As .'Iltioned 1%1 Chapt,r II and III. Olle of the priury u,ay for

BF/IL-2 activ1t7-lS the abillty of BF/IL-2 to trluer 2 0 .. Il ceU

prollferation (200). We t,oak advaDta.e of thu and used it as a

biolollcal screeZUD, assay for tybr Ido.a. ..hich .. y produee an

~UIlo.lobuliD with speclfieity direeted alainst HF/IL-2. (S ••

Chapter III anti-HP biolollcal ser.eniD, assay.)

H) Aati-Bf auay" inhlbiuop of hua, ,0 MLI-QIL asUYUlft. It

h .. ...u eu.abluhed th,t BF can Hi.nlate pr,---d eytolyue cell

proliferuioD lD the 20 Mli. (ZOO). Wc .. cre loOterested ID stndyuli

whether BS I1G1 caA ulublt BF/'IL-2 uulnced CIL actlYity.

LDto elch aierot1tre •• 11 wa, add.d 1 1 105 responde; ceIlI, 100

III IF au 100 pl hybr iÔOlla snperDa taDt. For rcatianI&tioD witk

;

1 -158-

sti.ll1&tor cells. 1 % lOS irradiated sU.ulators were added ill the

abs.nce of BF/IL-2. Ç}CL activities were tosted on days 2. 3 and 4.

To .ach weIl wu added 2000 (for a 50:1 ratio) or 1000 (for a 100:1

ratio) Cr51 labelled tarset celIs in a final 200 J'1 voluae. Alter a

4-6 hours incubation. 150 III of coll froo supe l'na tallt was a ... yod fJr

C1~ by tho roleasc of ly.ed cells. AU wells

quadrupl ica te at two killer ta tarie t raUos. C.11

r.sp~nder cells per w.ll was detor.ined by trypan b1ue dye e%clusion.

iH) Inhibitiop of tho la KLl. To test tho offect of hybrido.a

supernatant on haaan 10 JlLll. wo iDiti~tod 10 na in .icrotitre wells , with 1 % 105 respoader colIs. 1 % 105 irradiated sti.ulator$ and 50.

hybrido.a 11lperuatant in a final Tolmae of 200 Ill. This was incubated

for 6-7 da,.,. at 37 0 C ,1ft ~ Uld pulsod with 1.0 J'Ci of a3T for th,

last fi"e hours. The positive control w ••• 10 MLJt porfo:r1a04 in tlle

.bloace of the ,0. hybrido.a lupernatant.

To tost tho offect of the hybr ido.. 11lperna tut oa .OUM 10

11.&1, lOS respoaders were Iti.ala'od _!th .. % lOS irradiated

Iti.lllators in 200 1&1 tiual yolaao "itll SOI. hybrido •• sllperna'ant.

lolls w.re cultured for 4-S days\ aAd a3T pulsed for the lut 5-12 \ '

ho""rs. POlune controll w.ro ~rforaed ia th. abunco of 50ft

hybr ido •• 11lperu t&At.

i~) ~rldRa' IIPo".t.lt', .ff,ct. OR aito", .'twlilto' CIll, (1

.pd B etl1 .uo",,). To .... y for T etll .lto,!. Iti.""1&t10.1 ia th.

pr .... c.'·of hybricloaa' lupernatut. lOS cell, (htaaa PBL. or 80 ...

...

t ~.,

apleea. cells) were stiaul.tod with 5 J',/al COIl (Gibco), or 1 .. PRA

(Pha~.cia) in th. presence 'of 50tJ1. hybrido.. suporn.tant. Cell

proliferation was deterainod by tho uptake of 1.0 J'Ci H3T after 3 d.ys

incuba ti OD. PositiVIt cODtroh wore colIs stiaulated with T c~11

aitoloDI in the ablence of hybridoaa Iupernat.nt. A nelativo contrQl

w.. .lso perforaed with cells incub.tod in tho presence of 5~

hybridoaa supernat.nt. but in the .bsoDce of the .ito,enl. Hybridoa.

supernat.nts alone ... oro noi ther aitolenic Dor inhibitory for

UDlt1aulated cella.

The hybrida •• superut.nt was also Usud alainU two B cell

• itol.nl • Staph. Au.,.". aicroor,aAh.. and LPS • LPS (Si ... )

• tiau.lation of IlOnse spleen colis ..... perforaed w!th 1 J'I/al LPS. 105

.ou.. sphen c.ll a in a final ""01.. of 200 J'la Cell viability

(tm.a bln.e dye exclnsion) aad cell proliferation (B3T npt.ka) .... r.

doUraiaecl on clays 3. 5 ud 7. ~Inn., aic:roorlaaisa (in.actiYaU4)

il a f'kaowa hua.a. B ce 11 aitolea. na. batch of .1... .,r.,. usecl

(prO'l'idod by Dr. B. Rode) had pr'YiousIy been Iha...a. to stiaulate E-

P.8L (i.e. h ... a. B cel1s) prOliferation. but had ao effoet oa E+ PBLs

ce1h/al. ln tho presence of 7 s: 109 L IprtslI ateroora_Ai "I/al.

bcü.tlon .... s for ci,ht day' At 37OC. '" COz at .. hich poiat a3T

upt.k ..... deterainecl by a fiTe hour pnl •• lab.llinl.

To u.t the effeet of hybricSo •• supenaat .. t OD B ce11 altoloD

.tia~l.t.cl c.l1s. 50. bJbridoaa .uparnat .. t .as .ddod in tk. "r •••• c.

of tlt.. B c&11 alt0l.a. C.U proUfer.Uo ••• s 4euraiud by a3T

spt.ta at t~. appropri.te tt.e.

-...

1 -160-

v) . B' II~ inhibition of the murine co-stimu!ator assay. Mouse

thY1locy-te proliferation in response to 10. doses of the mitolen ConA

is dependent on the presence of 0%o,onou51y Ipplied IL-l. Therefore

B5 1,61 was tested alainst both human BF and lIlurine IL-2 in th.

aito,en stimul,ted costimulator assay.

1 % 105 Ba!b/c thymocytes were sti.ulated with 0.5 118 CanA in

the presence of 501 IL-2 and 5~ hybridom. supernatant. The IL-2 u.ed

".re either hmaan BF/IL-2 or ,murine IL-2. Positl"e controls wero

pertoraed in th, absence of hybrida.' supernltant. NellUve controls

".re performed in the preseuco of Con ouly. Thyaocytes cult~.d in

tho pres .. uco of Con and 5()1Jt hybrido.a lupernatant (no IL-2) cli4 not

.xhibit any cellular proliferation.

Ti) ,,&Ybr! •• sup'VlaUR" S "hct og IL-i .IP'Rd.eI cella aRd IL-Z

d,p.pd'Rt c'll lin ••• Sine. D.-2 d.peadaat calI Hllu 0,," n..-l

.s.p.aded cel1l are dopelld.llt Oll IL-l for s,,"ha! ucl prol if_raUoD.

a. anti-BF/IL-2 should bo able to 1.uhibit th ••• cell', proliferation.

na ~7Bl/6 uti-(C57Bl/6 x C3BlFl D.-l dopelld.nt c,l1 1 in. il po •

c1epeadellt Oll tha preselle. of 30 to '$0. frash n..-l tT.ry 1 .. ho.rs for

c.ll prol if,ratioll alld Sv.rTiTa1. l'bis clll lill'- caÀ b. ,row,1l iA th.

c.lls w.r. culturecl ill the pres'Ilce. of 100 pl r.t IL-l {CQaA

stla.lat.dl or h ••• BF' and 100 1&-1 of laybric10aa "uper ... tall.t. Cdl

prolifer.tioD. wa, clauraiud at 1. aad ... bours br t1le o.ptate of a3T. "

n. la_a. n..-l eapaAdecl cel la an dtpeade.t OD tlae pi .... c. of

1

1

!

t

(

-161-

frosh humaA BF or humaA mitogoA stilllo.lat.d IL-2 .vory 24 ta 48 hours.

1 x lOS and S x 105 IL-2 .xpand~d cells wero cultured in the presence

of 100 ~l human IL-2 and 100 ~1 hybridoma supernatant. Cell

proliferation "as determined at 24 and 48 hours by H3T upt-ak.e.

Hybrido.a sup.rna tant done (in the absence of, IL-2) , -had no .ffoct on

the cell proliferation, or survival of IL-2 dependent cells.

Pre •• gt.tion and Discussion of Results

Biolo.ical l'OIRon.!. igflgepcod by B5 l'Y1' Inhibi tion 'of 20

ULR c.ll proliferation.

A. shawn by Tables XI, XII, Dl, IllI and' nIIl, B5 I,Gl froa

culture suparnatant (wh.ther crude, ~n1ua sulfate 201 cone.ntrat.d, L.,.-

or s.r. frce s.pernataats) or laGl ISc'1tes fluid can inhibit th.

h'lllftn 20 'fUl c.ll proliferatioa in r.sponse tl) BF/IL-2 sUaulation.

Approxiaate ly '''' inhibJ. tion of BF actiT Hr wu lO~n Il 201

coae.ntrat.d BS cultur. lupernatant dilutions of 1:16 to 1:32.

corr.spondin, ta ail. uhbody titre b.tween 3.12 P' to 6.2' Piaf B5

I,Gl. Sow'Ter. th. d.,r .. of .. si .. 1 inh1b'itiOu "aries trOll ~iffer.ut

.... "s du ta th. di tf.rent 20 )ILl. colis .sed. Differ'llt 20 Ja.K

pr.paratioas r •• poAd to SF/IL-l dif~er'lltlr' d.peA~IlI· on th. nuab.r of , c,11s .xpr ••• ill' th. aF/IL-l r.ç.ptor. and the nuab.r of CIL.

sCia.lat.d to prolif.rat ••

la kinetic 'XP'l'iaellts perfora.d br th. addition of as IaGl at

.,ario.. tia.. followill' initiation of a BF stlaul,tloa, •• .'1" iat.r.IUd ia d.uraill!ll, th. ti .. ",q.ind fol' as 1161 to 'Urt: HI

-, ..

1 -162-

influence (T.ble nIV>. In tyO sep.r.te experiae~ts. it was shown

that .ddition of B5 IIG1 after 36 hours did not lo.d to an inhibition

of BF sti.ulated cell proliferation. Sinee our biololical assay

syst_ for cell proliferation is a coaplex systea involvinl nuaerous

cellular events. it is ditficult to detenaine whieh 'one' event was

iDhibited by the B5- 1&61. Bowever, this experiaent SUllosts th.t 85

acta in • specifie •• uner. If it is a noa-specific inhibition by A

factor in the concentrated B5 culture supern.t.nt. one wou1d expoct to

••• inhibition of the BF response at Any tilDe th.t 85 was added.

whether it be before or atter the initial 36 hours of incub.tlon.

le wore a1so interested in the effe~t of B5 1,61 on the response

of auriu- 20 !CLas to A, haaan and aurine n.-l. A C57Bl/6 Ulti-DBAl2 20

-.a was establ ished and rut i.ulatod wi th th. hua.n 8F /IL-2 .Ad a

aurin. CollA lti.a.IAud IL-l. As shawn by T.ble XIV. sianiflcant

)_ iDihibihon"as s.en At B5 dilutions of 1:6" and 1:128 (or

corrupondiD .• to 1.565 J" and 0.182 J1' la61). Bawn'er at tho hi,her

cone.ntrations of B5 1&61- (1:8 dilution. or 12.5 "' IIG1>. wo obserTed

a nontal. if not sliahtly iner .... d. prol if.rath. respons. to ,huaan

aF/IL-l. Thi. raiao. the 'possibdity that BF/IL-2 and 85 I,G1 fora an

u..a.ae co.ples .h~ch ... s aoro stlaulatory th&A BF/IL-2 alone. le .lso

ob.erT.d le .. l1bltl0n of aurue IL-2 at tht t"o hiper 85 laGl

Cbncontration. (1:8 and 1:16 d~lutions) ... hich .. y be partially due ta

ll5 I,Gl and IL-2 fontina a .t.1.ulatory u..tua. coaplex.

pral Heration iD re.ponu ta IL-l. Th. inhibition by B5 1,61 1S •

'peeifie .".nt which tzert. it'o.ff,ct withiu th, initial 36 hours of

,

t.

\

.'

-163-

/' IL-l Itiaulation. S ipif icant B5 1&61 aedia t,d illhibi tian olf IL-2

sUau1ated c,U pral iferation yas sean in the ranlo of 0 .. 782 t9 6.25

III of B5 1&61.

H) 20 !II, e.ll viabilitiu aRd bltst cel! tnufoAatiogs

inflp.nced by a5'. II~.

The screeninl assay ye have used had been ta aoni tOI' B5'. !ffoct

on BF stiaul.tod coll proliferatioD as d,toX1ain,d by 03T uptake. 'l,

yoro al.o interested lD' the reIationship betyocn coll viability, blast

cell transformatIon and cell proliferation as d,toraiDed by D3

thyaidine uptake WhlCh .as influonced by B' I8Gl'

As shown by T~ble XXVI, 20 MLR colis growD iD the presence of

Bf/lL-2 and BS IgGl for 48 hours .ill e%bibit decreased 03T llptake.

The dilloroncos iu 03T llptake •• 1', oven aoro dr .. atic at 72 hours. In

the s .. o cultures. there yere no dilforonces in coll viabdities

yhether the colls y,re It;0wn in tho presence or abseDce 01 B5 I,Gl' \

DoweTer. yhen co.parinl the perceDtaae of blast-like colls in these

culturo •• thorw .a. a 24 to 3" diff.ronce in the nuaber of blast-like

ceUs. Essontial1y 20 MLR cells stiall1ated by BF in tho presonce of

B5 laGl showed very low nuabers of blast-hko cells (13.2~ and 9.2 .... ).

yUre.. co Il s ,rown ln the absenco of B5 I,Gl

l , blast-like cells (48.DI' and 33.1'). "1

,%bibi tod aoro

., ':ï-

\

•

20 Ii..-colis uastiaulated by f/IL-2 ~ did DOt appear ta bo

iDfluoDced by B5 1161. This yas evide~ by the siailar cell viabllity ~!

&Ad percentale of bl.st-like c:olls iD II.Dstiaulated 20 ,.ta cells Ir~n

in th, p~"'Dce _~ ab.eDce of 8.5 IaGl.

/

1 -164-

t

Wa also studied the affect of B5 I,G1 on two IL-2 dependant cell

1 ines. Ala in a t 24 and 48 hour s • ce Il s groYIl in the pr e se ne e or

ab.ence of B' 11°1 exhibited the sae viabili ths. Bowever. cells

Irown in the presenee of B5 I,G1 showed sipificant decreasos in

blast-lib cell transformation and 83 thymidine uptake at 24 and 48

hous.

Therefore. it appears that culturing IL-2 responsive cells (of

huaan and lIlur1ne orilin) in the presence of B5 IgOl did oot see to

influence cell viabU ity in a oon-specific manner. BS IgGl did oot~

appear to influ.uce cells which have not been stimulated by IL-2. It

yould appear that BS IgGl exerts its effect predominantly by

inhibitin, the ,eneration of blast-like cells. This inhibi tian of

blast cell transformation yas also reflected in the inhibition of 83

thyaidina uptake.

iU) 20 MLll. CJIL responses influenced by BS h§I. It has ba en. we Il

establlshed by nnaerous ,roups that the major1ty of responding cells

in the 20 nas are the prUied CILs (200.28). Furthermore. the cells

respons1ve to BF/IL-2 are the primed CILs. /'

We next studied the effect of BS l,GIon the stimula tion of the

priaed CTI..s and the subsequent cytolytic activ i t les expressed (Tabl e

nvII) . A 20 MLll was restiaulated with BF/IL-2 or the orig10al

stiau1ator cells in the presence or abu'nee of B5 IgG1. By day 2 (48

hours) a 6-10 lold decrease in S3T uptake yas se.n with cells grown 1n

the presence of B5 IgG1. The inhibition of S3T uptake by cells ,royn

in the preseJlce of B5 IIGI was evtdent throulhout the subsequent four

-165-

days oC assay. In parallel cultures "e studied the stimulation of

crl.s. We sa" that by day 2 (48 hours) cells "hich were grown in the

presence of BS Ig61 "ere already substantial1y inhibited (by SQIJ&) in

their eytolytic activities d~rected against the specifie target ce11s.

The inhibition was more intense by day 3 in "hich cells grown in the

presence ,- of BS IgG1 exhibited a 95.. inhibl.tion of cytolytlc

activities. The same pattern of lÎihi.l,>l.tioD. of cytolytic responses "as

seen throughout the five days in cul turc. Once again UDstimul a ted

'" cells did not appear ta be lnflnenced by growth in the presence or

absence of BS Ig61.

The reults suggest that the inhibition of BF/IL-2 stimulatcQ

cclI proliferation by BS IgG1 is an inhibl.tion of the activation of ."

the BF/IL-2 responsive prlmed CTLs. Therefore we observed a decrease ~ ~

in cytolytic activity when cells "ere stimulated in the presence of BS

It is Dot surprising that CTLs s'tijlulated by the original

stimuIator colis were also inhlbited by B5 IgG1 (Table XXVII).

Restimulation of the 20 MLR by the, orl.ginal stimulator cell is

lIIediated by the restimulation of the T helper cells to produce IL-2.

The primod CTI..s were then stimulated by the IL-2 produced by the T

helper oells. If BS IgG1 is s-peciflc for the BF/IL-2, it would aIso

inhl.blt the IL-2 produced by the stululated T helper colIs. and th us

in.q.ib 1 t iene rat i on of pr l.JDed C'ns. ,

c "'Jr

Iv) llÙlibition of 10 na by the BS hfu.. It ls that

BF/IL-2 ls an ohliiltory $i11111 ln the MLRs (Chapter I). MLRs.

\

1

.. "

:-166-

init±ated in which there were poor T helpe'r cell stimulation and the

subsequent decrease in IL-2 production will result in poor MLR coll

proliferation (Le. poor T helper as weIl as paor pre-crL

prolifera~ion) • In such an MLR. the T helper cells have not been

stimulated, to prod'l1Co TI..-2/BF and as a result poor pre-CILs did not

receive the TI..-2 stimulus and thus thoy were not stimulated to

proliferate.

If BS IgGl is specifie for BF/IL-21 wc Tfould eXl'eet ta sec that

as IgG1 can also iuhibit the 10 MLR. As shown by Table XXVIII, B5

IgGl can indeed inhib~.t a 7 days old 'human 10 MLR and a 4 days old.l'

murine 1 0 MLR cclI prollferdion. In both 1 0 MLRs we sec a greater

than 90-. inhibition of the 10 MLll cell proliferation in the presence

of a 1:2 diluted BS IgG1.

If BS IgGl 1S indeed ,pacifie for the BF/IL-2 Molecule, this

" experiment would also confirm the cssenU.I role of BF/IL-2 as the

obligato~y stimulus in the 1 0 MLR. lt would also suggest that BS IgG1

reeognizes both the murine TI..-l and the human BF/IL-2. ~

v) IL-2 dependent and IL-2 expanded ce 11 1 ines. Recent ly there ':.

have been numerous reports of murine cell lines wh1eh are dependent on

exogenous IL-2 for grewth and prolIferation. As shown by Table XXIX,

we have developed'. CS7B1/6 .Jti-(CS7B1/6 % C3H)Fl cell 11ne which is

completely dependent on exo.genous IL-2. These cells did not grow lof t

TI..-2 was absent for as 11ttle as 24 hours. However. this cclI lino

<,

was maintained in optimal cell growt~ and cell proliferation in the

presence of mur ine or human IL-2. Thçse IL-2 dependent celis are

•

,

,

'\

-16i -

a..n.responS1VC to mltogen stlmulus. Addltlon of B~ IgG1 to these cells

abrog.ted cell proliferatIon ln response te Il-2 .. 'e se e a ~(JIII

Inhibitlon of murine IL-2 stlmulatlon and 80 to 9<J4f1 lnhlbitIOn of

BF/1L-2 medlated stlmuiation. As shenrn prevlously by Table Il'VI. BS

Ig61 the <.

of blast ce Il s IL-2 dependent ce Il lnhiblts gC;,neratlon lD Î

l i.ne s grown ln the presence of ll..-2. The ~nhib 1 t lon of b las t cell

transformatIon was reflected ln the dcereased H3T uptak~. However BS

• Ig61 did not appear to exert a tOXl.C effeet on the IL-2 dependent

\. cells since' there were no dlffercnces ln cell vlablllties of cells

grown in the presence or absence of B5 IgG1.

We sec a slmllar result wlth hwnan IL-Z ,

erpanded cells (Table

" llX). Human IL-2 dependcnt cclI lines arc dlfflcult to ~stabllsh.

Our human IL-2 e~andcd cells have been shawn to be dependent on human

IL-2 for cell proliferatlon. As shown by Table XX:X. these cells w11l

respond to alloantigen or mitogen stimulated human IL-2 (1. c. 8F/ or

PHA-S). These.,cells dl.d not proilferate IJ IL-2/BF yas absent for as

little as 24 hours. If the IL-2 erpanded cells were grown in the

cE presence of BS IgG1 we sec a slgnificant in,hl.bItl.On of ccll .~

60 to 90'lb Inhlbltion of fl

,proliferation. In tbree consecutive assays.

cell prollf&ration yas seen.

'" As previously shown in Table XXVI, 85

Ig61 affects 'the transformation of blast c,«;l1s and the subs.equent H3T

uptake and cell proliferation in human ll,-2 e:z:panded cultures. It did

not affect cclI viabllity in a non-specifie manner.

'iJlerefore. B5 Ig61 ean lnhibit cell proliferations of a murine

IL-2 dependent and hum.n ll,-2 expandcd cell 1 ine. It doc 5 not exe rt a

non-spe Cl f lC tOXl.C effect on the ll..-2 dopendent ce Il s. Furthermore.

/

/

t

i . .. ~,

-168-

BS 1,G1 can l.nhlbl.t both haman and lIIurlne li-l.

homololles betlfeen human and lIIurlne IL-ls. It 15 not surprlslng that

an antlbody dlrected a,alnst BF IL-2 can al~o recognlze lIIurlne IL-l.

IAhlblr1pn of the co-stlmnlator Assay bv BS's I,QJ. Thymocyt~s

cOntaln few .. ture T helper cells WhlCh are capable of produclna IL-l.

A. a re.nlt. thymocytes do not respond to Suboptlmal concentratlonll of

IIl1tolen. unless exo&enous IL-2 lS added. Paetkau's costlmulator Issay

takc. advant.se of the essentlal role of IL-2 ln ellcltlng a thymocyte

responsc to low concentratlons of ConA.

As shawn by Table XXXl. thymocytes dld not respond to ConA alone

ln the absence of IL-2. 'However. lf thymocytes were stlmulated with

IL-2 (Illurine or human IL-2) and ConA. an lntense pro}iferatlve

response yas seen.

low doses of ConA lS

Therefore. ,j.ymocyte prollferation in response

totally dependent on the presence of IL-2.

to

Our monoclonal antibody BS inhiblted thymocytes ~1!sponses to

ConA and IL-2 stilllulation. Wc sec a S04lt inhibition at a titre of 1:16

and 1:32. or the equl.Valent of 3.12S to 6.2S I1g of B5 19G1. Thi 5 i s

approximately the same antibody titre requircd to lnhlbit 5~ of the

BF stimulatcd 20 MLR cclI proliferation in the human ~ymphoeyte

syst cm. BS had no cffeet on thymoeytes: s'timulated by

mitogen-free IL-2 al one.

This cxperiment confirm's the essential role of IL-2 in the

costimnlator assay. It also confirms the abllity of B5's 19G1 to

inhibit cellular proliferation in responsc to lIIurine IL-2.

t -169-

Responslvcness to T and B cell mltoiens Influenced OV 8S', l,y!-

) It 11 we11 establuhec1 that the T IYlDPhoc:yte prollferatlve

response ,tlaulated by a T cell mitoien 15 dependent upon the

.ti.ulation of the T helper cell,. and the subsequent production of

U-2. Bowever, responSlvene ss to B cell lIuto,ens 15 ul.dependent of

ll.-2.

We 5tudiec1 the responslveness of hnman and mouse cells ,to the T

cell \:1to gen

aicroorganlSDs, and the murine B cell mItogen LPS.

cell mitogens and ConA, and h1llllan B aureus

As shawn by Table n.xI1, BS 19G1 Inhlblted hmun PBL responses

to the T cell mitogens PRA and ConA. There was a strongcr innibltion

of ConA stimulation (95' inhlbltion at a 1:2 dilution) relative to the

PRA nspouse (which exhibl ted a Sa.. inhibi tion a t a 1: 2 dil ution) •

Bowever BS 1961 did not exert an effect ou the B cell mitogen ~

aureus. In t'1ro consecutive experiments "ith different preparations of

hWllan PBLs and ~ aureus microorganism preparations, there "ere no

inhibition of proliferation when the cells "ere stimulated by ~

aureus in the presence of BS 1961.

As shawn by Table XXXII, wc obtained similar results when mouse

cells were stimulated with the T cell mHogens PRA and CanA, as weIl

as the lad. of inhibitiou "ith the B cell mitogen LPS. There "as

s ignificant inhibi tian of response s ta PRA and. CanA when the ce Il s

were stimulated in the presence of BS 19G1.

As expected. the BS Ig61 inhibits cell proliferative responses

which ar~ dependent on IL-2 as a Mediator (1. e. T cell milogons,

t -170-

... .lb). l t does not af fect the B ce Il 111 tOleD respollse 1 Whlch are

lDdepelldeDt of IL-2 as a lIedl.tor.

.. /

('

a

\

',\

-/\

-.... ...... ~

.' 1 Table XX. Biozzi in VlVO rmmunizat10n - Clone Testing

l1 --- "

InhibitIon of BF/IL-2 St~ulated 20 MLR Cell Proilferatlons ~

(Crude Culture Su~natants)

Clones 20 = 6165 (1) 6566x 3840 4044 , x x x

OC2 II ~ 4540 ± 144J2) 5020 ± 425 17724 ± 2899 9241 1 1262

002 II :

4582 ± 458 3780 ± 888 23223 :t 3054 137 42 1 1500 5505 ± 187 5568 ± 1058 30969 ± 1571 12460 t 2796 4024 ± 283 3236 ± 380 23072 ± 112 7595 t 165 1

~

A7 4231 ± 377 3443 ± 842 .49608 ± 2757 15110 t 17 lI} --J ->

AIO 6070 ± 385 7918 ± 1517 33335 ± 5216 11833 t 1'l6~) ,

III GIa 6013 ± 1435 7115 ± 1048 35065 ± 3977 149 n 1 101H AC4 l Dl 6441 ± 367 5116 i 220 3l~33 ± 1558 102fd ± 1140

E3 ~ ± 756 7222 ± 535 23733 ± 5371 9315 1 sn II A8 4773 ± 60 5385 ± 1006 20932 ± 972 11468 t ih'J ')

B5 5099 ± 1665 6361 ± 716 24260 ± 747 l 319') ! 2 1 1 () III A6 4165 ± 224 4048 ± 1209 22609 ± 1225 8946 1 ~.Hj'J ,. A9 4133 ± 1040 4576 ± 977 37925 ± 9732 14182 t 71'J

D2 6577 ± Q58 4664 ± 329 30075 ± 3449 12216 1 144! F2 5881 ± 1750 5373 ± 113 27354 1 4351 11888 t hO')

IV A4 3486 ± 1127 5279 ± 377 23720 :!: 9663 84BO ! 1116 ~ Af;I 4377 ± 918 5234 ± 1684 18868 ! 6 14537 1 282n

B4 4708 ± 133 7084 ± 1128 28426 ! 2988 16766 t 2 II') C4 3993 ± 642 4956 ± 1722 11169 ± 2b41 I02l" 1 1 J'JO E9 3072 ± 510 4534 ± 1812 4805 ! 483 Il ~4 7 t 1% 1 G2 4949 ± 825 7224 ± 288 32296 ± 2612 1 3437 1 314B

V F12 4867 ± 952 5226 ± 633 32747 ! 1566 lJ 128 t .47 jC)

H2 4061 ± 313 5079 i 940 . 25796 ! 1481 1171~ t IH2B

H~ 4160 ± 460 4801 ± 1983 32786 t 579 12117 14HIJ -~f4?OO) 7156 t 1643 5929 ± 2017 28748 ! 1 JOb 1 30U2 t nt)

1

912 ± 295 616 ± 115 986 t 107 IH99 t hOI "1

sF"61 7802 ± 608 -6626 ± 59!l 2 J 3H2 ! 124') 14423 t 2'1%

1

(

" .

-172-

Table ~

,1 J:oa.r 1

repre.eDta't"iTe ozperiaeDt. wHh four differèDt 20 JLll cell

preparatiOD.. Tho .... BP/IL-l preparatioD was cs.d.

2 auuh-.. are ezpressed as cpa of B3T uptake iIlto BF/IL-2 sti.1&ted 20

.. Il c.lla. 1 z 10~ 20 .. .1 colls, 100 pl BF/IL-2 and 100 pl of the

hy~rido.a supernatu,ts (l: 2 dil utitln.) • 3 days iDcubation. 5 hours

labellinl with 1 ~Ci alT.

3 Pl x 20 - spent cul ture superna tant froID tho P3 X 20 myelolDa.

posi Uv. control s.

4 Cells are not stimulated with BF/IL-2. Background cpms.

-1

S Cells stimulated with 100 ~l BF/IL-2 in the absence of Any hybridoma

supornatants or othot inhibitors'- positive controls.

--------- -- ----~

, -113-

Table III

Biozs! t-ua iz.. li OD

IDlaibi tioD I>.ta

Clo •• 20 • A(l) B

DD1U liO E(3) 2420 + 1452 3515 :t 193

AC4 1 El 1 210~ :t 203 4290 :t 393

II AS 1 2599 :t 455 4687 :t 42+

------II B5 1 239 ± 152 480 '! 26

III A9 E 2744 :t 346 3625 :t 85

III Dl E ; 1370 :!: 249 1 3797 :t 783

13637 IV CI 1 ' 2601 :!: 33 S- ± 204

Controls(S) , med 91 :t 18 104 ± 14 , .. BF/IL-2 4781 :!: 827 6061 ± 119

"

.' ) I!

... ~. 1

'.

(

t

(

,

) -174-

T.ble XII

1 ho repre.elat.th, ••••• y. wi th two pr.p.ration. of 20 ... Il c.lla.

Th ..... BF/IL-l w •• u •• d.

l Il ••• 1 t •• n .spr .... d •• cp .! SE of B3T .ptat. bl' 1 x 105 10 MUl

e.ll. .ti.vJ..ted with 100 \ Jl! BP/lL-l iD the presence of 100 JlI ---hybrido.. ..p.rDat~nt. 3 d.ys incub.tion, S hr, a3T pulse, 1

JlCi/well.

3 E - eAh.neement, 1 - inhi..bitory. Previoas testing of thie .erum

" cOJl,taining hybridom. su,pernatatJ,ts froll these clones h~d shown either

, eAhaneement (i. e. excess stimul atory response) or inhibi tian of the

BP/IL-2 stimulated response. See Ch.pter III.

4 Serum fr~e sapernatants were prodac~d by inoubatins 10 x 106

hybridoma_cells/m~ for 24 hours.

. S Controls. med = no BF/IL-2 or hybridoma sapernatant was added

\ ,

BF/IL~ ..... 2~'ML~ èells stimulated "ith 100 111 BF/IL-2 in

the absence of hyb~idoma supernatants.

-e

r

... .,..

\

,

·1 ~

"

, -

! 1

.-' :.

1

1 1

1 . -

i

20X CCIlcentrated '

culturé SUP.(l) A)

B)

l - .. Ascites flui&2)

- '1 (Biozzi x Balb/c) FI

Illalb/C nu/nu

l r-

I Control supérnatants ! 1 '(

',' 20 X <Xll1c. P3 x 20

20X canc~ anti SREC

., ~ .. " ..... :."- ~b, _......,oIt~ .. ~ ...

. Table XXI.

dilutions:

1:4

1951 ± 6613)

2098 ± 1536

dilutions

1:20

184'± 145

5623 ± 342

éiiiutions

'1:20

40915 ± 446

23992 ± 296

1:8

11538 ± 2927

9942 ± 1559

1:40

779 ± 82

5681 ± 2353

--,

-----/

:AC4 II B5 Clone .".

2D MLR Proliferation Inhibit~én by Various,Hybridoma Preparations

... -. 1

1:16 1:64 1:128

23125 ±"2598 [26928 ± 4819 27618 ! 3413

23426 ± 2723 28054 ± 2690 25800 ! 7913

1:80 1:160 1:320

.. 1159 ± 461 4730 ± 491 9199 ± 748

1:256

32510 1 3025

28765 t 547

-'

1:640

8431 .t 121

16660 ± 3073 1 22015 ± 1527 1 20640 ± 3798 1 18552 ± 1849

....

no u14 ) 1 ....

"'" VI

35924 t 3502 1

30749 ! S.()87

I~ ns

12410 ! 1569

35Y lli 1 809b

..

.....

, (

-176-

Table nI

1 AC4 II BS hybrido.a sup.rnatants _oro concontrated '20 1 by ..-onia.

sulfate precipitation. Lite_ise the P3 1 20 spent culture superuatant

and the .0llDcloual anti-SUC hybridoaa superua tant _ore al so

--------- -concentrated 20 1.

2 Ascitos fluid collocted from (Bio%%i x Balb/c)F1 or Balb/c nu/nu

.ico. ....onilla sul fato proci·pita lod and reconsU tuted to i ts original

volume. .'

3 Results are oxpressed as 'i1J1Il ± SE of H3T uptake by 20 MLR ce11l

stimulatod by 100 ~1 BF/IL-2 in the prosence of different dilu~ions of

BS •

4 Positive contraIs. 20 ML~ cells stimulated by BF/IL-2 in the absence

of any BS product.

stimulated _ith the

These are four different 20. MLR preparations. but

• .... BF/IL-2. )

"

/ '- .

'" •

1 J

Table XXII. :Two dimenslonaL neutralization assay of RS hybrldoma land BF/IL-2 as measured by IL-2 dependent stlmulatlon of thymldine uptake.

B5

BF 1:2 1:4 1:8 1:16 l, 1: 32 1 :64 1:128 1:256

1:1 4721 ± 96P.) 12813 ± 2220 116628 ± 3173 12455 ± 1540 19971 ± 3522 16582 ± 5163 13876 ± 5245 14739 i 6809 . 1:2 6529 ± 994

61BB '1903 6833 ± 4009 13834 ± 1726 11723 :t 1172 14852 :t 1244 7598 ± 2740 10274 i 3883 1:4 3490 ± 352 5449 ± 154 7212 ± 1416 9433 ± 2152 9041 ± 340 8684 ± 1358 6213 ± 1613 8220 ± HU 1 ~

J5721 :t 2362 -J 1:8 2621 ± 364 ·2767 ± 575 8743 ± 4487 4908 ± 455 6119 ± 48~ 2303 ! 1240 2592 ! 1476 ~ . ' 1 1:16 2581 ± 2012 3540 ± 1615 2651 ± 688 2273 ± 484 4879 ± 1026 3225 ! 72B 1980 ! 330 3129 ! 20BO

1:32 2480 ± 999 4065 ± 931 1543 ± 466 1788 ± 296 2579 ± 294- 2901 ! 245 2234 ! 469 41~7 ± 591 1:64 1967 ± 465 . 2290 ± 828 2611 ± 489 2854 ± 1481 2266 ± 401 1816 ± 820 2183 ± 580 3175 1 l)3 i~-

1:.] 28 t 1153 ± 222 2631 :li 1409 913 ± 389 1059 ± 411 1296 ± 445 976 ! 2 H 1OS4 ! S22 Ji -

1065 500 , - >

1:1 1:2 t 1:4 'l:y 1:16 1: 32 1: 64 1:12B

Bé) 17774 ± 2411 14345 ± 1527 10086 ± 2534 1 4948 ± 316 1 2769 ± 1324 1 2620 ! 296 1 27B 3 ! l JH j l ')Y2 t ~24 (no B?>

• \r-

"

-.. ~

Table YXIII.

B5

BF 1:2 1:4

1:1 4839 ± 316 8365 ± 2958

1:2 3174 .:!: 1192 6206 ± 4266

1:4 2292 ± 745 5145 ± 781

1:8 . 1666 ± 283 4223 ± 619

1:16 4367 ± 4618 12309 ± 782

1:32 5223 ± 4640 ,J 4167 ± 696

1:64 6884 ± 5630

1: 128 3184 ± 1985

ued ,1 1011 ± 344

BF (5) dil't1

1:1

1 3834 ± 2044

11761 ± 299

1:2 ~

~ ~

Two dimenslonaL neutraLi~atlon assay of ~5 hylrldoma ~

and BF/IL-2 ps measured by IL-2 dependent stlmulatlon of thymldine uptake.

1:8 1: 16 1:32 1:64

19242 ± 1641 15360 ± 1940 15927 ± 639 18499 ! 1938

11438 ± 2665 14360 ± 6206 12230 ± 1011 11966' ! 481

5953 ± 978 7285 ± 612 9472 ± 851 9085 ± 735

3981 ± 7r 4532 ! 531 5416 ! 327 5851 t 830

2477 ± 2 4 3184 ± 1085 2895 ± 488 3626 ± 144 . 3380 ± 1 3 3180 .1 543 2296 ± 786 2942 ! 1405

1400 ± 64 11698 ± 444 11644 ± 277 11B51 ± 421

1576 ± 405 11866 ± 84 11173 ± 183 117711622

~ 1:4 "~ 1:8 1:16 1: 32

00 135 17291 ± 1820 1 14277 ± 29151 9423 ± 401 7077 ± 758 3403 ± 55 2257 ! 500 l'

.......

.'

1:128 1:256

15203 ! 1085 19407 ! 1433

12776 ± 1751 16766 ! 1059

8698 ± 442 9753 ! 960

5095 ± 178 5225 ! 721

3503 ! 604 3186 1 120 l 1

..... 3226 ! 1210

-."j

3004 ! 838 ;! 1

2096 ± 33B 2680 ! %6 r

/'

1802 ! 242 160 l ± 7

1:b4 1: 128

2492 ± 'l60 2305 ! .U5

(

-177-

Table IllI. XXIII

1 Various dl1utions of BS product (20 X concentrated ammonlWD sulfate

procipitated aaterial) .cro tested a,ainst various dilutions of

BF/IL-2.

2""iteSUlott.- are expressed as Cpll .!., SE of H3T uptat.e by lOS 20 ~R ce Ils

stilDuiated "ith 100 111 BF/IL-2 ln the presence of 100 111 BS products.

Throe days incubation. S hrs H3T pulse 1 I1Ci/wcll.

. ,

3 Posl.tivc control. the ability of diffcredt BF dilutionS"'to stimulate

20 MLR cells in the absencé of BS IgG1.

..

..

)."

1

l

-178-

Table XXIV

Kinetics of B5 IgG1°Addition

Inhibition of 20 MLR Proliferations

------ 1 Time of B5 1

i

addi tion(1) A B

b 1

0 4149 + 1347(2) 1 4021 + 371

'~

12 hrs 2401 + 158 4832 + 288

24 us 5234 + 2756 7066 + 741

36 hrs 6817 + 907 10199 + 458

48 us 173~1.±. 1577 26665 + 433

~ • Confro l s: me d (3) 3817 .±. 145 2880 .±. 494

1

BF/1L-2 l 14692.±. 1723 :~81 ------ ----"-- -

~_ 1 100 ~l of 201 concentrated B5 19G1 was added at the times shawn.

(Corresponding to 50~ of the final volume.)

2 '!'wo repres~ntative assays. R~sults are expressed as cpm ± SE of H3T 1>

uptake" by 105 20 MLR ce 11 s stimulated by 100 ~l BFtIL-2 in the • ,

presence/ absence of BS Ig61. Cell S w~re incuba ted for 3 days. and

labelled 1f ith 1 ~Ci H3T/well for S hours. . cl'

•

•

\.

1 3 Controls.

1

t,

(

-179-

aed • 20 XLi cells UDstiaulated

BF/IL-2 - 20 XLi cells stiaulated by BF/IL-2 in the

absence of 85 IgG!.

1

-..., "

\

-180-

Table XXV

EHect of AC4-II-B5 ft Murine 20 IILR 'r -'

B~ dilutions -...

\ Stiaul us 1:8 1:16 1:32 1:64 1:128 ï 1

---_.

1

!Huaan BF/IL-2(1) 4063!,51~ (3) 612!48 771!164 98S!,173 1690!,181, 1 IlluriDe D.-2(2) 6390!,163 5839!140 383Q!328 4464!,1520 8337:!:,lOl;

" . 1

1

1 17~127 \ ICo1ltrols lDed ~

1 BFl.D..-l 3747:!:,351

" 81U'i1lO D.-2 1382~352 • •

1 Huaa1l BF/IL-l • G75 c01lcentrated BF/IL-2 100 ~l/well.

2 Murine ConA stiaulated Lewis rat spleen IL-2 100 ~I/Yell.

, 3 Results are expressed as co. :!:. SE of HlT uptake by 5 x 105 .urine 20

XLi cells (CS7BL/6 x DBA/l) ati.utated b7 IL-2 in the preseDce/abaence

of B5 1&61. Cells "e\e pulaed "ith 1 ~Ci HlT for S, brs after 3 clays

.incuba ti on.

l'

,- - - ----------

-, ... l,

j

Table XXVI. Effect on 85 on Cel1 Viabi1ities, B1ast Cel1 Transformations .1

/ and 113T uptake.

1

1 t-b. of Viable Ceuf)

Tine CeU T.ypes " Blast(2) H3T uptake/105 ce11s(3) x 106/ml

+838) -85 +B5 -BS " +85 -B5

24 hrs muri~ Ur2 dep.(4) 0.775 0.750 19.8 76.0 2479 t 274 3709 ± 206

hlJllaJl Ilr 2 dep.(5) 1.125 1.190 24.0 63.0 6558 t 1137 8005 ± ~96

48 hrs murine IL- 2 dep. 1.075 1.05 8.6 41.2 2897 t 1163 4376 ± 794

hunan Il.-2 dep. 0.70 0.69 5.5 26.7 2798 ± 1938 5826 :t 304 unstim. 2° rruJi 0.42 0.39 9.3 11.2 2856 t 208 9188 ± 316

BF stim. 20 J> 1

0.52 0.57 13.2 48.0 2664 t 265 7762 ± 101 ~

Cl . ..... 14 hrs unstim. ;p MLR 0.35 0.29 0 0 4540 t 322 4835 ± 11 1

BF stim. 2D MLR 0.575 0.650 9.2 33.1 2923 t 232 13738 ± 756

,

.-

"

j !

t

.'

,

Table In'X

. . 1 ne Illmber of viable cella .. ere de terain.d by trypall blu dy.

esc1uaioll. The re •• lt. are .spr •••• d a. Iluab.r of viable ce11, s 106

cella/al.

l Percentale of bla.t-like cells al'. dete~i~.d by ce11 -arpholqcr and

siaa after Turks staillinl.

3 B3T .ptake 1 ~Ci/ .. ell 105 cal1a/".11. ,

.. A .urilLe IL-l d.pelld:.nt cJt1 Un. aailltainad by 30i auiA. U-l

evary l4-48 hous. 104 cel1a/ .. ell.

.JI 5 H_all ·IL-l espand_d calls .. hich are depend.nt on BF/n.-l for caU

proliferatiolls. -105 ce11s/w.11 with 5~ RF/IL-l.

6 H_an la MLR c.11s, DAstiaul.ted 105 c.l1a/ .. e11.

7 H_all 20 MLR c.11s sti.ul,ted with !~ Br/IL-l. 105 ce11a/ ... 11.

8 AlI cella ,ro"ll ill th. pres.llce of B5 bel 501 B5 1,01 added ta tha

cultures at th. initiatioll of cultures •

. _-..--".,-.--"'-------------------------_-:...._-,-

•

.. -t ... ,' ....

.... ,~ _, l ,,_'" ,.., ,_ ~ .. , " ~ .... ~

---If, •

.',\ .. , . .'

il' . , "

Resti.nW.iltion

Qf 2° MLR

niltl )

BF/IL-~4~ (-a5}

, StÎlmllator oellS~) . . -

1 . • r' 1\11 1

,-; lF/IL-2 (+85)(6)

'... . Stfnn:ùator cells

1

, " , . . 1" , li ... ~,,~

.-~.

~. )-~ fl. /

~

t· .

\.

Table XXVII.

.. ;

2

"H~-Uptake ,

Cytolysis

1010 t 17JD 11. 91<2) ~

30412 t·2043 49.54

61695 t 17'2 30 .• 81

.'

553 ± 227 , , ,::;

7.39

5252 :t 151 23.25.

6678 i 238 - 15.69

:

"

•

, fit

Effect of l!C4 II B5 on the Generation of CI'Ls fran a Stinulat.ed 20 MLR

Days

3 4 5

HlT Ofo

HlT %.

HlT °10

CTLs CTLs CTLs

664 ± 140 56.33 3121 ± 153 Il.:30 2009 t 798 5.67

38033 :t 1945 100.00 41633 1 1364 39.47 22623 ± 4089 17.90

85678 t 4811 100.00 68930 t 1951 58.29 37985 :t 5118 34.06 1 1

1 .... . C3 562 t:87 54.56 538 :t 1B8 5.27 935 :t 109 0 ~ 1

8758 :t: 1&6 2.60 7179 'j 925 9.56 9482 t -1439, 3.05 .-J

11996 :t 697 9.40 15016 t 643 7.03 19392 ± 1979 0.88

'-

~ • 0

~ Î

/ /

) . J.

..

; ... .

l '

,. r •

. t·

Table nvII

• 1 ".ultl are expre •• ed a. B3T uptata by aotiyated hua .. 10 ILl cell ••

,

1 _Cl B3T/we1~. , br. pul.e. 10$ oell./well.

% ... 1Ilt. are expresaed as .. lyd. of the .,-olfie tarllt celh at a

50:1 kil1er-tar.et ratio.

3 %0 JILJl lUlsti.u1atecl. but oldtvecl' in the pr •• ellc. or ab.elloe of as

IaGt.

, '

~ ~" ...

. " %0 lILas ,ti.utata" witIL ,()II' BF'/n.-1 iD the pre.ellce· or ab.e,llo, of B5 ,

'rlol.

'5 20 lLb sU."laUd witl> a ~ati. Gf the orillut .taurator oeIl.

1. the pre.ellce or asellce of B5 1161.

'-JJ>r ,; e.u •• ero cultured in the pre.lllcl' of '" B5 (%0% o01lCelltratld

c:1Ll tue 111"1''' tut) •

(' ./ /'

/

/

/ !

'1

, .

\

c

'c

, ,( E

" " ,

, ,

, '"

:- 'r

""/ 'c , 1 .. . .1 1 •

,

-185-

Table nvIII

Bfl.ct: of 85 IaGl, 011 the '10 MLJl !~ '( '. .

, f 1 , ..

Il . , ,

, B$' dila.tiolla(5) .

, . ..:.

1:2 1:. , 1:8 1:~' .'

01

. ~ .. 10 JlLK 13319 :t 358~~) 4J'J7 :t 339 8113 + 1200 - ,

26411 :t 517.5 J.

lI1lI:iu 1° MLKI 889 :t 321(2) 1476 :t 248

Co_trola

. . \ . '.

,-. ,

f' ... '

Ju.aa A (r •• poAder)

Ils (sti.al~tOl:)

A + B%(3)

lI1ü"iu C57Bl/6 (re.poDder)

DBAll (stlaalator) . C57Bl/6 +'DBA/2(4)

'r { l ,

t, .~. 'l)

i =

"

!. . . , , ';

l t.' " '

,

3196 :!: 323 4133 :t 688 . " ,

, . • J " '"

16$ :t 63, ,i

806 :t 72

36690 + 1296 - . , 9i.:z :t 91

192 :t 89 t ) ,

.4427 t: 198 , J,

JO , 1

"

~ 1 l

f ' .. ,

t • ~ •

, ' J '

J • .' "

, , '

..

, ,

, . , . , 1 " ' " ~ " l '

J ., - ~ '"f

... ~- .. .

,

,-f

" '

" 1

"

. ,

"

"

- .

,1

" ,

, .' , " . ',(" " 'l .

, . -l'

. .

1 ae.ul t. aire expressed as H~T uptake by lOS tesponde:r: CIUS aftelt 1 7 ~

day. incubatioD: 1 ~Ci S3T/I0' ce11./we11.

2 I.lu1t. are .• xpres,ed as H3T uptab by 2.5 %10S rUponder cella

aft.r .1 • dlYs incubation. 1 flei H3T/2.5 z 105 cells/well. ,

3 H~an ro JlLll. established' It 1 1:1 respo,nder:stblulation ratio. 1 %

105 1: 1 IZ 105 Sz. Stiaulator c.11s Ir. inactivated by y irradiation •

o '

. " ,4 Jlu:r:1ne 1~ NLll •• tabli.h.d ",:t a 1:4 r"poDd.,r:Sti.llltt~~ r.aUo. 105

l: 4 z 105 Sz. Stianlator cel1. wete· irradiated DBA/2 ,ple.n c.l1 ••

5 ,0. B5 I~i YII added to .Ich culture. \ ..

oo.c.~tr.t.d cul turf ~u,.:r:JUltlJlt.

" '.

1 -"",

"

BS froa 201

'. .. .

. ,

'. 1 - • , . '\': , .

"

, , ,~

\

.. _~_.~. - .... 1 _. ____ -' . ; ,-, -

l' ( ..

.'

, ,

1,'

: :

, " . , .

. ...

"

-187-, , II "',

, f J "

, \, Table ~. Effect of B~~ on Mur.iile lL-2 Dependent cells, '

. , ,24 MS

48.hrs,

'1

-', ,

i· -.

"

l'X? ,IL-2

murine IL-.p.)

llum9n aF /IL-~P )

no IIr2 . : .. rnun.ne . IL-2 '

human BF /IL-2 ' .

. "

no-BS "

1:.2

25045 ± 2235 l ,12174 ± ·937

6241 ± 316 ,929 ± 164

73 ± 20 77 ± 47" '

800,70 ±' 2902' ,

45637 ± 6003 ,

12243 ± '4162 2234'± 2509 \

1:4 .

1153 ± 544

21855 ±' 2451

5368 ± 1560

118 ± 52'

'77B36 ±. 653

5272 ± 366

i . ~, . 3 ';, )4" ,/1,

Results are expressed~ ± ~ of li ~ ~e by 10 ,~cti-

~ted mutine' IL-2 de~,ent c,t Une ~~. Tœ C57~1/6, : anti-'(CS7Bl/6 x C:3H)Fl'~ly ~ef( t:cotally' ~~ent ~ the .

presence of IIr2' for œil ~Oliferation. ' . \ . ..

•2 ltJrine 'CollA ~teà rat spL, J.tr!2. Cells, ,were st.imuJ.at:ed 1

. with 301 IL-2. ~. T4

• r'-

"

.~

3 H\mIan G7S BF/D:,-2. Cells were st1nulated with 50% BF/IL-2.

./ If 4 "

B5 fran 20X c:onQ!!ltrated et4ture supe:tl'li!.tant. , .

• .'

'lO, 1

" '1

"

~ ,

, ' \ . 1 ,

. 1 " '" • .. /" ,1 1 ...;~ ,

,. 1

l'

l '

. ,1-

., ,

" , ,

, .

1 \ . '\

1'W.iorJ...:·"i">r ... ~ ... ~ ''''l'~'!.1:"I'#M .... ~~'Lf'..i&II1~o\J...t'-' ........... ~..aiIoooo::JoJN~''''III-I' ... ,,,.,....---.......... _ , ....... _ ~

,

t \ f (

t

)

j

(

\

Tabl. XXI

'f -188-

, .

l ltalul t. aro .%pr .... CS as CPN ± SE of 113T uptab by 5 z 105 leti •• t.el

IL-Z o%paDel.el ceUI. l "Ci BST/s z 105 cella/.ell. Cel 1 a "'1'0

0111 tlttod for 12 hoUI.

2 B5 elilutioDs of 201 CODcentrat.el cuJture 11IpOrDatlnt.

3 B_ID BF'/IL-2 prod1lceel by a1101e1lie .• tialllationl of hmau PIL,

(c1'u4e BF'/IL-2).

4 B_u. .HOIID atillalate4 IJ--2 prodllce4 by PB! ItfiulaUoIUI of h.ut

toaail ce11, (crud. P.KA-S).

,

) .

~ .

l'

. ,

->

"

\

o

-190-

Tabie XXXI. Effect of B5 CIl the Costimulator ~ay .

L~4) Stimulations~

'Ccr1A + IL-2 . Ccr1A + II.,-2

canA + IL-2

canA + IL-2

,

B5 diluticns P)

1:8 7075 ± 1188

nil 1:2 16592 ± 25611 ) 793 ± 500

1: 4 2623 ± 153 .

1:32 1:64 1:128 1:256 -10987 ± 1232 ll530 :f liS1 10940 ± 7U 13103 ± 769

154 ± 14

nil 1:2 1:4 1:8 1 2934 ± 310 61 ± 15 7~ ± 16 548- ± 375

1:32 1:64 1:128 1:256 2016 ± 712 3127 ± 1056 4046 ± 268 3848 ± 264

261 ± 7

1 1 x lOS Balb/c ~s 'Nere stimulat~ with 0.5 lJg CanA

and 100 lJl ImJrine ll.-2 (COllA stimulated rat spleen IL-2)

cell.s were cultured for 72, hours am. the results are expressed

as H~ uptake by 1 x 105 cells after a 12 hr p.tlse-. 1 wei/lOS

ce1ls;'well.

2 20X concentrated culture superna~ts B5 IgGl.

3 '!hyroocytes st.imulated with 0.5 ug ConA alone' in the absence of'

B5 IgGl and IL-2. .

4 ~ representative experiments.

-----------_Al~m4;--.--------------------------~--------------T_----------------

/. i 1

,1

~ t-t ~ ~

Table Y.XXlI • (Effect of BS on Mitogen Stinulation of Hunan and Murine Ce1ls.

• 1

•

. Hunan PBL stimu1at€d with

CanA

PIfA

• Staptl A.

Stafil A .

.. Balb/e spleen eells stinulated with

PHA

-CanA

LPS

c , -IJ ,

",' ,-

.. , , ,

..

, '"

no BS

- 64836 ± 3J2)

o 68154 ± '7Jl) (4 )

21998 ± 336

S d '1 ' (5) B 1 utlons

noBS 1:2 1:4 1 1:8

44339 ± 5747 ,1996 ± 471 14020 ± 1123 23945 ± 2472 1 , 31154" :t 170~2) 16535 ± 1219 1 45661 ± 3026 46329 :t 2146 ...

62858 t 1704~3) 5656~ J 3744 -i 35331. ± 7531

38830 Î 4038~1 56103 Î 16986 36744 ± 6031

1 52858 ± 6430

36482 4 2151

B5 di1ptiorJ5)

1:2 1:4 1:8 - 1:10 1:32 1:b4

'. 11882 ± 6752 16394 + 5718 9961 ± 349 68032 ± 3354 80127 ± 6620 92466 ± 3203 .

11H61 ± 1020 68671 ± 5365 134030 ± 1436 131897 2507 88197 ± 6709 87586 ± 1396 .

45197 ± 1086 53752 ± 1546 46996 ± 6247 58379 ± 5187 41694 ± 6705 39098 t 6549 .

r .. 40

1 -" -0 -" 1

oj

-

,

(

\ ~

-192-

Table IXl.II

1 Baman PBL. or murine spleen cells wero stimul1ied with 5 ~g ConA/.I,

106 colls/ml. Cells ';ero incubatod for 3 dayt, and lab,lled with 1

~Ci B3T/10S cells/will.

2 ColIs wero stimulatod .. ith lllt PRA. Culturo conditions àre the same

as for ConA stimulations.

3 Baman PBLs .. ere stimulatod with 7 x 109 ~ aurous microorganisms/ml,

5 x 106 human PBLs/ml. Cells lf'oro culturod for 8 days and labelled

.. ith 1 ~Ci B3T/S x 105 co1ls/ .. ell. • ho represoDtative oxperimonts shown using, t .. o different prepara'iODS ,

of human fresh PBLs. The same ~ aureus microorganism preparatioD .. as

uaed in both assays.

4 Murine spleen cella were stimulatod .. ith 1 JoLI/ml LPS per 105 spleen·

colla. B3T uptake .. as de tormined follow1ng a S days incubation

5 BS preparation was 20X concentra ted cul turo super. tant •

...

•

--- -- - --- ----: '

t

\ f' t \

-193-

Chapter VI

AC4-II-B5 IrG1 absorption aAd bindin, Itudiol

WAat il the anti,enie deteraià&nt reco,nized by the aonoclonal B5

I,G1 &àtibody1 As we "ished to rule out the possibility that B5 IgG1

recoinize, a lymphocyte cel1 aembrane component. we attempted ta

absorb BS 1,61 onto lymphocytes at various stages of cell activation. \

To visu.alizo the absorpttoa. of BS I,G1 oa.to eo~ surfaces. wo 'iltilized

a fl:a.orescea.t antibody bia.dia.g technique. Je also aa.alysod the loss J

of B5 I,G1 froID the supernatant' a.fter pre-absorption to colls.

An ÙIIIIllUloabsorbant collJllln of cross. linked hlUllan serum was

propared to study 'Whether BS 1&61 was diroctod a,ainst a huaan serlml

cOlDponent. A BS IaG1 iluaunoabsorbant col UIIln was lDado to study whether

BF/n.-2 ean be retained onto such a colUlllD. Libwise an ELISA wa.

establishod to observe for the bindinl of BS I,Gl to immobilized IL-2.

Finally we "ero intel'èstod in studyinl whether BS ,<1,G1 and

BF/IL-2 -Can f01'1ll an illDlluno cOlDple:. in 501 utioa. and thus remove 'froe'

BF/IL-2. IndeoAi such an ÙIIIIlune complox Dlay bo stiJlulatory or

inhibitory in a biologiea! assay.

\ IlATEJUALS AND ME'DIODS

i) Immunoabsorbant columns

Various iIIDIlunoablorbant col1Dll1s were us iDg hybrid91la

antibodies. BS IgGî~ obtained concentrated , culture frolll 20

/ .

j

1

•

(,

• -1"-

supernatant and iro. ascites fluid of the Balb/e DD/nu .iee were

coupled to Ilutaraldehyde aetivated AH Sepharose (245). Approxi.ately

1 al of BS 1&61' w.s coupled to 4 al of activUed sels. To each

coh.n, 8.0 arJ. of eoncentraU,d G75 BF/IL-2 yas applied. Followin, a

30 ainute iDcub~t10D. the unbound .aterial was eluted with '0.5 K NaCI

in PDS pB 1.3. Attempts wero lIIade to elute the COlumD bound BF/IL-2

wlth 0.2 1( glycine BCI pB 2.4 or 1111 SDS (246). A 1 .. SDS solution was

heated to 70·C and immediatoly usod to .1ute the colman bound .aterial

at 37·C. The eluted aaterial yas collected on ie. and diluted to 0.1'

SOS with 4'C PDS. Free SDS wa$ precipitated at 4·C, and biologieally

active BF/IL-2 was recovered in tlLe superuatant. FractioDs elut,d

.ith 0.2 Il ,lyeine BCI pB 2.4 were illUDediately neutrali,ud w1th 3N

NaOU.

A aODOclonal anti-SRBC izJDunoabsorbant eolUlU1 .as ilia de as a

control column. An equivalent aJDount of anti-SRâC I,G1 wa. eoupled to

glutaraldehyde activated AB Sepharose. A thyroglobulin cohmn

(provided by Dr. K. Abikar) wu •• de usinl the SUle proceciures. This

thyroglobulin colOilln haci previously beon shoyn Dot ta retain Any

BF/IL-2 aetivities specifically or nonspacifieally. AU frlCtions

collectod from the elution of .. ch cohUlln wcre di.lysed. oonceDtratod

and assayed for BF/IL-2 biologieal ~ctivi~y.

ii) lmaunoabsorbant IDatrix of cross linked human s~rua prateins

To test whether BF' s IgG1 was, r~.ctive toyards a human serlUll

cOllponent •• e ilDmobilized hua,n serlDll by cross 1.inking Sertull prateins

with glutaralodehyde (~47)." Ten ml of human serum was dia~sed

f

t

(

\ -195-

ov.rniJht at 4·C against PBS. So the dialysed,human sdrum. 1.0 ml of

1.011 sodium bicarbonate pH 8.1 was added followed by the dropwhe ...

addition of 3.0 ml of 2.S~ alutaraldehyde. The s.rum proteins gelled

in approxi .. tely ten .inut05. , To saturate biDding sites. the geUed

ser,. proteins .... re resuspendod in 100 ml of 1.0 JI ethanolamine pH 8.5

and homogenized in a looso fitting Potter hOlllolenber. The

1

homogenized gels were filtered through a glass funnel and resusponded

in 0.1 AI ethanolamine to be kept overnight at 4°C on a rotating

platfona. The goUed proteins wore washed alternately ... ith 0.1 M

sodium bicarbonate. 0.5 M NaCI pH 8~1 and 0.1 M $odium acetate, 0.5 M

NaCI pH 4.0. It was finally ... ash~d ... ith 0.2~ glycine Bel. O.SM NaCI

pB 2.S'and PBS pB 7.3.

To study ... hethor BS IgGl biDds to this immobilized human serlllil

column .... e chose to apply H3 leucine labolled BS 1861 onto the column. • ~

The UDbound material ... as oluted w!th 0.5 M NaCI in PBS pB 7.3. The

Ulount of radioactivi ty recovered in the. el utod fractio"n was

detormiDed in a ~ 'counter. Binding of ilS huei .. e BS IgGI to the

col umn ... ould b~ refleeted in a decreasod reco~ery of radioactivity

from the co 1 umn ru through.

Ui) Applioation of 'proiJ1cubatod BS II61 and BF/IL-l' OJ1to a

precalibrated P60 colman

.To observè whether B5 Ig61 and BF/D.-2 cali fona an iDlllUllO co.plu .............

in solution. "e promixed equal vol 'QIIles of BS IgG1 and BF~IL-2 (4 ml "

e4eh) for 4 hours 4t 4°C. This ".a per~ormed at a BS IgGI

.. ooncentration "hich ~ad proviously b.en shown to inhibit 90'1t "BF/IL-2

(

• -196-

sti.ulated cell proliferation. This mixture .. as thon appUed to a

preFalibrated P60 column.· AlI fractions collected were as.ayod

independentIy for BF/IL-2 acthi ty and anti-HF inhibitory biololicaI

activity. If BF/IL-2 and BS IIGl caa fora an immune complex ... e would

either see a decrease in 'free' BF/IL-2 recoveries. or a shift in the

BF/IL-2 elution profile. A radioactive BS 1161 .. as nsed as a IUrbr

for the BS IaG1.

Iv) Fluorescent antibody bindinl 'to cells

AlI anUsera used in fluorescent antibody billdial assay. Yere

airfuged iÀ a Bectmaa airfule prior ta use so as tO$remove allreaates.

!WO methods ~f cell treatments 'yere used to study the surface biadinl

1

,and/or i'1ltrace"llular content of D.-2. SeTera! cé.l.t types .. ere used,

1) fr •• h ..... tià~ ... d post ficoU PBL., 2) 24 JaoU? old la JILl c.U •

.. hich may contai1n the putative BF/D..-2 producia, cells. 3) 2· lILll

cells .. hich have a~sorbed BF/IL-2 onto their surface <absorption of 8F 1

.. as for 60 minutes \at 37·C) •• ) an D.,-2 proc!ucial coll liae tJCD 1"., 1 • '

5) 24 hours oid T ce\l aitosea sti.ulated cells and 6) IL~2 depe~eD.t cell lino Irown and o~.aded in tho presence of D.-2.

1

For observation 01\ s~face bindinl. 106 coUs were iacubated Yith ,

100 "lof BS hybridoza ,antibodies for 60 ainutes at "·C. This' ..... 1

followeci by three .. asho\s .. ith cold 1" BSA-PDS pli 1.3 The FITe \

cODjulatod rabbit anti-"'ouse baunoalobulin .. as used At a 1:10, \

dilution (dU uUons .. ere .~de in 1 .. BSA-PDS) 1 100 "lof Rail laG "as

added to the ce1ls and iDc~ated

final threé .. &sh Ylth 1 .. BSA\PBS.

\

for 60 lIliDutes at "·C. FollcwiDI a , -

the colts woro resuspeDded in 0.5 ~l

~ ........ '----'-"'-----'\._" .------.---.-,- ji •

•

('

-197-

1 .outinl aoeliaa ud cell fluor.scenc. deterainoel the s.ae ela,.. AU

sa.ples were .aintainoel at "IC to eliscoura,e cappln, anel

intorl1&ll%atlon or shedeli~1 of tho surface bOUDcl antibodies.

For obson'ation of intracell1llar eOlltont of IL-2, colls woro

f irst f ixoel Ollto a aicro.cope si ide. Tho s1 ldo s woro prewashod and

procoatod with 0.2 al 3' BSA-PBS. For each spot. 2 % 105 colls/O.S ml

wore spu OlltO -t~e" .iero.cope al ide in a cyto.pin centrif1llo. Cells

woro fhed for 10 minutes in .IC 5~ acotic asciel-ethanol.

Subsoquolltly tho .lidos wore washod with threo changes of .oC PBS and

elried carof1llly. Tho hybridoma antibody wu applied directly to the

coU spot in 100 to 200 Jll voluao and allowed to bind for 30 to .,

millutes at room teaporature •

~ This was followéd by one yash with PDS.

The FITC conjuatod rabbit anti-mQuso Ig was applied ta tho same cell

spot and incubatod for anothor 30 to ., minutes at room temperat1lre.

Fo~'lowinl thre,.e washos with cold PDS. tho spot was mOUJlted with

mOUAtinl medium. The samples were read within 24 hours in a ,

fluorescellt microscope.'

ne control antibody u,ed was a 1Il01lSO anti-S~C monoclonal

autibody. Normal mouse sera were not usod as the y gave very intense • backgroWld b,indlngs. Sinee tho second anti'body was a rabbit

, . anU-mouse Il. we were restricted in terms of which ce Il type ta

1 study. Therefore all, fluorescent JIli~roscopy e%periments ,were

performed with human or Gibbon cells.

V'>, "l!!nz.:rme Li nked Immunoaltsor,~ant As Bay

f

An enzyme linked immunoabsorbant ass~y was performed by the

1

,

, '

.. "

-1'8- ,

41~.ot hiA4iA, of the a.t~.eA, BP/ÏL-2 to plastic (Liabro E~

1638104). It ha. bee. reported that IL-1 in a ai%ed protein solution'

.111 preferentially biad to thé plastic. For optimal biadinl of IL-l,

, Ir ft'Ye units of n,-l per .. ell .ere incubated on the plastic for two

houri at l7·e. or; overni,ht at "·C. Prior to use, the plates .ere

•• shed oace .ith the .ashin,' buffer, 0.0" T.eea 20 ia 10 mM Tris pB

8.0 (149) and 1~ BSA-PBS .as used to saturate unbound sites.

Monoclonal ,ntiboaies ycre applied in 100 pl volume. AU,-

d~lutions of Intibodies "ere aado, iil PBS. The monoclonal anUbodies

.. ere incubated on tho plastic plates for 30, to ,60' ainutes at 37°C

followed by threo .ashes "ith tho y.shin, buffér.

Our second antibody .as an alkal ine phosphatase ,coupled

1 anti-aouse laG (TAGO) .hich .. e used at a 1:200 dilution, 100 pl/weIl.

1

:, '1):11ut10ns of TAGO goat anti-mouse 19 .ore

o

made in a dilution buffer of i

~'0 mM HaCti, 0.1 mM ZnC12, Tween 20 0.0s.., Tris 10 mM, ,pB 8.0, NaCI 1

O.85'rt at pB 8.5 (249). The plates yere incubated "ith tho' TAGO

lantibodhs for 60 minutes at 37°C folloyed by throo ".shos. The

ilubatrato, phosphato, yas addod at 1 as/ml; 100 111/"011. :Tho

substrate d'il utions "ero a.de in tho substrate buffer of 1() mM AMP in

1 aM HgCl2 pB 10.2 .. The reaction "as 'developed at roOlD tcmperature in •

the dark for 15 .to 30' minutes. This, reacUon w~s terminated by the '

addit,ion of 4 N NaOR, 15 pl/well. Color determination of posi Uve

reaction was read at 405 ttm in a spectrophotométer or a Flow

Mul tistanner. Positive contrats for the ELISA ~sisted ,Of mousé

anti-BSA antibodies. or the 4iroct binding-of mouse I~t~ tho plastic

~làtos.

1 l,

(' ,

_ 0

J i ;r

1

1

. ,

-199-,

PRESENTATION AND D~SCUSSION OF USlJLTS' 1

( - - - r _ _

1) Absorption and: bind.1nl stui!_, \

Can BS IaGl b'e absorbed ont'o cell surfaces?

- ' , la i.t possible that B5 1,01 was specifie 'for i cell sùrfac •

••• brane eompollOnt? If 'thi. is truo, then.e should be able to-abaorb

C1ut th. B5 1,01 as "eU as the inhibitory Icti~ities usina' the BF

-tarlel 2° MLR colls.

A. showD in Table IrnII: in three conseoutive' aslays, ,

pro-absorption of B5 Ig01 oato 2°, na colfs did, not removo' the

Q "

iDhibitory component •• Full, biological inhibitions "ere recovered in

aIl thre. asslys. Furthe1'1llore, "e

rel a Uve to the non:"absorbed BS IgGl_

sa" a more intense, inhibition '\ .... -\

Je attributed th!s phenomenon

,to the removal of various mitogenic substances present in the 20:1

coac~ntrated BS culture superaatant (i.o. co~centrated FCS).

Howover "0 were UIlsucce ssful in absorbing out B5' ~ inhibitory

.cth1ty onto l'o MLR colis "hich had previously absorbed BF onto their

cell sûrfaces. 011% inability to ab~orb out BS IgGl onto (20 MLR + BF)

colts lDay reUect the quantitative expression of -SF/IL-2 on the ceU

surfACes. Perhaps the BF/IL-l wu rapid1y. internalized follo"inl

" . absorption to their -respective receptors. It"as also possible that

BS 1,01 only recognized BF/IL-2 in H'. 'free' fOrll in solution,

thorefore it did not recognize BF/IL-2 "hich "as surface bound. . . Je ".re also unable to absorb out the BS 1,iGl onto control- RUes.

Thereforo "0 do not believe B5 IgGl is directed towards cell surface

co.ponentS found On l~phocytes or RBCs. Bo"evor "0 cannot ,r~le ollt

,1

, '

" ~-~- -,~--'------

-20~

. the poaaibiUty that B5 1&61 il .Ur.ctecl a.aiaat a .. turation or

, differ.atiatioa tntil.a pr ••• at on aetivat.d T oeIl. 0.1y.

",

ii) la B5 IIGI .pecific for a hUllan •• rua co.pount?

3. la it pos.ible that B5 liG1 n.utraliz.s ah_an •• rua ooapouat

( , ,

( r il JEI

aacl thua pr.vent a hua an 2- MLlt 0.11 prolif.ration due to the

d.pletioa of a vital serua ca.poDtnt. Ta atady wh.th.r BS IraI binda

to a hatan .erma compount. .e imIobil !zacl hUllan sarUll by the •• thoda

of Au_.as and Ternyck (2<41) usinl ,lutarafd.hyde croas BAkin, of

.erua proteins •. le then p •••• d a pr.:det.r.ained aaount of uS 1ab.llld

B' I,Gl throu,h the imaobilized huaan serUll colUllJl and oolleoted

fractions to cl.termine wh.ther th.r. were BS I,G1 bindin,s' to th.

J.aobilized .erUll. We did not oba.r:e any hindrance -in U3 BS I,G1 1

milration throu, the huaan serUll colUlln. i.e. total radioaotiYity was

recovered in th. coll_n fla-. throulh. ~.r.fos:. U5 1.61 doe. not

,r,colnlz. a hUlll&D l.rlDl compon.nt. 1re do not believi 1t reoolniz •• a ,l..

FCS oomponent .ithlr ainet th. US hybrido.a h ,rown in 1~ FCS-IlPJII.

iii) B5 IIG} x.Bunoabsorbant colman

Sinee D5 IIG1 can specifioaUy inhibit BF/II,-2 'ti.utated 0.11

proliferation. is it pouibl. that imaobUized BS la01 oan b. us.d a.

~ a tool ta obtain puifi.d DF/IL-2?

iK

Thre. bmnllloabsorbant col_na wer.1 .adl, BS I,G1. • .oaoGlo .. 1

&Dti-sec. and • thyrollobulin .c01uan. As sh~n by' Table nIIV,

DF/IL-2 was not retained

two control' colum~s of

on a B5 IIG! ilIImunoab.orbant 00 I1111n, nor th

Ulti-SBBC oAd t)l7~.ll.1 ... 1iA. ci:.. f1l11 1. '\

(

\

J / .

• (~ .'-'

t S4

/' "

-201-

, biolo,ical aoti.itiol •• ~O ~.co •• ~.d f~o. aIl tb~ •• ~olmanl. It woald

.• ~ •• o.t that B5 laOi CIAllot be 1I •• d .s • tool ta ObtaiA 1»'1010,10.11y·. \

acti.e parified BF/IL-2 aiace BF/IL-2 .a. DOt ret&ia.a OA the B5 laOl _

col..a.

The%'o .~. no PO' lib le o%plautions ta .CCOUDt for the l.ok of \

biAd1a'l boty.ôn DF/IL-2 to the B5 laOl coll111D. 1) Perh.p •• e h.d

~UiDOd the .ati,onic biadial dtos of .,the B5 I,G].. la tho l process ot , .

laaobil1zial. tho .atibody onto Soph.dez '01,. 2) Pe~h.p. iIIIlobil hed

85 I.Gi ClaBot bind 'f~~e' DF/IL-2 in 101ut10a.

i~) The P60 ,el prec.libra~ed colu.a

Ta fiad out whethe~ BF/n.-2 ad B5 I,Gl caa fana 1 eoaptez in

.ol1Ltio~ •• decided ta preiacubate BF/n.-:2 lad BS liGl and pass this

.i.xtve ~roulh a procalibrated P60 lel cohan. This P60 col1DllA hld

'b.ea previously precalibràted with DF/IL-2 sa .0 bow the onet

frl;Uoa ia .h1ch BF/n.-2 activity .i11 appear. If 'BP/n.-2 and BS

I,Gl Cln fora a c~mplez~ we .ould expect to shift tho olution pro(ilos~

qf DF/n.-2 acti~e fractions. The elution p~ofil. (Figure VI-and Table

XXXV) showed that the poak H3 (BS I,Gl) radioactivity Ippea~ed~ in 1

fr.cUoa. 15 ta 25. Thil corroapoads with the B5 mediat.ci biololi"ca,l

iAhibitory aotivity which Ippe.red in fractions 16 to 25. Bowever tho

. p ..... k Do-2/BF activ'ity o,-f this P60 co hum ,~4llPeareci in fraoUoas 24 1:0 . /

32. le did not observe a sfiit ia the olution profile of the BF/IL-2

activity, aeither was there co-ai,ratioll of BF/n.-2 and B5 'IaGl

activiUe.. Full BF/n.-2 biololical a'Ctivity and B5 I,Gl inhibitory

activity .ero recovored in .eparate fractioDJ off the P60 colœan.,

/ / ,

l' -

"

l i

;

--

( 1

t

, ,

\ ....

-202-

nenfœ. it .ould appear that the BP/IL-2 and B5 IaGl. did not

t,ona u t.81Ule coaplex in .olution. If .uch a coaples "as fom04. it

"o1Ilcl npre.ent a very a.table coaplu: "hich can be e •• Uy'

dissociatod followin, chro •• to,raphy OD P60 ,ols.

y) Fluore.coat aatibody biD4in, studios

We,att .. ptod to visualize BS I,Gl bin4ial to cell. "hich expross

tho BF/IL-2 rocoptor.. or colIs .hich are aciively pr04ucinl BF/IL-2.

Our appro.eh .as to uso ~ sandwich te'clutique .ith • fluoroseein

conjlllilUd rabbit, aati-mouse l, aatib04y.

PreU.illary exporwnt •• oro SUllotUve that a 24 hourI 014 l'

JILll coU pOPUlatiOD "as capable of ~iDdinl tho B5 IJGl.s. Tho

correapoDdial cODtrals. UDstbl1zJ. atocl 24 hours 014 colis clid DOt bin4

85IJG1. Bowover. 24 ha~s 014 .itolon stimulatod, coll. did uot bind

B5 IiGl eithor. UD.tilllulat"04 2° JILR coll. and 2- MLR colla

pro-absorbod "ith BF/IL-2 4i4 uot biud BS I,Gl oither.

We bellov. the 24 hourI 1°, JILK. c.lls coutaiu activat.4 T

h'1.".r/IL~2 produciul colis thordo'ra thoy .ould biD4' an nU-HP

autibody outo thoi~ cell surfacos. UDstiJiulated priaary ~ells cio not

po ... ss tho SUle papulatioa of stiJDulated BP/IL-2 producer cell ••

Bowever th,oretically 24 hours old aitolon activated colIs shoul4 al.o

contaia .cao activated IL-2 proclucer colIs. We .ere also 1Ul&ble to

o~taiD B5 1161 biudinl to au IL-2 producor cel1 line-UCD 144. , "

It is not surprisiul that unsU.utated 2' KI.Jl cel~. did' uot biDd

B5 1&61 siuee th ... cells do Dot express n.-2 ou th.ir é.u surfaè.s,

uither are they aetive1y produci11l' IL-2. Bmrover. ,the 2' lILll cel1. ",

1

-103-

which had BF absorbod onto thel':r cell surfaces did DOt bind BS I,Gl.

Again this •• y be due to the quanti taUve expression of BF/IL-2 on 1-

KLR colIs absorbed with BF/IL-l.

We believo that the BS IgG1 antibody has a very low affini ty for

BF/IL-l. This is based on the observltion th.t wo need in exeeas of

.icrolr .. s quanti ties of BS IgG1 to produce 5()ti inhibition of

biololical activ ity. Therefora our inabil i ty to surface label n,-l

producors or IL-l tarlet cells .ay be a rofl.ction of the 10. affinity

monoclonal Intibody. Alternatively it .ay Ilso rofl.ct the 10. j

expression of cell surface bound SF/IL-l, and the insensitivity of our

antibody bindin, assay.

vi) Enzyme Linked Immunoa~8orbant Assuy

FollowinS the report by Ilobb that IL-l can se1ecti~ely bind to

ELISA plastic plates. wo attempted to look at BS IgG1 bindins to

t-obil bed IL-l. A monoclonal anti-n,-l suppl iod by Dr. 2.. Rob])

serTod as a 'positive control'.

As shawn by Table XxxvI. wa sa'; th.t BSA coa tcd pla tes -il1 bizul

• m011sO anti-BSA antibody. Likew ise direct b lndinl of mouse

i_11Doglob11lin to the plates will dicit a ,positivo l'esponse O. e. the

bindin, of the loat ',Inti-aouse antibodies .hich has the cnz,.e ,

alkal~n~ phosphata.e conju,ated to the antibody) • . , Bow,",or. in various combina tions of IL-2/BF and 'anti-BF'/IL-2'

( preparations. w. observed only one positive response.

antibody does bind to • proparation of human BF/IL-2 produced'in serum

, . froc ITS medium. The anti-IL-l provided by Jlobb .as essantially . ,

~-~,---. .. -._ ... ---- . .---. ... ~----------.,..,..-_._~-- ,

t -204-

..... Uve in aIl .... y ••

Since our positive and ne,aUve contraIs performed .s e.çected.

ye yere confident th.t the ELISA results y.re r~al phenomena and nôt

d .. ta .n artef.ct. The ELISA observations suggested th.t BS lIlipt

react .ith IL-2 produced in serum frae œedina.

k j LI lA

-205- *

Table XXXIII.

t ,.

Pre-absorption ofB5 19G1 onto targetcells.

85 mediated inhibition of ce11 proliferat;onJ1) ,

~per;rrrents

1 2 1

, 3

Pre-absorption of 85: cpm ~2) cpm • % ~ cpm %

No absorpti Q~3) 2411+130 2Q.5 9883+874 87.Q 14567+610 28.n - -Rsd4)

<

1 2976:!:222 36.4 4109+420 36.4 1 ND - 1

1

20 Ml~5) 351"'156 4.3 772+314 11.8 3451+158 21.8 -

(2° MlR + sd6 ) i NO NO 6796+166 42.6 (

1

f , 85}n: Control s (no

med. 1313+16.-4 7~2+415 1901 +783 1 -BF 1 Il-2 AI61 +825 100 11263+1319 100 15926+1923 100 -

t

:

1

\. , ,

". "'4,"

-206-

1 Table XXXIII

1) RenI ts are expressed as the IDlOunt of B5 mediated inhibition of

BF/IL-2 st imulated 2° MLR ce Il prol iieration. Three represeDtative

experiments are shawn.

Assay system a 1 X 105 human 2° MLR cells. 100 ~l BF/IL-2, 100 ~l 85

19G1 (absorbed), H3T pulsed for the last S h6urs.

2) ~ of the control 8F/IL-2 response remaining.

3) B5 Dot preabs.orbed.

/ 4) B5 abso'rbed with 1 volllllie of packed fresh 1œC. 3 hrs. 4°C.

f.~, '} y S) --a5"'absorbed with 10à :z: _6 2° MLR cells/1.0 ml BS.

6) 2° MLR cells were preabsorbe4 with BF/IL-2 (100 x 106 cells/1 ml •

8F) 2 hrs. 37°. The cells were pelleted and used to absorb 1.0 ~ BS

...

7) Controls med = 2° HLR cells not stimulated with BF/~-2.

BF/IL-2 = 2° MLR cells stimnlated with 8F/IL-2. •

No BS was pres~.

(

r , -.

-207-

Tab 1 e XXX IV. , "---~-' Recovery of BF~IL-2 f~om immunoabs&rbant columns.

<;,i Immundabsorbant columns

65 19G1Y) anti-SRBt2 ) hyroglobulih3 ) {}

. FractionJ4) ,.

% % % ) cpm cpm c~

1 5529+140~5 ) ~.4 2358+210 16.4 756::,873 5.2

2 2973+487 20.6 2809+136 19.5 1860::,63 12.9 .. 3281 +813' 3 22.8 2410+275 16.7 2797::,202 19.4

• 4 8160+646 56.4 7431 +134 52.0 6089:,894 42.3

5 14104+5976 98.1 ! 17464+3370 l,OO 10375::,5523 72.2

120706+1651 6 18381 +432 100 100 13225:,1421 92.0 1

7 17950+6220 100 118652+5.623 100 10223+746 71.1 1 -

8 21389+1867 100 ' 5467+2060 38p 4355:,715 30.3

/18808+1148 9 21399+3111 100 100 1978+742 13.7 1 ,

10 19110+3133 100 /12576+2594 87..5 ~423:,380 16.8

11 13369+1472 93.0 !16873+5478 100 no 1

12 11487+3495 79.9 18599+6415 100 no ..

13 8820+477 61.3 10018+1122 69.7 no 1 /J

14 5284::,194 36.7 1 5162+735 35.9 no

15 3495:,555 24.3 l 'l)Zl+1222 '30.0 no

Contra {6 ) 14369+2414 #

BF:

(

.~ -- .. --~--- ------ ------_...:.._~~-

...

-208-

Tablo XXXIV

1) An immunoabsorbant column of ÙDDobilized BS IgGl.

2 D' B5 IgG1/4 IIÛ gels.

8.0 al 8F/IL-2 applied to the colamn.

2) An immunoabsorbant column of UDaobilized anti-SRBC IgGl.

2 D' Anti-SRBC/4 ml gels.

3) An immunoabsorbant column of UDaobilized thyroglobuline

4) 1.0 ml fractions collccted and tcstod for BF/IL-2 biological

activitics r~covored.

~ssay system • 1 % 105 2° KLR colis + 100 ~l test sample from

o.ch fraction. ColIs .oro incubated at J'OC

3 days.

;V

) 5) R.esu! ts aro upressed as cpm of H3T uptake of BF/IL-2 sUmulatod

2° MLR cells. and '.of control responso. 0

f

6) Control, the orilinal unfractionatcd G7S BF/IL-2 •

•

--~-~---------------------------

-209a-

( Optical Density

L!1 0 Ul 0 . . (\J (\J

\ . 5 Cl c.. . (J a

5 ;'(1

.' 'J .

----------.. ~ - - --

1

11

<!l--- __________ _

'-

( OOS! OOt"! 002 t aoo t'ODe 009

wdo

_-4

DOt"

IJ') . a

.1' ..

0 . 0

1

~ 1

.(J

ii

002 a o

L!1 (T)

0 CT)

U1 (\J

fi) c: 0 ,-..., 0 a L ~ -

0 ru

....

o -

'1

...... ,.

J -209b-

r T ab 1 e . XXXV • , Biologieal activities recovered from the P60 column.

BF activi tieJl) 85 activitieJ2)

Fraction, % of % of control stimul a tian control stimulation "-

12 210+263) 0 no

14 453+201 3.6 12532+196 102.3

16 166+60 1.3 5705+112 46.5

/1a 169+56 1.3 5367+671 43.8

20 440::268 3.5 ! 7414:,1423 60.5

22 658+122 5.3 1 0887:t.70 7 88.8

24 1444+188 Il. 7 14850+2049 121.2 -, ", f

26 t 7236+932 "59.0 0 16437:,807 134.2 1 - 1 "

'28 1 9595+684 78.3 no , 1 1

! 30 4136+109 33.7 no

32 .. 689+152 5.6 no -34 384+50 3.1 no

36 357+18 2.9 no

Contl'OlJ4): Bf BF+BS medium

12247+150 4764+266 491+58 - -<1:'

, 1 , ! j

( 1 ,i l

" 1 q; \~

, . î - l

" 4 . , . .. .. ~

.'

i -

1

•

( ,~

l'.

-,210-

Table nx'V ,

1) To Assay for BF acU'Viths", 100 ,,1 frolll each fractiôn "a. added to

105 2. MLR cells. Coll proUferation was detonained alter 3 days in,

c'4ltuS'o.

2) To' assay for B5 bioloSical inhibition., 100 p.l froll oach fraction , .

wu addod to 105 2° MLR co11s 'and 100 III BF/IL-2. Colls wore

incubated for 3 days and coll proliferations dotormined by UST uptako.

3) Re.ulta are oxpros.sod as CpIIl of JI3T lI.ptako, and" of pOsitivo

control.

-4) , COJ;ltrols:- ilF - 1Uifractionatod DF

BF ~ B5 ·'unfractionate4 BF and B5

..di .. • No BF, BS or othor stimulus add.d.

Backaro.1Uld U3T uptake by 20 )(LR cell,s.

,"

- , ,\ .

- ' " .

0,

.' ' t \. " , '

l, , ,

'.

, '" ~ $~F E cl fi • -'-, -, -"'-"'~-~.I' .~ . .". '.

·r (~", ! ~~

, ,

J f

,/

•

','

~" ' e 1

-211-(

Tabl.e XXX"/L . ' .

Enzyme 'l.inked' ttnltlUnOassBy, .01 Varions "Anti-lL-2" Prf!p~rations

We 11s- coa ted ':

BSA

BSA

aSA

6 BSA

mouse Ig (NMS)

BF/IL-2 (ITS~5)

BF/IL-2 (ITS)

• G75BF /IL-~6)

G75BF/IL-2

, '

, ,

" '

, l ,,-

, Antibodies added

l '

"

pss

anti -IL-f2}

85 IgG{3)

anti-BsA4)

anti-IL-2

B5 IgG1

anti -IL-2

, ,

/,

"

/--. -

.,

Colorimetrie,changes {OO}l)

0.015

NO

0.016

0.182 " ; l "

0.129 /

0.025

·0.104

0.,024

0.046

) . \ ' , , , • 1

J

"

',- .' : .'

- ,

,

-.

" , ! , r

c , ,

, '

, ,

" '

1 •

f -212-

l, • rable lDYI

1) ae'ulta are ezprea.ed a, the optical aenaity at 405 Da. " .

2) Aati~lL2 provided by Dr. Richard Robb 100 ~1/wel1.

3) B5 IsG1_ 100 ~l of a 20% concentrated cutture ,upe rDa tant ,

... tpv'iUed 011 DEAl iOIl e:z:ch&1lle col1mJ1.

\

, ..

.) Aat i BSA, ~1Ul. .e ru. f ra. a BSA Uua1Uli:z:.d Bal b / C IDOUSO •

Previollaly shown ta bo anU BSA .po c if ic due to i ta ab il ity to l'y ••

BSA coatocl SUC. U.ed at a 1:5 dit"lltio'u. 100 J'l/well.

5) Bli'/IL-2 producod under .0rlDl froo conditi,ons in l'I'S .odiva (fro. 1

Dr. 1:. Ahibr).

6) G75 HP/IL-2. The ... 0 BF{IL-2 u.ed to lmaunize the Biozzi,lDouse

" ••• 4 for hybrido •• fudoll •• " ~, ;t:I:',

, "

, ,

, ' , , ,

, . -,

,.

)

,. ,

"

-213-

CONCLl1SION

\ Tho aim· of this york YU to prodùce. a 1I0Jl0010_1 antibody to the

~

hUll~ lymphokine Blastogonic Factor (BF). ,\fo boUeve that Blasto.enic

Factor is a human Interleukin-2 equiTalent. The reasons for producinl'. ,

:monocl.onal antibody directed against BF/IL-2 are numerous:

1) Anti-BF/IL-2 can be used as a tool to purify IL-2 on immunoabsorbant

co 1 umn chroma to gr a'phy • n,-2 preparations an often " coUection of

lymphokines of 'Which 'one is n,-2 (133,213,214,215). E.xisting n,-2

purification schemes are inefficie~t and often represent .. five step - "'~ d~

sequential purification procedure. It invu-1ves l ,!,ery largo startina

volume_, and a very p'oor yield of 'pure' IL-2'. 'ETen sa. this 'purified

n,-2' is not homogenous sinee it represents lIlore ~han one protein band

1 on SDS gel electrophoresis. "Üh an immunoabsorbant column of

monoclonal antl-BFïIL-2. .. one step purification of ll.-2 yould be

possible and perhaps yield a more homogenous preparation of n,-2.

2) The aTail abU Hy of • monoclonal anti-BF/IL-2 'Would allOlt one ta

study, the n,-2 producer_ cell and the tt.-2 responder coll il1d~pendent1Y'. i

It provides for the potential to separate. the n..-2, produc.rs from, the

n,-2 rosponders (i. 0 FACS) and thus the pote~tial ta clone the n,.-2

producer/rosponder yith alms of stûdying the finer characterisUcs of

J ,1;' each cell type. fi

3) "ith a 1Il0nocionai anti-BF/n,-2. a more son si tin assay may be

àvailab,le' to studr the oarly evellh in ,the' activation of n,-Z prodllcer

cells. In teras 'of kin.tics it 'Would be posSible to deteot n.-l

1-.-----.' ...... ...,j..,.i& __ ._""' ..... fj .-.----,-,...) -~ ~ , ....... -- .,- ---'.~..,...-:--~-'-------------

. ..:

C, '".

(

(

~, .. f

;

-214";'

production It the cellular lev..! rather than ta wait for snffiaient IL-2

release and lecuanlation in the culture supernatant.

4) Final 1,.. it may be possible w1th a sensitive radio-illUll1lll0assay ta

detect in vivo cireulatin, levels of IL-2. Thus one can monitor ehanles

in cirenlating IL-2 levels during tissue transplant leeeptanco or

rejection.

Initial attempts ta obtain illllllunhed serUDJ from BF/IL-2 immunhed

ani.als have been suceessful. There are various pJ!'oblems which may

hinder our attempts ta illllllunize "ith BF/IL-2 as an antigen •. The poor i

Ittti,enicity of BF/IL-2 may be due ta it', poor inherent ÎlDlD1Ill0genieity.

Thit ean be "attributed ta the similar al1tigenic structu.x:e of all ~~wn

IL-2 species (human, murine and rat) the short half life of IL-2 in vivo

CT 1/2 of 84 seconds). the law concentration of IL-l produeed in eulturo

supernatant (pi'eomolar concentrations), as weil as the lack of pue

hOIIIogenous 11.-2 to be used as ln ilIIIDUDolen. Therefore we chose ta

ut.ilize the hybridoma technique ta optimize the chances of obtainin&

a sp~cific clone which produces an antibody directed'alainst BF/IL-l.

The in vivo illUll1llli:ution of the (CS7Bl/6 .x C3H)Fl manse. the in Q

vitro illlllluuizÀtion of the Balb/e mouse lnd the adoptive trlDsfer

iœm1lllizaton of the Biozzi mouse gave disapPointing results. ,. yere DOt

able ta recover any immUDoglobul in producin& cells from these throo

illDlluni:utions and hybridoma fusions. - Perhaps the (C57B1/6".x C3lUFl d'id.

not have the potential ta recogni:. the la" concentration of huaan IL-2

which we used for UmaUQization.

Luben and Mueller had shawn the advantages ~ the in vitro

..J. 1 ...... ----·..,.·'O\"Oht"' .. Jf-' ... Lt ... '-,.---~----------- .. ~ ~~.~ ... -----___________ _

l ,!.,

...

(

(

-215-

t..llJl!zaUon lIlethod to i_lUlize mouse celIs to the lywapholtine OAF.

Boyever OAF doe. Dot have the potentia! ta Itaulate T lymphocytes

yherea. huaan n.-2 aan staulate .urine r cells as a seconda.ry signal.'

le u.ed the in vitro i .. unization tech~ique of Laben and Iluelle~ to

avercom. the short half life of IL-2/BF in vivo, and the lower antigenic

concentrations required for ill vitro imIltmization. To overcome the

utilization or degradaUoll of BF/IL-2 by murine T ce1ls in vitro. ".

used an e:.leess of murine D,.-2 ln hopes of saturating the murine T cell .

n.-2 receptor. w. "ore unablo ta generate anti-BF/IL-2 specifie

hybridoma. frolll in vitro taDunization. \

Our failure may b. attributed to \

1 the utilization of the alltiaens (BF/~~y the murine splenic ~ cells.

Thul little of the antiaen, BF/IL-2 "as left to stillulate the ,

Ùlaunoeolllpetent B cells. Stadler .fi.!l. (50) "a. dso una~le to

produce an allti-human IL-2 specifie hybrido.a usina the in vitro

imaunization protoeol of Laben and Mueller.

A. shown by Fox .!.! Al. (242), the adoptive transfer of immunÎ%ed

cells into an irradiated hast can enbance ·the stimulation of the anti,en .~

.pecific clones by 10 ta 50 fold. Bawever. using the Bio%zi outbred

hi'p antibody responder lIlice. "e "ere unahle to senerate anti-BF/IL-2

specifie clones froll adoptive transfer of ilDannized cells. Since "e

"ere transferrina potentially alloreactive .cel1s froll one outbred Bioz:i . into an X-irradiated Biozzi host. it .as p,ossible tbat & Iraft verlus

host reaction "as elicited Yh~ch interfered' and yeakened our chances of

, oht~ininl an &nti-BF/IL-2 specifie clo~e. Bawever. in viey of the short

-imaUDization period. and the monoclonal nature of the immune response

~---------j~,,-'--~----------~--------------

, ...

"

(, . "

-l16-/

a4d hybridoaa cell fusion tochniques. it i. ualikoly thst a GVB response

CS4 ' ... k' aD on-Ioin. antibody response.

The in vivo~ Balb/c i .. ua1zation lave tho firat indicatiou of aD

anti-BP/IL-l specific hybridoms. Wo obtained 23 clones which secutod .

• ou.e 1,G1s lnto the spont culture supernatants aud woro able to iDhibit

BF/IL-2 stimulated 2° HLR coll proliforation. (At. 1 z2 dilution. we

obvaine~ approxiillate ly 60 to 7a iDhibi tion of ma xima 1 cell

'proliferation.) Furthormore, the spent culture sup.rnatants were ablo

ta inhibit 1 0 allogenoie MLRs and T coll -it080n stiaula tions. but had

no effect on B cell mit080n modiatod stimulation. Succossful 1° MLR and

T coll aitolon stiaulations are depoadont on IL-2 as a secondary sisnal >

thoroforo it would be oxpoctod that an anti-BP/IL-2 antibody would block

or aeutralizo these responsos. The lJ. cell mitoloa, PW1f is a T cell

dependont B coll mitoloa (251). Boyover. it is uuknoyn whother tho 1'010

of T cells iD a FOI stimulation can be replaced by D.-2. Our Balb/c

hybridam. data would suuest th,t the .t~.tiOJl of B cells by PI){ is

ilI.dependont of IL-2. Other B coll mito,e s, LPS and Staph. Auroas, are

totally iadopendtJnt of D.-2 and thus are 40t affected by aD

&Dti-~ IIL-2. " '

It ia uafortuate that .os~, of the Balb/c anti-BF/IL-2 hybrida ••

clones ceased .... UOllobulin ~roduction alter six aoaths. It wOuld

su"ost that we have obt.iaed fused hybrido.a cells of a hiShly unstable

phenotype. lndeed hybrido.a fused cells ca4 be vory 11llstable

phenotypically and aay uderlo chromoso.al aborrations (252).

To opti.be our in vivo ÎJIIIIlunizations, we usod a straia of Ilice

J... __ ".. ...... __ ........ ,......_, ..... __ ..,..,. _________ .~ _,*"

1

"

t -117-

selected for Hs high titre aDtibodr rosponse s. Fr~ .. the Bioui iD vivo

~uaization. we obtained the AC4-II-B5 clone "hieh had. eonsistently ,

emibited areater than 90\ inhibitioD of BF/IL-2 stimulated 2' HLR ceU

proliferation. The AC4-II-B' cIano la cODsidered monoclonal sinee on

several subeloninls, "e have obtained a 2~ eloninl"efficiency of "hich

84 to 98 .. of the clonos eùibited IgG1 secreUon, "ith correspondinl

inhibition of BF/IL-2 stimulated 2° MLR cel1 proliferation. , The AC4-II-B5 clone socretes a mouse IgG1 as defiDed by

imauaodiffusion. Furthel'lllore on illlDlUDoe1ectrophores h. tho B5 product

miar.tes, .s one major ,protoiD band corresponding to the mouse IgG1

relion.

le uo confident that B5 IgG! h specifie for BF/IL-2. This is

based On the ob.ervations that 85 ISG1 CUl inhibit variou. biological

•••• ys which aré dependent on n.-2 as • Mediator. B' IgG1 (in the range

of 3 .. 12 Jil to 6.25 Ill) c.n givo 5o-At inhibition of human 2° MLR coll

pro1iforatiou. stimlllated by 100 111 G75 BF/IL-2. BS IgG1 can also

inhibit IL-2 (of lIlurine or hlUllan orisin) stimlllated murino 2° HLR colls

(the 50-. inhibition ".s fOUDd in the ranse of 0.782 ILS to 1. 565 111

I,6t). It ~s weil established that tho BP/IL-2 rosponsive cells in the

• le Jà.Jb .re the primed Cl'Ls. le "ere able to sho" that BS ISG1 cau.

iDhibit the ,ener.Uou. of pri.ed cytolytic lymphocytes stiJllulate.d with ....

BF/IL-2 or the original st~u1ator colis. nèrefore BS IgG1 can inhibit

hUllaa aad murine respoDses which are dependent ou. n,-2 as .n o~lig.tory

stimulus.

SU.au.1ation. by T cell aitosens (CanA and PRA) are n,-2 dependent

1

f ft , !

-

1

('

N,B - ?

-218-

whéreas stimulations by B cclI mitogens are IL-2 lndependent (i.e. LPS

and Staph. Aureus microorganisms). As expected BS IgGl cau inhibit the

T cell lIIitogens. but have no effect on the B cell mitoSens stimulated

cells. Paetkau's thymocyte costimulator Assay is dependent on IL-2 as &

costimulator in eliciting thymooyte rosponses to CanA. Again BS laGl

cau Inhibit thymocyte responae s in a costimul 'a tor assay. Th" d-ef'ini tive

results were provided by the abiliJY of BS IgGl to inhiblt cell

proliferations of two IL-2 dependent cell lines (both a murine and human

IL-2 dependent cell line) , BS IgG1 can inhibit human and murine D..-2 \,

depdndent cell proliferations in response to hum an and/pr murine IL-2s.

We are confident that BS IgGl mediated inhibition of coll

proliferations is a specifie event. This is basod on the observations

that. 1) Cells (unstimulated or stimulatod) when ,rown in the presence

or absence of BS IgG1 exhibited the same cell viabUiUea after throo

days in culture. Rowever. there were significantly more blast cells i,n

cul turos grown in the absonce of BS I,Gl. Therolore BS I,Gl is not

nonspecifically to:dc for stimulated or unstimulated cells. 2)

Inhibition of BF/IL-l stimulat04 coll proliferation is associated with

the BS clone only. Sovoral control hybridoma or myeloma culture r

supernatants (i. e. IgGl secreting anti-SEC hybrido_a, or the, pano

myeloma) did Dot affect BF/IL-2 stimulated eoll proliferatio.n. J) BS °l

IIG~ exerts its effect on BF/IL-l stimulated 20 HLR cell proliferation

i~ tho first 36 hours of culture. Addition of BS IgGl after th. ~nitial

36 hours had DO effect On .cell proliferation. Therefore BS IgGl must

influence a cellular event "b.ich occurs within the first 36 hours of

-

-

t

.. -219-

stimuLation. However ceLl proLiferatlons ln bulk cuLtures are complex

syst~ms lnvoLvlng numerous cellular events. Therefore lt iS' dlfficult

to determlne WhlCh one event was; lnhlb,lted by 8S IgG 1 •

We are confident that the biologlcal lnhibltlOn of ce~l prollferation

is mediated by the IgG1 fractlo~ of the BS cuLture supernatant or ascites

fluld. We have shown that onLy the IgG 1 positlve fractions from a DEAE

lon exchange coLumn, and from lsoelectrlc focuslng wrLl lnhlblt ceLLular .. proLlferatlon. Furthermore, 8S medlated lnhlbltlon can be removed on

a rabbit antl-mouse IgG1 lmmunoafflnlty column, but not on a controL

rabbit antl-mouse IgG2 coLumn.

However~ uSlng numerous ~Lution methods we ~ave been unabLe to

recover bioLoglcaLLy actlve aS IgG1 from the rabblt antl-mouse lmmuno­

affimty coLumns. Perhaps by the hars,h eLutlon treatments, we have

destroyed the antigen blndlng site on the 8S IgG1 antibody. Indeed, with

the use of a radioactlve tracer, we were abLe to recover approximately

2 Si:: of the column bound 8S IgG~, however the e LLlJ:ed 19G1( di d not 1 nh, b, t -ce LL proLlferatlon. ~'î>

What is the speclfic;ty of the aS IgG1 antlbody? We onLy have

lndirect eVldence that BS ;5 ~ot dlrected against a ceLL surface antigen.

We've shown that upon pre-absorption of es wlth RBC, 20 MLR ceLLs and 7

o days oLd 1 ceLLs, we did not remove 85's inhibltory activlty.

ceLLs are known to express IL-2 receptors, so the Lac~ of~bsorPtlon onto

2° MLR ceLLs would ruLe out the poss,biLity 0 ti-IL-2 receptor.

However, we can not ruLe out the posslbiLl

against

ceLLs. The specificlty

di rected

d on act i vated lffII

directed against a human serum

prote;n and most LikeLy not directed against a vlt~l·FCS·component. Pre-

t

-220-

l1minary ELISA experiments suggested that a5 IgG1 can bind to a preparat10n

of human IL-2 produced in serum free ITS medium. However, we have been

unable to remove 8F/Il-2 on an immobilized 85 IgG1 Sepharose matrix. 8FI

IL-2 and 85 IgG1 do not form an immune comPjex in solution either.

Can 85 IgG1 recogn1ze BF/IL-2 immobiL~ed onto cell surfaces? To

answer thlS quest10n, we used fluorescent antibody binding studies. In

prelim;nary experlmènts 1t appeared tnat 85 IgG1 will not recognize Bf/IL-2

expressed on aLloantlgen activated IL-2 produc;ng cells. We were unabLe 4

to find fluorescent antibody binding to BF/IL-2 absorbed 20 MLR celLs.

Perhaps 8F/IL-2 was trans;ently expressed on celL surfaces of 20 MLR cells.

It is possibLe that 8F/IL-2 is rapldLy internalized or degraded following

absorption so that ;t is no longer recognized by the 85 IgG1 ant1body.

Studies by Robb et al. (209) suggest that IL-2 does undergo an enzymatic

degradatlon foLlowing binding to their respective receptor. Alternat1veLy

1f G1lL1S et at.'s (212) estimation of IL-2 bi~logicaLly active range of

'" -12 10 M lS correct, then it ls-possible that ouf Low affinity B5 19G1"

1 antibody cannot quantitativeLy detect so few molecules of 1L-2 bound to

ceLL surfaces. However, Ste;nmann et aL. (253) recentLy reported that ~

a monoclonaL ant1-murine 1L-2 (withi'n a biological active range of 10urg/mL)

was abLé to surface label IL-2 producer celLs as well as IL-2 dependent

responder celLs of human and mur;ne origine The pattern of sta1nlng they

observed was compatible with our observations. Indeed the IL-2 producer

and responder cells exhibited dlstinct characteristic stalning patterns ....

,and may be a usefuL tool in future dlfferentiat;on of IL-2 producers ana----.---

responders.

We believe that the es 19G1 antibody has a very Low affinity for the 'r ~

antigen BF/IL-2. This;s based on the observation that we require in

excess of 3.12 ug of 85 19G1 to inhibit 50X of the control response

--_._-- '-"-' ( -

-221-

in • BF/IL-2 stiaul.tod 2° )fI.Jl coll proliferation as say (this

corresponds ta 15.6 pl IIG1/ml). A la.. .ffini ty anti-IL-2 may be •

characteristic of all anti-IL-2 antibodios Dawn to date. The series of

anti-h .. an IL-2 clones described by Stadler II Al. (250) and Smith li

.!.l. (257) appeared ta be 1011' affinity also. Stadlor.!.!.!l.' s clones

have a 501ft in.hibitory dose in the rUle of 3.5 PI ta greator than 20

PI/al. Smith.!.! Al.'s anti-hnman IL-2s have • 5~ neutralizatioo in tho

range of 8 PI ta 250 PI/ml. whereas Gillis II al_'s .nti-murine IL-2 has

a biololical aeU.,e rUle of 10 pg/ml. A la.. Aff ini ty anti-BF/IL-2

antibody would o%plain some of the inconsistenc ies of our fluores,eent

antibody bindinl assays. It is possible that BF/IL-2. onco bound ta its

specifie IL-2 rocept or. .ay 1lllderao conf igurational ehanaes and thus

bloek. or change the antilenie site recolnized by the aonoclonal

antibodies Ci. e. the anUlenit:: si te may possibly be the IL-2 biololieal

aeU." site). It is, aIso eoncoivabIe that the a.,idi ty of the IL-2

recoptors for BF/IL-2 is much stronser than the af-finity of tho

anti-BF/IL-2 antibody for its specifie antiaon-BF/IL-2. lhereforo in a

coaplez biololieal •• say. a larle aIIlount of uti bodies was required in

arder to black a BF/IL-2 stimulated response (i.e ta bloek the bindinl

of BF/IL-2 to the IL-2 receptors).

Robb ~!l. (248) had sUllested that IL-2 specifie hybrido.a clone.

deri.,od fra. one sinale fusion exhibited differont biololical fun~tions.

Soeo anti-D..-2 clooes ean inhibi t n-2 depeudent biologieal assays

(neutral haton assay). qther elonos can detect .urfaee boud IL-l. and

~ers cau preçipitate soluble IL-2. B01I'ever. a 'au1tifunctional' cIano

.' .

1 -222-

which exhibits all of the above flUlctions has not bun found. Perhaps

IL-2 undergoes aolecular confiprational chanles when 1t is coll surfaco

b01lllcl. thorofore clifferont anti-a-2 clones wouId recoln1zo oi ther the

coa.fipraUon of the froe anbouncl IL-l. or the receptor' bound IL-2.

Al ternati'Yely the omonoolonal aatibody may rocolnize the IL-2 lIlolocul. at

its active site or at an epitope adjaceat. to the activo, site. Siace

monoclonal antiboclies are moaospecific. thon differont cloaos would

rocoanizo d~fforont IL-2 epitopos.

Of tho four Dowa anti-n.-2 latibodies reported to date, they .. ro \

aIl cross reactive botween the h.aa ancl aurine IL-2. Wlith the possible

exception of Saith .!.1 .!l.'s D_S-1.2.3, aIl ucnrn anti-IL-2 were also

cross reactive with rat a-2. Gillis.!1 .!.l., StadIer .!oS Al. and our

AC4-II-B5 anti-IL-l wero initially selectecl based on their ability to ,

ilÛlibit IL-f dopoaclent T cell proliferl,tio~ (250.2.5$,256). 'Illerefore

thore is an antilenic site 011 the differeat spocies of IL-l which is

recOlnhed by 85 liGl as well as by Gillis et.!.l. ancl StaclIer ~ .!l. ' s

aati-IL-2 antiboclies • Thîs i5 Dot surprisinl coasideria& the central .

role of IL-l in the ~une response. In evolntionary te~s. it wouId be

beneficial to conserve the aolecular structure of ÎL-2 in ordor to

.ail1tain a funetio.l imaune response throulhout evollltionary changes. 1

Saith .!1 li.' s series of uti-IL-2 were initially selected by an.

ELISA iaaunoassay. Of interest is the DIIS-3 moaoclonal antibody whicla

exhibited very poor bioloaica1 noutralizltion of IL-2 (5~ inhibition at

250 Ill/al) yet i tisa very efficient imauaoabsorbant for the one step

pur if ica tion of IL-2. (Coaversely the DJ(S-1.2 which &%'11 ,Jood

"

----------------------------------~.-----~---

,1

.

-223-

neutralizing antibodies, are poor immunoabsorbants). -GilLis et al.,

Stadler et al. and we had experienced the same difficulties in preparing

an immunoabsorbant matrix using monoclonal antibodies which are good

neutralizing antibodies. Perhaps:;""he neutr,alizing antibody recognizes

an epitope adjacent ta, or which is the IL-2 biologically active site,

whereas the DMS-3 recognizes an alternate epitope which ;5 more readiLy

acc.essible on the IL-2 mOlécule, and thus DMS-3 has greater affinity for

Il-2 <similar findings have been shown witb other polypeptide hormone

monoc.lonal antibodies) (-254).

We have successfully shown here that BF is indeed a lymphokine of the

IL-2 category. In this report, we have shown that BF can support the growth

of IL-2 dependent, cell l ines of mouse, -and human origin. 8F can provide the

co-stimulator signal lin a mitogen stimulated thymocyte co~stimulator assay,

and as evidence provided by Uotila et al. (55), BF can trigger the

/primed CTLs to proliferated'and ex~ibit ,their cytolytiè ac~ivities.

~ In summary, we have produced ~ monoclonal antibody with specificity

directed towards the human IL-2 lymphokine,Wlastogenic Factor. This

antibody <AC4-II-B5) car inhibit numerous biological aS5ays which are IL-2

dependent but has no effect on IL-2 independent cell proliferation. The

AC4-11-85 antibody can also recognize murine and rat IL-2. Preliminary ELISA

and fluorescent antibody'binding studies suggest that AC4-II-BS can detect

immobilized 8F/IL-2. Unfortunately AC4-II-85 cannat be used as a tool ta

obtain purified IL-2 as we were not able to retain BF/IL-2 o~ an immoabsorbant'

matrix of AC4-1I-B5. Furthermore, 1 feel that Many questions concerning the

specificity of 85 can not be answered due to the limited availability of ..

high titre 8F/IL-2. Also it is difficult to control the variability of each

preparation of 20 MLR ceLLs 1 respor;1se to BF/lL-Z:' fThi s is a reflection

i' (:

--:;~'~--~--~_~\--~II*~----------~----------~----~-~~--~~------------,,----__________ ___

1

1

, ~ r

-

'of the activation of ~L-Z respbns,ive ceLLs.> , Usi09 an IL-2 dependent 1 /

~.

cell l ine ,can overcome th; s probLem. However at the time, .ti1ese exp~'riments

wer_e performed, our ayaitabLe source of Il''2 depenaent celt.line was very

limited and inconsistànt.

, '.

/ . "

'-'

. ' , .. '

!.. .~ .,.~ \

<,

t

"

( .l,

l..'l.-' '><,. ....... ,.,. . ~' . • l"

, .

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