Blood-1988-Delia-241-7

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1988 72: 241-247

D Delia, G Cattoretti, N Polli, E Fontanella, A Aiello, R Giardini, F Rilke and G Della Portasubsetactivated, and malignant human B cells: identification of a new B-cellCD1c but neither CD1a nor CD1b molecules are expressed on normal,

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Copyright 2011 by The American Society of Hematology; all rights reserved.20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DCBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

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lood Vo l 72 , No 1 Ju ly ), 1988 : pp 24 1-247 24 1

CD 1c bu t N eith er C D 1 a n or C D 1b M o le cu les A r e E xp ressed on N or m alA ctiv a ted an d M alig n an t H u m an B C ells: Id en tifica tion of a N ew B C ell S u b set

B y D o m enico D elia , G io rg io C atto retti, N iC o le tta P o lli, E nrico Fon tan ella , A n tone lla A ie llo , R oberto G ia rd in i, F ra nC o R ilkeand G iuseppe D ella Por ta

The CD 1 c lu s te r o f m ono c lo na l an tibo d ies M oA bs ) C D1 a .CD 1 b . and CD1 c , id en tifie s m ole cu les th a t a re d iffe ren tia llyexp re ssed on hem a topo ie tic and nonhem a topo ie tic tis -su es . O u r ea r lie r fin d ing th a t th e m an tle zone M Z ) bu t no tth e germ in a l cen ter GC ) o f no rm al lym ph nodes IN ) isC D 1c+ . C D 1a-. and CD 1b - p rom p ted us to fu r th e rin v es tig a te th e exp ress ion o f th e se m o lecu les on no rm a l.ac t iva ted . and m align an t B ce lls . W e rep o r t tha t b loo d an dsp leen con ta in CD 1c+ B ce lls th a t ac coun t fo r 49 20 .4% (m ean S D ) an d 50 .9% 4 .4% of the to ta l B c e llpopu la tio n . re spec tiv e ly . C D1 a - and CD1 b -sp ec ific M oA bsare un reac tiv e w ith bo th B and T ce lls ; th ese la tte r a reC D 1 c - as w ell. W hen CD 1c+ and CD1c - B ce lls areac t iva ted in v itro . th e C D1 c m o le cu le is u p regu la ted in thefo rm er su bset an d ind uced d e no vo in the la t te r . C o nvers e -ly . ac tiv a te d b lo od T ce lls rem a in C D1 c - . Ne ith er C D1 a no rCD1 b m o le cu le s are de te c ted on ac tiva ted T and B lym pho -cy tes . A t u ltras tru c tu ra l lev e l. th e C D 1c+ B ce lls e xh ib itd is t inc tiv e fea tu re s . nam ely . co nd en sed ch rom a tin w ith o rw itho u t a n uc leo lu s and a u n iq ue c lu s te r o f cy top la sm icT H E C D 1 m olecu les are g lycopro te ins tha t w ere first

iden tified on th e su rface of cortica l th ym ocy tes2 andthough t to rep resen t th e hum an coun te rpart o f the m ouseT la .3 Im m uno prec ip ita tion s tud ies w ith sev era l C D 1-spec if icm onocb ona l an tib od ies (M oA bs) h av e rev ea led th e ex istenceof th ree m olecu les o f 4 9 K d, 45 K d , and 4 3 K d non co va len tlyassoc ia ted w ith f3 2-m icrog lobu lin (f32M ) an d bearing d istinc tse ts o f e pito pe s.4 5

M oA bs spec ific fo r these m olecu les have b een c lus tered asC D 1a , C D 1b , and C D 1 c, resp ec tive ly (T h ird In te rn a tion alW o rkshop on H um an L eucocy te D iffe ren tiation A ntigens ,O x ford , 1986 ). T here is a d iffe ren tial in terac tion of C D 1m o lecu les w ith 2M as revea led by the fact th a t C D ia an dC D 1b bu t n o t C D 1 c h eavy cha ins partly d issoc ia te from it.4T he recen t c lon in g and ana ly sis o f th e C D I genes by a cD N Ap robe spec if ic fo r C D 1a (H T A 1 )6 su ggests the ex istence offive hum an H T A 1 cross-hy brid iz in g genes tha t, un like them ajo r h istocom patib ib ity com plex (M H C ) lo cu s genes , a reloca lized on ch rom osom e 1 .

H om olo gy s tud ies6 7 ind ica te tha t C D is a m em ber of theim m un ogbo bu lin supeng ene fam ily an d shares a low degree ofh om ology to b o th hu m an an d m ouse M H C class I a and c lassII /3 ch ains . T he b io log ic ro le o f C D 1 is so fa r unkn ow n;h ow ev er , its he te rog en eity m ay sugges t a m atu ra tion -lin kedfunc tio na l ro le since the th ree m olecu les a re d iffe ren tia llyexpressed in norm al tis sues . T hu s, co rtica l thym o cy tes a reC D 1a+ , C D 1b +, and C D 1c+ , and L angenh an s ce lls a reC D 1 a + , C D 1 b - , and C D 1c + / - ; in te rd ig itating re ticu lu mce lls are C D 1a+ , C D 1b+ , and C D 1 c+ ; eccrine ce lls a reC D 1a+ , C D 1b+ /-, and C D 1c-; and m antle zo ne (M Z ) Bce lls a re C O la- , C D 1b-, and C D 1c+ .8 9 T h e b ind ing ofC D 1c M oA bs to B ce lls is no t du e to cro ss -reactiv ity s ince theappro pria te m olecu la r w eigh t can b e prec ip ita ted .#{1 76}

W ith regard to th e fo llicu lan m antle B ce lls , it m ust beno ted tha t these ce lls resem ble, m o rphob og ica lly an d p heno-typ ically , b lood B ce lls (IgD + , Ig M + , C D 2O + , H L A -

ve s ic le s an d org an e lles ; th e n um be r o f nu c le o la te d ce lls ish ig he r in th e sp leen 95 ) th an in the to ns il 40 ) o r b lo od( 5 % ) . These fin d ing s fu rth e r con firm th e s im ila rity b e tw eenb lo od an d M Z B ce lls . Th e C D1c exp res s ion a ss es sed o n 27B -ce ll c h ron ic lym phocy tic le u kem ia s B -C u ) and 46 Bnon -H odgk in s lym phom as B -NH L ) w as de tec te d o n 41an d 32 of cas es . re sp ec tiv e ly ; the la tte r com prised fo u rfo l lic u la r a nd 1 1 d iffu se h is to typ es . T he Bu rk itts lym pho -m as w ere C D1 c -n ega tiv e . T h e B -c e ll n eop lasm s w ere a llCD 1 a - and . e xcep t fo r fo u r w ith a w eak cytop la sm ics ta in ing . a ll CD 1 b - as w e ll. T h e c le ar -c u t C D1 c d is tr ib u -tion in norm al LN M Z + GC - con tras ted w ith th ee v idence th a t som e B -NH L ce lls o f G C o rig in eg . fo l lic u la rw ith p red om in an tly sm a ll c le av ed ce lls ) w ere C D1 c O ve ra ll. th e fin d ing th a t C D1 c exp re ss ion is res tric te d to af rac tio n o f B ce lls p re sen t in lym pho id o rgans an d inp eriph era l b lo od ind ic a te s th a t CD 1 c is a p ow erfu l m arke rfo r th e id en tifica t ion and d is se c t ion o f B -c e ll su bsetsw hose fun ct ion a l p rop er ties c an now b e eva lua ted .S 1988 b y G ru n e S tra tto n , In c .

D R + ) and are thou gh t to m ig ra te , a fte r an tigen ic stim u-lu s, to the germ ina l cen te r (G C ) w here th ey pro life ra te andacqu ire fu nc tio na l p roperties (m em ory , sec re to ry ). T h is s tepis acco m pan ied by th e acqu is ition of the peanu t bec tinrecep to r2 and by the loss o f su rface IgD (Sm Ig D ) and ofhigh endo the lia l venub es (H E V ) ho m in g recep to rs.3 In th iscon tex t, C D lc cou ld be an app ropria te m arker fo r assessingth e B ce ll d iffe ren tiation and fu nc tio na l even ts occu rring inprim ary and second ary lym pho id organs . In add ition , C D 1cM oA bs co u ld a llow iden tifica tion of th e B -ce ll ly m phom as ofM Z d er iv ation . T here fo re, th is s tudy w as undertaken tode te rm ine , in norm al B ce lls , th e linkag e of C D Ic w ithd iffe ren tia tion and /on ac tiva tio n and ev a lu ate its exp ress io nin h is to lo g ica lly and pheno typ ica lly defined B -cell non-H odg k in s lym pho m a (B -N H L ).

M ATER IA LS AND MET HO DST issu es a nd d iag nos is . N od al and ex tran oda l ly m phom a b io psy

sp ec im ens w ere su rg ically rem oved from patien ts fo r d iagnos is; fo r

F rom the D iv isio ns o fE xp er im en ta l O n co log y A a nd A na tom i-ca l Pa tho log y and C ytopa tho log y , Ist itu to N a zion a le T um or i,M ilan o , Ita ly .

S ubm itted Ju ne 1 1 , 19 87; acc ep ted F ebruary 25 . 198 8 .T h is w o rk w as su pp orted in p art b y C o n tracts N o . 8 5 .02 16 3 .4 4an d 8 6 .0 04 55 .44 o f the Ita lia n N ationa l R ese arc h C ou nci l, P rog etto

F ina l izza to O n co log ia . an d b y the A s soc iaz ion e Ita lia na R ice rch eCa n c ro .

A d dre ss re prin t req ues ts to D om en ico D e lia . P hD , D iv O nc S pe rA , I stitu to N aziona le Tum o ri, V ia G . V en ezian 1 , 20 133 M ilan o ,I ta ly .

T he pub lica tion costs o fth is a r tic le w ere de frayed in p art by p agech arg e pa ym en t . Th is art ic le m ust the re fo re be hereb y m ark edadver t i semen t in a cco rda nc e w ith 18 U .S .C . s ec t ion 1 734 so le ly toind ica te th is fa c t.

1 9 88 by G run e S tra tto n , Inc .0006 -4971 /88 /7201 -0059 3 .00 /0

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242 DELIA E T AL

such purpose a portion of the biopsy sam ple w as paraf f i n em beddedand processed f or routi ne hi stol ogi cal ex am inati on. T he nonneoplas-ti c l ym phoid ti ssues w ere obtai ned f rom pati ents undergoing tonsi l -l ectom y or splenectom y f or therapeuti c purposes. T hese and arem aini ng portion of the bym phom a biopsy specim ens w ere partl ysnap-f rozen in l iqui d ni trogen, stored at -75 { 176} C,and partl y teasedw i th a surgical scalpel to obtai n cel l suspensions. T he l ym phom asw ere classi f i ed according to the K iel and W ork ing Form ulation(W F) 5 cl assi f i cati ons; f or thi s study the f orm er and the latter w i l l bes h o w n .

H epari ni zed peri pheral bl ood (PB ) obtained f rom norm al donorsand f rom pati ents w i th B -cel l chronic l ym phocy ti c l eukem ia (B -CL L ) w as centri f uged on Ficol l -H ypaque (Flow L aborator ies,I rv i ne, U K ) gradient to i sol ate the m ononuclear cel l s (PB M C).T i ssue l ym phocy tes and PB M C w ere w ashed tw i ce in RPM I 1640cul ture m edium (Flow ) supplem ented w ith 10% heat-i nactiv atedf etal cal f serum (Flow ), 2 m m ol /L glutam ine, 100 U /m L peni ci l l i n,and 100 ig/m L streptom ycin (R PM I -C).

Reagents. T he m ouse M oA bs L 161 6 C D I c, N A I /34 CD 1a,N U T -2 6 CD 1b, H D 37 7 CD 19, H D 39 7 CD 22, and M HM 6 8C D 23, w ere obtai ned f rom the T hi rd I nternati onal W orkshop onH um an L eucocy te D i f f erenti ati on A ntigens (O x f ord, 1986). T heC D 3-, CD 5-, C D 14-, and CD 38-speci f i c M oA bs U CH -T l , 9 U CH -T3 , { 1 7 6 } C H - M l 2 and A lO ,22 w ere k indl y prov i ded by P. B everl ey(I C RF, L ondon) and by F. M alavasi ( I sti tuto di G eneti ca, T onino).T he M oA bs iS23 and D A 224 w ere used to detect the CD 1O and theH L A -D R m olecules. T he C D 2 and T ransf erri n receptors w ereidenti f i ed w i th the M oA bs O K T I 12 5 and O K T92 (Ortho Pharm a-ceuti cal Co. Rari tan, N i ). A l l M oA bs w ere used at saturati ngconcentrati ons. T he f buorescein-i sothi ocyanate--conjugated Fab2goat antim ouse I g (FI T C-G A M) (T echnogeneti cs, 5. M auro T ori -nese, I tal y ), the phycoery thri n- labebed goat antim ouse I gG (PE-G A M ) (B iom eda, Foster Ci ty , C A ), and the FI T C- labeled Fab2f ragm ents of goat anti hum an im m unoglobul i ns (FI T C -G A H u)(K al l estad, A usti n, T X ) w ere used as speci f i ed by the suppl i ers. T heal kal i ne phosphatase (A PA A P) im m unoenzym ati c stai ni ng w asperf orm ed w i th a com m ercial k i t (D ako, Gbostrup, D enm ark ).Fifteen n a n om e te r s o f gold-l abeled goat antim ouse I g (GA M I gGG I 5; Janssen Pharm aceutical s, B eerse, B elgi um ) w as used f orel ectron m icroscopy (EM ) studies. T he phorbol ester 12-0-tetratde-canoy l phorbol - 1 3-acetate (T PA ) (Sigm a Chem ical C o. St L oui s)and the C a2 ionophore lonom ycin (H oechst-C albiochem , L a Jol l a,C A ) w ere stored at -20 { 176} Cn dim ethy l sul f ox ide and in ethanol atconcentrati ons of 1 and 5 m g/m L , respecti vel y . Staphylococcusa u r e u s C ow an strai n I (SA C) (Calbi ochem -B ehri ng Corp. L a Jol l a,C A ) w as used at 1:15,000 (vob/vol ).

Im mu no enzym a tic a n d imm un ofluor esc en t la bel ing . flo w c ytofluo r im etr ic a na lys is a nd cell sor t ing . A PA A P im m unoenzy -m atic stai ni ng of acetone-f ix ed cryostat ti ssue secti ons w asperf orm ed as prev iousl y descr ibed.27 L abel i ng of ly m phocy te cel lsuspensions w as carri ed out by standard indi rect im m unof luores-cence (I F) procedures on 96-w el l m icroti ter pl ates (Stenibin, L td.,Febtham , U K ) . B ri ef l y , 100,000 cel l s i n 45 L RPM I -C w ereincubated f i rst w i th S jL of M oA b (or the appropri ate di l uti on ofnorm al m ouse serum f or the negati ve control ) f or 30 m inutes on i ceand then w i th 5 zL of FI TC -G A M f or an addi ti onal 30 m inutes.A fter w ashing in RPM I-C the cel ls w ere transf erred into m icrotesttubes and analy zed by f l ow cy tof l uorom etry (FA C S-4, B ectonD ick i nson, M ountai n V iew , CA ). Steri l e cel l separati ons w ereperf orm ed on the FA CS-4 w hose f lu id i c system had been steri l izedw i th 70% ethanol and ex tensiv ely ri nsed w i th sal i ne soluti on. T hesam ples used f or the separati ons w ere labeled by indi rect I F w i thazide-f ree steri l e M oA bs. D ouble l abel i ng w as accom pl i shed by f i rstincubating the cel l s w i th the CD 1c-speci f i c M oA b and then w i th theappropri ate di l utions of the FI T C-G A H u and PE-GA M reagents.Cross-reacti v i t i es betw een these tw o reagents or betw een FI T C-G A H u and the m ouse M oA bs w ere not observ ed. T he tw o-colorf l uorescence anal ysi s w as perf orm ed on the FA CS-4 equipped w i than argon ion baser (output pow er, 400 m W ; w avelength, 488 nm ) andw i th a 525-545B P and 600L P set of optical f i l ters f or the sim ubta-neous detecti on of the green and red em issions, respecti vel y.

C e ll c u ltu r es a nd a c tiva tio n . T he cel l s (unf racti onated orsorted) w ere resuspended in R PM I-C and seeded (50,000 cel l s/SOjL /w ebl ) i n round-bottom ed m icroti ter pl ates (Costar, Cam br idge,M A ). T he m i togens w ere added at the tim e of seeding; the f i nalconcentrati ons of T PA and lonom y cin (alw ays used together) w ere Ing/m L and 0.5 g/m L , respecti vely . SA C w as used at 1:15,000(vol /vob). T he cul ture plates w ere kept at 37 { 176} Cor 72 hours in ahum idi f i ed i ncubator contain ing 5% CO 2.

U ltr a str uc tu r a l a na lysis. For thi s study norm al PB M C, tonsi l s,and spleens w ere used. FA C S-puni f i ed CD lc+ B cel l s w ere f i xed in3% gluteral dehy de in 0.1 m ob/L phosphate buf f er, post-f i xed in 1%osm ium tetrox i de, preem bedded in 3% agar, and em bedded inaral di te accordi ng to standard techniques. U ltrathin secti ons w erestai ned w i th al cohol i c uranyb acetate and bead ci trate and v iew edw i th a Phi l i ps 410 T EM . For the im m unogold m ethod, the sam plesw ere processed as prev iousl y descni bed. B ri ef ly , 5 x 10 6 cel l s (Tcel l s and m onocy te depleted) w ere incubated w ith the CD lc-speci f i cM oA b L 161 f or 30 m inutes at room tem perature (R T ). A f ter tw ow ashes w i th phosphate buf f er contai ni ng decom plem ented hum anA B serum , 1% bov ine serum album in and 0.2% sodium azide, pH7.4, the cel l s w ere incubated (30 m inutes at RT ) w i th 20 L of

Table 1 . C D1 E xp ression o n P urified B lo od and S pleen N orm al B and T C ells:E ffects of A ctivation and C orrelation W ith O th er S urface M a rkers

S a mp l e CD1 c CD1a CD1b CD2 CD5 CD19 CD2 2 CD25 I F iB lo od B c ells:

Control 49.1 20.42 0 0

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C oC

B

B c e l l s T c el ls

Co n t CD1 c

C D 2 5 {1 49 } C 02

L Jk JL I

C D 1 c + B C E LL S U B S E T 24 3G A M IgG G 1 5 . A f te r tw o w ashes the sam p les w ere f ix ed an d

processed as d escribed earlie r.

RESULTS \O )

S n i I g C D 1 c ---S m Ig + C D 1 c

Evidence that C D Jc blood and spleen cells are Bcells. S ev eral sam p les , im m unoph eno ty ped o n ce ll su spen -sio n , con tained C D 1c-p ositiv e cells that acco un ted f o r 3 2 in the PB M C , 2 6 .8 8 .06 in the sp leen , and18.57 3 .04 in non neop lastic ly m p h nodes (L N ); thepercen tages o f to tal B ce lls (C D 19+, C D 22+) w ere 9 6 , 43.5 8 .50 , an d 42 .14 8 .87% , respec tiv e ly . T od irec tly dem on strate the B -cell natu re o f the C D 1c + cells,an im m uno pheno ty p ic analy sis w as carried ou t on FA C S -purif ied B ce lls (C D 2- , C D 3-, C D 14-) and T ce lls(C D 19-, C D 22 -, C D I4 -). T he data, lis ted in T ab le I(con tro l row s) , in d icated that b o th b lood and sp leen B ce lls(>95 , C D 19+, C D 2 2+) con tain 49 .1 20 . 42 an d50.9 4 .47 o f the C D 1c-po sitiv e ce lls. C o nv ersely b lo od(and sp leen , n o t show n) T cells (>97 CD 2 + ) w e r eC D 1 c - . B oth B an d T ce lls w ere n eg ativ e f o r C D 1a andC D 1b . T h e C D 1c+ ce lls o f L N h ad also a B -ce ll ph eno ty pe(no t sh ow n). T hese data ind icate th at, accord ing to the C D Iex press io n , B ce lls are C D 1a-, C D lb -, an d C D lc+ o r -,whereasT ce l l sa reCDla - ,CD1b-- ,andCDlc - .

In vitro B- and T-cell activation and C D J m odula-tion. T o de te rm ine the e f f ec ts o f activ ation on the ex p res-s ion o f th e C D 1 m olecu le s , purif ied B and T ce lls w ereph en o ty ped w ith the C D 1 - and o ther lineage - on ac tiv ation -spec if ic M o A bs af te r in v itro ac tiv atio n f o r 72 hou rs. T heresu lts (T ab le 1 , ac tiv atio n row s) sh ow ed that, u pon ac tiv a-tion , the f raction o f b lood and sp leen B ce lls pos itiv e f o rC D 1c in creased (as com pared w ith the co n tro ls) to 64 .0 18.5 an d 71 .7 6.65 , resp ec tiv e ly ; an increased an tigen

dens ity w as also m easu red . In con tras t, T ce lls rem ainedC D 1c- and , lik e B ce lls, C D 1 a-.- an d C D 1b- (Fig I) . T hestatu s o f ac tiv ation o f T (C D 2 +) and B (C D 19 + /C D 22 +)cells w as ind icated b y the reac tiv ity w ith C D 2 S and T F-r

. C o n t {149 }C D 1 C

L : C D 1 a {14 9} C D 1b C D 1a C D 1b

, - C D 2 5 C D 2r,

_ _ _ _ _ _ _ _ _ _ L I - j__ JF i g 1 . IF p r o f i les o f C D 1 C D 2 , an d C D 25 an t ig en e x p r es s io n

o n ac t i v a te d T an d B c e l l s . T h e f lo w c y t o f l u o r im e t r i c an a l y s iss h o w s th a t n e i t h e r C D 1 a n o r C D 1 b is p o s i t i v e o n B a n d T c e l l s ,w h e r e as C D I c is p o s i t i v e o n B c e l l s o n ly . T h e ac t i v a t i o n o f t h e c e l l sis d em o n s t r a te d b y th e s t r o n g e x p r e s s i o n o f C D 2 5 o n b o th T - C D 2 + ) a n d B C D 2 - )-c e l l s u b s e t s . T h e h is t o g r am s w ere o b ta i n e df r o m th e an a ly s is o f 2 .00 0 c e l l s /s am p le ; t h e x - an d y -ax is r e fe r t oth e r e l a t i v e f l u o r es c e n c e in ten s i t y (lo g s c a l e ) a n d c e l l n u m b e r ,r e s p e c t i v e l y .

F lu o re s c e n c e in te n s ity

F i g 2 . T w o -c o l o r a n a ly s is o f s p le en c e l l s d o u b le l ab e le d f o rC D 1c a n d S m Ig (A ) an d C D 1 c m o d u la t io n o n C D 1c - a n d C D 1c +ac t i v a te d f r ac t io n s (B ). T h e d u a l -p a r a m ete r h is t o g r am (A ) w asg e n e r a t ed o n F A C S -4 ; t h e d a ta , p r es en ted a s a t h r e e -d im en s i o n a lp lo t , s h o w th e S m Ig a n d C D 1 c f lu o r es c e n c e s an d t h e c e l l n u m b e r(y -ax i s ). T h e S m Ig - f r a c t io n i s n o t v is ib l e b ec au s e i t h a s b eeng a ted o f f . T h e d o t te d l in e in d i c a t es t h e th r e s h o ld fo r C D 1 c -p o s it ive a n d -n e g a tive flu o re s c e n c e s . T h e tw o i d en ti f i a b le p op ul a -tio n s w e re s o rte d o u t. a c tiv a te d fo r 7 2 h o u r s , a n d r e tes te d f o rC D 1 c ex p r e s s io n . It ca n b e s ee n (B ) th a t t h e o r ig in a l l y C D 1 c f r ac t io n b e c o m e s C D 1 c + w h er e as th e C D 1 c + f r a c t i o n r e ta in s i t sreactivity.

M o A bs . T o d irec tly p rov e a de n ov o ex p ress io n o f C D Ic onthe C D 1 c-negativ e B ce lls, the batte r an d th e C D Ic+ su bse t,iden tif ied by tw o-co lon IF (Fig 2A ) , w ere sorted an d rephe -no ty p ed af te r ac tiv ation . I t w as f oun d (T ab le 2 ) th at b loodand sp leen C D 1 c- B ce lls b ecom e C D Ic+ af te r ac tiv ation(60 .6 6.0 and 52.6 7.5 , respec tiv e ly ) (Fig 2B ).A n upreg u lation o f the C D 1c m o lecu le w as observ ed in theC D Ic+ ac tiv ated subse t. S im ilar data w ere ob tained w iththe B -ce ll m itogen S A C (no t show n). T he au gm en tation o f

T ab le 2 . A c t i v a t io n - In d u c ed C D 1 c M o d u la t io n o n C D 1 c + an dC D 1 c - B C el l S ub se ts

Subse t S a m p le C D 1 c C D 2 C D 5 C D 1 9 /C D 2 2

C D 1 c- B l o o dS p l e e n6 0 . 6 6. 05 2 . 6 7 .5

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244 DELIA ET AL

CD 5 expression (consti tuti vely present on less than 20% of Bcel ls), w as al so observ ed. CD 5 posi ti v i ty w as evenl y div idedbef ore and af ter acti vati on into the CD I c+ and CD 1c-f racti ons (not show n). These data suggest that bl ood andspleen B cel ls contai n f our di sti ncti ve phenoty pes: C D 1c+ /CD 5+ , CD I c+ /C D 5-, CD 1c-/CD 5+ , and C D 1c-/CD5- .

EM Studies The ul trastructunal anal ysis w as pen-f orm ed on norm al bl ood and spleen B cel l s. A dual approachw as f ol l ow ed to identi f y and characteri ze the CD 1c+ cel l s.T he f i rst w as based on an indi rect im m unogold label i ng ofthe B -cel l populati on w i th the CD 1c M oA b; the secondconsi sted in sorti ng the CD 1c+ subset pri or to EM analy si s.Essenti al l y , both approaches bed to i denti cal resul ts (Fi g 3).

b

Fig 3. E M p ho to graph s o f norm al blo od an d to nsil C D 1 c + and C D 1 c - lym p hocytes. A ) P eriph eral lym p hocytes. A n on rosettingP B M C is sho w n; th is cell is C D1 c reactive as ind icated b y the nu m ero us colloid al-go ld particles attach ed o n its surface arrow s). Th epresen ce of cyto plasm ic vesicles em p ty arro w ) app eared to be a p eculiar featu re of th is cell sub set P bU a stain ing ; o rig inal m a gnificationx 1 7.000) . (B ) T onsil lym p hocytes. C D 1 c-po sitive. no nrosetting . no nadh erent ton sil lym p ho cyte ch aracterized b y cyto plasm ic clusters o fvesicles an d g ran ules em p ty arrow ) P bU a; o rig inal m a gnificatio n x 13,000). C ) P erip heral lym p ho cytes. P BM C w ith lym p hop lasm a cytoidfeatures is sh ow n; th is cell is C D1 c-negative P bU a; original m ag nificatio n x 1 5 ,000 ). D ) T on sil lym p ho cytes. F A C S -pu rified . C D 1 c-po sitive to nsil lym p ho cyte. Th is cell has a nu cleus w ith eviden ce o f a nu cleolu s an d cytop lasm rich w ith g ranules and vesicles P bU as ta in in g . o rig in al m a gn ific atio n x 1 7.7 00 ).


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Table 3. C orrelation B etw een C D 1 c an d O th er S urface M a rke rs in H is to lo gica lly D e fin ed B -N H LHistology No. of

WF Ki d Ca s e s CD1 c CD1 a CD1 b CD5 CD1 O CD1 9 CD2 2 CD2 3 CD3 8 K LA CLL 6 2 0 0 4 0 5 5 5 5 2 3 1 3 3 : 1B C b-C cF 3 3* 0 1 1 2 2 /2 3 0 /2 0 /1 2 :1C Cb-C cF 2 0 0 0 0 1 N T 2 1 1 NTD C b-C cF 2 1 0 0 0 2 N T 2 2 2 1:1E Cc 3 2 0 0 3 0 3 3 0 2 1 1 0 : 3F C b-CcD 8 4 0 2 2 1 4 /4 8 1 /4 0 /4 2 :3G C b 12 2 0 0 0 5 8/8 8/9 0/3 2/2 6:2H 1 mb 4 1 0 1 0 3 0 3 1 1 3 3 NT 1 2 1 : 1J Burkitts 6 0 0 0 0 3 6 6 0 NT 3:3The numbers indica te the rea ctive ca ses, immunophenotyped on ce ll suspensions and/or tissue sections. T he neopla stic ce lls of the cases we re a ll

unreac t ive with CD 2 and/or CD 3 T -ce II-specific M oAbs. N onneoplastic LNs pre sented an average of 1 8 .5 CD ic-positive ce lls.Abbre via tions: K /L, and A immunoglobulin light cha ins. NT , not te ste d. A , sma ll lymphocytic; B, follicula r, predom inantly sma ll cleaved ce ll; C ,

follicula r, m ixed sma ll cle aved and large ce ll; D , follicula r, predom inantly la rge ce ll; E , diffuse , sma ll cle aved ce ll; F . diffuse , m ixed sma ll a nd large ce ll; G ,diffuse , la rge ce ll; H , la rge ce ll; immunobla stic; J, sma ll, noncle aved. Kie l cla ssifica tion abbre via tions: C LL, lymphoma of chronic lymphocytic leukemiatype; C c, C entrocytic; C b-C cF , centrobla stic-ce ntrocytic follicula r; C b-C cD , centrobla stic-ce ntrocytic diffuse ; C b, C entrobla stic; 1m b, Im munoblastic.

C ases tw o per histotype or the single one) w ith less than 30 C D 1 c-labeled neoplastic cells. C D 1 b staining w as faint and cytoplasm ic and appearednon-specific.

CD1c+ B C ELL SUBSET 245

T he cy top lasm ic o u tline of the C D 1c-po sitiv e ce lls (F ig 3A ,B , and D ) w as s ligh tly irregu la r bu t never v ilbou s, and th enu c lea r to cy top lasm ic ratio w as re la tive ly h igh . T he nuc lea rch rom atin w as u sua lly condensed a t the periphery , an d aproportion of ce lls con ta ined a sm all n uc leo lu s. In th ecy to p lasm , the G olg i appara tus w as n o t deve loped , b u tc lus te rs o f vesic les and sm all g ran u les w ere presen t; th epresence of th ese o ngane lles w as res tric ted to the C D 1c+frac tion since they w ere absen t on the C D 1 c- B ce lls (F ig3C ). D iffe ren tial p ropo rtio ns o f n onnuc leo la ted v nuc leo -la ted C D 1 c+ B cells w ere foun d in the b lood (95 :5 ), to nsil(6 0 :40), and sp leen (5 :95) . In th is s tu dy , no ha iry ce ll-b ike orlym p hop lasm acy to id ly m phocy tes w ere de tec ted in theC D Ic+ frac tion . In sum m ary , C D 1c app ears to be a m arkerfo r a B -ce ll su bse t charac te rized by a un ique cy top lasm ic se to f ong ane lles tha t en co m passes, accord in g to the absence onp resence o f the nuc leo lus , tw o m atu ra tion s tages.

CD J expr ession on B-NH L and B-CLL : cor r elati on w i thother sur face marker s. F orty -s ix B -N H L (inc lud ing s ixsporad ic B unk itts lym p hom as) and 27 B -C L L patien ts w ereim m u nophen o typed on ce ll su spens ions (3 7 B -N H L and theB -C L L ) and on cryos ta tic tis su e sec tions (22 B -N H L ); 13ly m phom as w ere tested w ith bo th p rocedures and gav es im ila r resu lts. T he B -ce ll o r ig in of the neop las tic ce lls w as,in a ll cases, ind ica ted b y th e reac tiv ity w ith on e on m oreB -cell m arkers , C D 19 , C D 22 , and C D 23; a lack ofthe T ce llm ark ers C D 3 and /o n C D 2; an d expression o f H L A -D Rm olecu les . T h e da ta co ncern ing the lym pho m as are reportedi n T a b l e 3

C D 1c w as pos itive in four o f sev en (57% ) fo llicu lanly m phom as inc lu d ing th ree cases w ith pred om inan tly sm allc leaved cells (W F :B ) and one case w ith p redom inan tly la rgece lls (W F:D ). E leven of 39 (2 8% ) d iffuse lym p hom as w ereC D 1c+ : tw o w ith sm all lym p hocy tes (W F :A ) , tw o w ithsm all c leaved ce lls (W F:E ), fou r w ith m ixed sm all and bargece lls (W F :F ), tw o w ith la rge cells (W F:G ), and on e w ithlarge im m u nob las tic (W F :H ) ce lls . T he low es t num ber ofreactive cases w as o bserv ed am ong the latte r tw o sub group s(16% and 25 respec t ive ly ) .

C D 1a w as alw ays nega tive , w hereas C D 1 b fa in tly reac tedw ith one fo b licu lan an d th ree d iffu se sub types . T h e re la tion -sh ip be tw een C D 1c and o ther re lev an t B -ce ll d if fe ren tia tionm ark ers w as a lso in vestiga ted . O f ten cases pos itive fo r C D 5,one w ith predom in an tly sm all c leav ed ce lls (W F:B ), tw o w ithsm all cleaved ce lls (W F:E ), and tw o w ith sm all an d la rgece lls (W F:F ) coexpressed C D Ic. C D IO (C A L L A ), reportedto be preva len tly reac tive w ith fob licu lan lym pho m as , w asp ositive in five of seven cases be lo ng ing to th is h isto typ e asw ell as in n ine (23% ) d iffuse sub types inc lud ing th reeB u rk itts cases (W F :J). C D IO /C D 1c coexpression w as seenin tw o fo b licu lan cases and one d iffuse case . O f 1 7 cases, e ig h tw ere po sitiv e (five s trong ly ) fo r C D 38; th ree of these w erealso C D 1c+ .

T h e B -C L L sam ples inc luded in th is s tudy w ere C D I9 + ,C D 2-, C D 3- , and excep t fo r tw o , C D 5+ . E leven cases(4 1% ) w ere C D 1c-po sitiv e ; four o f them had bess than 30%labe led ce lls (d ata no t sh ow n ).

D I S C U S S I O NT he d issec tio n of th e C D 1 clus te r d iffe ren tia tion in to th ree

d is tinc t subg roups is based on b iochem ical and sero lo g ica lfind ing s. T he u n ique d iffe ren tia l pa tte rn of ex press ion of th isfam ily of m o lecu les in v ar io us tis sues is fu rthe r ev iden ced inthe presen t study w here it is show n tha t o n ly C D Ic iscons titu tive ly expressed on b loo d , tons il, and sp leen ce lls .M o reo ver, the find ing th at C D 1 c reac ts w ith a B -ce ll frac -tion presen t in lym pho id tissues an d charac te rized by d is tin c -tive u ltras truc tu ra l fea tu res sug gests tha t it is a m arker fo r aB -ce ll su bse t. U ltrastruc tu rab ana lysis h as recen tly a llow edthe iden tification of five b lood B -ce ll subse ts o f inc reasingd eg rees o f m atu rity th a t a re p resen t in d iffe ren t p rop or-tions30 ; in te resting ly , tw o o f th ese , sh ow n to have B -ce llch ron ic lym ph ocy tic leukem ia (B -C L L )-lik e and pno bym ph o-cy tic leuk em ia (P L L )-like featu res , s trong ly resem ble theC D 1c-p ositive b lood and fo llic le B ce lls. H ence , the u ltra -s truc tu ra l and phen o typ ic sim ila rity o f P B an d M Z C D Ic+B cells len ds fu rthe r ev id en ce to suppor t the co ncep t tha t M Z


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R E F E R E N E S

24 6 DE LI E T L

areas of by m phoid ti ssues f orm pant of a pool of reci rcul atingcells.3

T he acqui si t i on of CD lc m olecules by CD 1c- activ ated Bcel l s w as unexpected since the repl i cating or acti vated G C Bcel l s of norm al and reacti ve L N are consistentl y negati ve asshow n by highl y sensi ti ve A PA A P im m unohi stology .9 I n v i voand in v i tro di f f erences in the am pl i tude of the B -cel lacti vati on response could ex plai n thi s f i ndi ng. W e have al sof ound that f our of si x B -CL L cel l s (CD 1c-, CD 5+) becam eCD lc-posi ti ve af ter i n v i tro activ ati on (not show n) .

I n v i ew of these resul ts the expression of CD lc on B -N H Lm ay ei ther ref l ect the anatom ic si te of deri vati on (that i s,M Z ) or an activ ati on status of the tum or. T he f act, how ever,that som e of the C D 1c+ N H L of G C ori gi n w ere negati vew i th C D 38, an activ ati on m ark er reacti ve w i th GC andplasm a cel l s, suggests that the expression of CD I c in thesel ym phom as i s not activ ati on but rather di f f erenti ati onlinked.

I n addi ti on to CD lc, other prev iousl y reported M oA bssuch as L 30,32 K B 61,33 and SN 334 exhibi t the sam e M ZB -cel l reacti v i ty al though each of them recogni zes a uniquem olecule. A l though K B 6I i s unreactiv e w i th the GC -deni vedB -N H L , SN 3 i s, l i k e C D I c, posi ti ve on som e of them . T hesedata w ould im ply that som e B -N H L of GC ori gi n hav e aninterm ediate phenotype of M Z /G C; w hether they ari se f romnew ly arri ved M Z B cel l s i s a m atter of speculation.

I . M cM ichael A i , Pi l ch JR. Gabf re G , M ason D Y , Fabre JW ,M ibstei n C : A hum an thym ocy te anti gen def i ned by a hybri dm y ebom a m onoclonal antibody . Eur i I m m unob 9:205, 1979

2. C otner T , M ashim o H , K ung PC, G obstei n G , Strom inger J:H um an T cel l surf ace antigens beari ng a structural rel ati onship toH L A anti gens. Proc N atl A cad Sci U SA 78:3858, 1981

3. B ernard A , B oum sel l L , H i l l C: I n B ernard A , B oum sel l L ,D ausset i , M ibstei n C, Schbossm an SF (eds): Joi nt R eport of theFirst I nternati onal W orkshop on H um an L eucocy te D i f f erenti ati onA nti gens by the I nv estigators of the Parti ci pati ng L aboratori es i nL eucocy te T yping, vol 9. B erl in, Spri nger-V erl ag, 1984

4. A m iot M , B ernard A , Raynal B , K napp W , D eschibdre,B oum sel l L : H eterogenei ty of the f i rst cl uster of di f f erenti ati on:Character izati on and epi topi c m apping of three C D I m olecules onnorm al hum an thym us cel ls. i I m m unol 136:1752, 1986

5. B oum sel l L , A m iot M , R ay nal B , G ay-B ebi l e V , Cai l bou B ,B ernard A : Epi topic groups of CD I m olecules, i n R einherz EL ,H ay nes B F, N adler L M , B ernstei n I D (eds) . L euk ocy te T yping I I ,vol 2. B erl i n, Spri nger -V erl ag, 1985, p 289

6. C alabi F, M i l stei n C : A novel f am i l y of hum an m ajor histo-com pati bi l i ty com plex -rel ated genes not m apping to chrom osom e 6.Na t ur e 3 23 : 54 0 19 86

7. M arti n L H , C alabi F, M ibstei n C: I sol ati on of CD I genes: Af am i l y of m ajor hi stocom patib ib i ty com plex -rebated di f f erenti ati onanti gens. Proc N atI A cad Sci U SA 83:9154, 1986

8. M urphy G F, B ronstei n B R, K now les R W , B han A K : U l tra-structural docum entati on of M 24l gl ycoprotei n on dendni ti c andendothebial cel l s i n norm al hum an sk in. L ab I nvest 52:264, 1985

9. C attoretti G, B erti E, M ancuso A , D A m ato L , Schi ro R,Sol i go D , D el i a D : CD 1: A M HC class I rel ated f am i ly of anti gensw i th w idespread distr i buti on on resting and acti vated cel ls, i nM cM ichael A J, B ev er ley PC L , Cobbold 5, Crum ptom M i, Gi l ks W ,Gol ch FM , H ogg N , H or ton M , L ing N , M acL ennon 1CM , M ason

O nly a f ew blood B -cel l subset-speci f i c m ark ers hav e beenreported; one of these i s CD 5, w hi ch i s reacti ve w i th 18% ofblood and spleen B cel ls.35 CD 5+ B cel l s are regarded as aseparate B -cel l l i neage that i s im pl icated in autoim m uni ty .36Our study conf i rm s the presence of C D 5-posi ti ve norm al Bcel l s, 40% to 60% of w hi ch coexpress C D I c (D .D ., obsenv a-l i on), and prov ides ev idence f or the ex istence of f our pheno-ty pi cal l y i denti f i able subsets, nam ely CD 1 c - /CD5 CD 1c+ /CD 5-, CD I c-/CD 5+ , and CD I c+/C D 5+ , ofunknow n f uncti onal si gni f i cance.

A l together, thi s study has conclusi vel y show n that CD 1cis, am ong m ature l ym phocy tes, posi tiv e on B cel l s onl y . Sincethis m arker al l ow s the identi f i cati on and i solati on of di screteB cel l subsets, a f urther advance in the study of B -cel lf unction and regulati on m ay now be achieved. CD 1c couldal so have a potenti al appl icati on in the m oni tori ng of B -cel lsubsets f or cl i ni cal evaluation. Final l y , thi s m arker, al thoughunable to unequi vocal ly di scrim inate G C- f rom M Z - deri vedB -cel l l ym phom as, m ay f i nd a place in the classi f i cation oft hese m al i gn an ci es.

ACKNOWLEDGMENTW e w ish to thank D rs P.C.L . B everl y , U CH , L ondon, and F.

M alavasi , I sti tuto di G eneti ca, T ur in, f or the gi f ts of U C H -T1,U CH -T 3, U C H -M I and A lO M oA bs, respecti vel y .

D Y , M ibstein C, Spiegebhal ter D , W abdm ann H (eds): L eukocy teT yping I I I . O x f ord, O x f ord U niv ersi ty Press, 1987, p89

10. Sm al l T N , K now les RW , K eev er C , K ernan N A , Col l i ns N ,O Rei l l y R i , D uPont B , Flom enberg N : M 241 (CD 1) ex pression onB -l ym phocy tes. J I m m unol 1 38:2864, 1987

I I . H su SM , Jaf f e ES: Phenoty pi c expression of B l ym phocy tes1 . I denti f i cati on w i th m onoclonal anti bodies i n norm al l ym phoidt i s s u e s .A m i Pathol 1 14:387, 1984

12. W einberg D 5, A ul t K A , Gur ley M , Pinkus G S: T he hum anl ym ph node germ inal center cel l : Characterizati on and i solati on byusing tw o-color f l ow cy tom etry . i I m m unol 137:1486, 1986

13. Reicher t RA , G al lati n W M , W eissm an A L , B utcher EC:G erm inal center B cel l s back ing hom ing receptors necessary f ornorm al l ym phocy te reci rculati on. i Exp M ed 157:813, 1983

14. G erard-M erchant R, H am bin I , L ennert K , Ri l ke F, Stansf eldA G, V an U nni k iA M : C lassi f i cati on of non H odgk in s by m phom as.L ancet 2:406, 1974

I 5. N ati onal C ancer I nsti tute sponsored study of cl assi f i cati on ofnon-H odgk in bym phom as. Sum m ary and descri pti on of a w ork i ngf orm ulati on f or cl i ni cal usage. Cancer 49:2112, 1982

16. B oum sebl L , B ernard A : H igh ef f i ci ency of B iozzi s hi ghresponder m ouse strai n in the generati on of antibody secreti nghybnidom as. i I m m unol M ethods 138:225, 19801 7. D orken B , M oldenhauer G , Pezzutto A , Schw arz R , K ieseb 5,H unstein W : A naly si s of the B cel l / l eukem ia w orkshop m onoclonalanti bodies using an im m unoenzim ati c stai ni ng assay and a radioim -m unoassay on cel l s, i n Reinherz EL , H aynes B F, N adler L M ,B ernstei n I D (eds) : L eukocy te T yping I I , v ol 2. B erl in, Spri nger-V erl ag, 1985, p47

18. N adler L M : B cel l / l eukem ia panel w orkshop: Sum m ary andcom m ents, i n R einherz EL , H aynes B F, N adler L M , B ernstei n I D(eds): L eukocy te T yping I I , vol 2. B erl i n, Spri nger-V erl ag, 1985,p3


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CD 1c+ B CELL SUBSET 24 7

19. B everley P C L , C allard R E : D istinctive functio nal character-istics of hum an T lym phocytes defined by E rosetting or a m ono-clonal anti T cell antibody. E ur J Im m unob 1 1:329, 1981

20. B everley PC L, C ablard R E: R edefinition of hum an T cells bym o noclonal antibodies. P rotides B iol F luids 29:653, 1981

21. H ogg N M , M acD onald 5, Slusarenko M , B everbey PC L:M onoclonal antibody specific for m o nocytes and other m y eboid cells.I mmu n o l o gy 5 3 : 7 5 3 1 98 4

22. M abavasi F, C aliganis-C appio F, M ilanese C , D ellabona P.R ichiardi P, C arbonara A O : C haracterization of a m urine m ono-cbonab antibody specific for hum an early lym p hohem o poietic cells.H um Im m unob 9:9, 1984

23. R itz i, Pesando iM , N otis-M cC onarty i, Lazarus H ,Schbossm an SF: A m onoclonab antibody to hum an acute lym pho-blastic antigen. N ature 283:583, 1980

24. B rodsky FM , Parham P. B arnastabbe C i, C rum pton M i,B odm er W F: M onoclonab antibodies for analysis ofthe H L A system .Im m unob R ev 47:3, 1979

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Blood-1988-Delia-241-7

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