Functional Characterization of Infiltrati~g T Lymphocytes in Human Hepatic Allografts*

John J. Fung, Adriana Zeevi, Thomas E. Stanl, Jake Demetris, Shunzaburo I watsuki, and Rene J. Duquesnoy

ABSTRAcr: w, b4", nn~ rrmlily dnotltl/H" IIChniqllu in T ~,II (11III1rin, Itl 1111", 1M nllill" lind IlIn(litl" 01 infill,.IIli", Mp6li( ""tlIt'41 T ClIIs. Usin, 1M ,.III;tlnlll,lha, ;nl,.II,,.1I/1 T Clils II" IIetiNltti tlllrin, cJJ _ill"" "" .. ,t Itl lIN II11t1,,.41. '" _ IIb/t 10 SMw lhal IMIt (tlls WOII'" prop6p" "'" ,.,..i" IlIn(I;O_II, II(li", in 1M prrltnct 01 1M T ~tli ,rtIUIlh 111(10,.. 1 L-2. In s"",.J inllll,,",. p •• "pi( II-',siJ 01 "lis ,rown in Ihil "",nn". WIIS M'J si",il",. 10 lhallollntl wilhin lIN ,,,"/1. Bolh pro/il".IIliflt lin" cyloloxi( f'tSponm (011'" IN tk'telttl lro", Iht (III 111m! (til lints. TIN _jori/, 01 lIN prolilt,."';fIt "sponsts _ titJno,..J;rrr,," IInti i",,,,lIno­,mtli( IIIIIII,sis (011'" tkfi", "o".,..Jirrclt" HLA ""tl;,,il]. I. tilhtr c"'ss 1 or (lass II IInl;gms. orbolh. Mon«/o"J "",i-liLA "nliboJiu inhibilion profilu ,,";fit" Ihtllpp6"nl HLA ,.,.tl;"il]. I" " s"",II" jJtt'r,nl"",1 auu. Dnl, 1 L-2 "spons;fltnm (011/4 IN tk,te,,". IInti no HLA ""tl;,,il] CDII'" IN MI".",i",". Cylfluitily COli'" IN Mlteutl II,IIinsl bolh class I· lin" elIISS 11 IInl;gms. howtwr. IhDstttils which _,,,II,.IIlttl II ,,.,11'" "",gn;IIIM ,1 tltJnor.Jirrr,," cy/o/oxicily IIP/HII"" 10 btJirrct,tI "pi"sl class 1 "",i""s. A si,,,ifit,,nl cDrrr"',io" 1N,wtt" titJno,..Jirrrltti pro/;I"'"liDn DI biopsy tlll,""tI 1].ph«yItJ """ C,I/II"',. "jteliDn WIIS IDIIII". This ",tJtklllPJHII,., ID IN IIstllll in ./i"""i", "",eti.", ,IIIN ;",,.11,,.4' T ~,I/ poP""";o" ""rint. "jtel;O".

ABBREVIATIONS 11,2 locerieukin-2 MHC . major hiscocompatibility

HLA PHA

complex human leucocyce antigen phytohemaglutin-M

INTRODUCTION

EBV LCL TCM VEC MoAb

Epstein-Barr virus lymphoblastoid cell line tissue culture medium vascular endothelial cell monoclonal antibodies

Hepatic allograft transplantation has become an accepted form of therapy for treatment of a variety of Ijfe-threatening liver diseases [1-4]. The indicacions for this procedure range from end-stage liver failure, due to a variety of causes, to patients with inborn errors in metabolism and hepatic malignancy. The success of this procedure has been well documented. With the advent of cydosporine immunosuppression. 60-70% ~-yr survival rates are being achieved [1]. Re;ec-

F,.. ,1» 0,,. .. _, .,s-"r,-". ,IN Oil/iii ... 1 Cli"ic'" I •• II".,.,bJ.C!. U.ifIMi".1 Pi1ll611rr.h Sc6.oI.1 MtJKi. _ .. ,1» C.,rIIi 81 ... 8_" • • 1 Pitlsb",."h. Pimbllrr,h. P", •• ,/N"iII.

ItIUms .. /Hi,,' ,.,."",U Ie: )"',,}. FII.,. M.D •• Ph.D .• U"ifIM;', ,1 Pitl.bllrr.h. Scheel .1 M,Jiti,,~. Dqtm_, .1 SII,.""". 406 Sc_il, H_U, 3"0 Ttrr_(f S,rw,. Pi1lS611rr.h, PA 1'261. ·S.,,.,.,.., GNW' Al-21410 _", AI· 18923 In. NIAID. Gr.", M93 IrrnII U,,;,tJ W_, H,_I,b R_rrhSnri(f F.II1111M_.G .. ", AM·29961. _"tI_ 1'-'" IrrnII,lN C_/lt,il;w M#tIiI'" R'ItII .. b FII.tI­RitlMM 1Ci., Mill." F .. IIIIM"".

182 0198-88)918611}. ~

Human Immunoiotly 16, 182-199 (1986) C) EI_~r ScicDC~ Publilhina Co., Inc .• 1986

)2 Van.J"rbilc A~ .• Nrw Yon.. NY 10011

Lymphocytes in Hepatic Allografts 183

..

tion continues to be a major cause of graft dysfunction. in spite of tech'lological and immunosuppressive advances (1,4].

While little is known about the immunobiology of hepatic rejection, several observations point out possible differences from other allograft rejection models. The ability to transplant cadaveric liven in spite of a positive lymphocytotoxicity cross-match and across ABO incompatibilities highlight possible differences in allorecognition [5,6]. Whether this reflects the unique anatomic architecture of the liver, or whether there are differences in the expression of alloantigens. is not known. Several studies have demonsuated disparity of expression of class I and class II MHC antigens on normal liver vasculature when compared to other vascularized organs [7.B].

While most current models of aIlorecognition and subsequent rejection assign T lymphocytes a central role [9]. little is known regarding the function of these cells or their contribution to the severity of graft rejection. Immunohistochemical staining of organ transplant tissues with monospecific cell surface marker anti­bodies has given conflicting data on the CD4 and CDB marken l of infiltrating T cells [11-14]. These studies have other limitations: (i) inability to correlate cell surface markers with functional characteristics of the ce[s in question. and (ii) presence of "irrelevant" mononuclear cell infiltrates in the absence of clinical rejection within the allograft (15.16). Several models have therefore been ad­vanced to study the functional characteristics of infiltrating graft cells and their role in rejection. In vitro functional assays of enzymarically isolated lymphocytes from rejected organs have demonstrated allospecificity (17]. The sponge-allograft model has been employed to study the kinetics of graft infiltration (lB.19J. Recent advances in T-cell culture technology have enabled the propagation and expansion of activated T cells from allograft biopsies. Kim et aI. have shown that cloned noncytotoxic T -cell lines from mouse skin allografts could mediate rejection when reinjected into naive animals (20). Both Moreau et aI. (21). and Mayer et al. [22]. have described isolation of functionally active allospecific human T -cells lines propagated from either percutaneous biopsies or rejected renal grafts. We have recently described the allospecificity of T cells grown from serial enda­myoc!Udiai biopsies from heart allograft recipients. and demonsttated both class I and class II HLA recognition (23).

We are interested in undentanding the mechanisms of allorecognition and hc:patic rejection. Because immunologic monitoring of peripheral blood has 1;­itUions in these patients (24), we have routinely obtained liver core biopsies during an episode of hepatic allograft dysfunction for histologic confirmaticn of cellular infiltration. Utilizing T-cell culture techniques. we report the functional character~zation of expanded T cells from these biopsies.

MATERIALS AND METHODS SDllrn """mal. Samples of hepatic allografts were obtained from clinical material taken from percutaneous liver biopsies. intraoperative liver biopsies. or allograft hepatectomies. The patient profile is shown in Table 1. All transplant recipients were placed on post-operative intravenous cyc1osporine A and steroids, as main­tenance immunosuppression. Indicanons for sampling were derangements in liver function tests and bile composition via T-tube drainage from the allograft [2H

IThe nomenclarure CD3. CD4. and CDS refer (0 n. T4. and TB. respecrivel7. IoCcordil18 ro rhe 1984 ~po" by the Commirwc on Hwnaa Leucocyre DifTerenriuion Anri&enl: IUISIWHO No­mendaru~ Subcommiuee (10).

; I

1&6 J. J. fU118 et at

TABLEJ Patient and allograft profile

Parienc . " l..ivn number A&e Sex Primary d;'posis biopsy

I 2 3 <4 ~ 6 7 8

9

10 11 12 13

1<4

19 M <4<4 f ~ f 27 M <42 N 7 F

<48 f <42 N

36 f

17 N 17 F 15 N <43 f

9 N

cIuoaic bepIIids ~ bepIIic arciDaaIa Carob .. , Diteae ~ biIiIry aaaia ....., biIiuy cirrhosis poll bepecicic cirrhosia

ar .... - cirrboaia

WiIIoD', Diteae h',..... ......... ICIeroaiaI cboIenpris primary biIiuy cirrbosia

AIIIiIIea Syadrome

l.78 2.16A 3.32A U2A 5.3008 6.73A 7.~A

8.9A 8.BA 9.9A 9.118 9.398 10.~9B II.M 12.3A B.~A 13.288 14.3A

DO

DO

DO

DO

DO

DO

DO

DO

yes

AU material was caken in • sterile manner for propagation of infiltrating cells and for histologic evaluation.

.... His"". Samples seat for histolog were sectioned and stained with (i) he­matOXylin and eosin, (ii) recicuIin, aocl in several instances (iii) immunohisto­chemical stains,~. ana-T ceD.1Ilrl-8 cell. and ana·DR. These slides were then evalualed in a blind manner by one of us (J.D.) using previously defined criteria for liver rejectioa [25-28].

Plltul ails. Lymphocytes were obc:ained either by mechanical disruption of donor spleens, obcained during ocpo procurement, or from periphenl blood' from normal healthy adult volunteers. Cells were isolated by centtifuption over • FacoD-Hypaque sradienc (sp gr 1.077) (Picoll.Paque, Phannacia. Pisc:auway, NJ) and washed sevcnl times with Hank's Balanced Salt Solution (BacBlSC01T, FISIceviIle, R1). Vaability was determined with trypan blue and the cell aliquou were frozen in 20% hwnan AD serum aod 20% dimethyl sulfoxide. and stOred in liquid nirrogea. EBV -umsformed lymphoblasroid ceD lines (LCL) were pre­pared from doGor spleaoc:yrp usiaa Epstein-Barr virus and cyc1osporine A [29], PHA-uansformcd lymphoblast culaues were prepared by incubating lympho­cytes with phytobemaglutin-M (PHA) (DIFCO. Decroit, MI) for" days prior to use.

Gtllmlli .. '11~bot,k tltiums tm-1iM- u.pks. Percutaneous aod iocnoper­acne core liver biopsies were divided into sevenI smaller fnsmeau, senenIly 4-12 in nUIDbu. AUosnfc hep.lectamy samples were similarly seccioDed bur die yield ·wu .ubaaaa.lly grearer aocl • coal of 96 ffl&lllelltl were proceued. for purposes of chis paper. aIl1iver samples wiD be designaced as ""biopsy." AU fnpleGtI were individually pLaced into 100 1'1 tissue culture medium (TCM) supplemented with 100 '" recombiNnt 1L-2 (Sandoz Pharnulceuticals, Basel) (final dilution 1:10,000) in 96-weD microculture plates (COSTAll. Cambridge,

"'1:f;~.r,l:,. ~l.~~>.;'". .. r_

.'

Lymphocytes in Hepatic Allografts 185

MA). TCM consisted of RPMI-I640 (B&B/SCOm. supplemented with 10% normal human AB serum, 4 mM L-gluwnine, 24 mM HEPES buffer, and 60 p.g/ml of gentamycin sulfate (GIBCO, Chagrin Falls, OH). .

Liver sample cultures were incubated at 37°C in a humidified 5% CO2 atmo­sphere. After 3-4 days culture, supernatants were replenished with fresh TCM supplemented with recombinant 11.-2. The cultures were observed daily on an inverted stage microscope for cellular outgrowth. When growth was observed, the contents of the wells were pooled and transferred into 24 well culture plates (COSTAR) and again supplemented with IL-2 and TCM. After approximately 2 weeks, sufficient cells were obtained for functional and phenotypic studies as well as for further propagation.

PrimtJ Iymphocytt Itslin, (PLT). The PLT activity of lymphocyte cultures grown from liver samples was measured in 3-day proliferation assays, as previously described for alloreactive T-cell clones [30,3 I]. Prior to testing, the lymphocyte cultures had not received IL-2 for 3 days. This was necessary to reduce back­ground proliferation of these cultured cells. In each PLT assay, 5 x 103 cultured cells were incubated for 72 hI' with 10% AB serum, IL-2, or 5 X 1<r irradiated stimulators (2000R) in 96-well round bottom microculture plates with TCM in a final volume of 200 pJ. During the final 20 hI' of incubation, each culture was pulsed with llLCi of3H-thymidine (specific activity, 20 mCilmmol, New England Nuclear Products, Boston, MA). The cultures were harvested with a multiple sample harvester (Sutron, Inc, Sterling, V A) and uptake determined by liquid scintillation counting (LKB, Gaithersburg, MD).

PLT inhibilion with monoclonlll anliboJies (MoA.b). Inhibition of proliferation of • • bulk cultured biopsy T cells to irradiated donor lymphocytes, by monoclonal

anci-class I and anti-class II antibodies, was accomplished according to previously described methods [32]. Briefly, the same number of scimulator and responder cells as for PLT, in 100 pJ TCM, were coincubated with 100 pJ of various MoAbs. The following MoAbs, with their corresponding HLA molecular specificity, were used: SG 15 7 (anti-DR) and SG465 (broad anti-class II) (5. Goyen and J. Silver [33]); L243 (anti-DR) and Leu 10 (anci-DQwl + w3) (Becton Dickinson, Moun­tain View, CA (34-36]); and PA2.6 (anti-HLA-A,B,C) (P. Parham (37,38]). The PLT activity was measured in a 72-hr assay as described above.

Ctll meJialtJ cyloloxicity (CML). The CML activity of lymphocyte cultures grown from liver samples was measured in 4-hr 'ICr release assays, according to previous descriptions for alloreactive T-cell clones (31] with slight modifications as noted. Briefly, targets were 4-day PHA stimulated spleen cells (class I targets) or EBV­transformed spleen cells (class I and II targets) from the donor or other HLA­typed panel cells (LCL). An effecror:wget ratio of 10: 1 was used to measure release of labeled chromium from 2 x 103 labeled target cells. A measured aliquot of supernatant was mixed with a high-d'6ciency aqueous compatible scin­tillation counting mixture (READY-SOLV HPIb, Beckman Instruments, Fuller­ton, CA) and coueued in a liquid scintillarion counter. The percentage of CML expressed was calculared from a formula which represents a ratio of experimental to total <determined by lriton-X release) "Cr release (subtracting spontaneous release) [32].

CtU sur/au phmoIJPi( "",,/ysis. Lymphocytes grown from liver samples were tested for various T -<ell differentiation antigens using a modification of the avidin-biotin­immunoperoxidase technique (39]. The following differentiation markers were

..

186 J. J. Fung et aI.

. analyzed: CD3 (pan-T cell), CD4 ("helper/inducer T cell"), and COS ("cyto­toxidsuppressor T cell") (OKT series purchased from Orrho Diagnostics, Rantan, N); Leu series purchased from Becton Dickinson. Sunnyvale. CAl. In addition. anti-DR staining was similarly done using a monoclonal anti-HLA-DR (Becton Dickinson). Briefly, cytocentrifuge smears were prepared. using 2.5 X 1 ()4 ceIWslide, and fixed in acetone immediately after preparation. A total of 200-400 cells were enumerated.

HLA phmotyping. Peripheral blood lymphocytes, donor spleen cells, and Iym­phoblastoid cell lines (LCL) were typed for HLA A, and B antigens by the standard NIH microlymphocytotoxicity technique. Serologic typing for HLA DR was done by prolonged incubation microlymphocytotoxicity test using enriched B-cell preparations obtained following carbonyl-iron treatment and Ficoll-H ypaque sed­imentation after rosetting with neuraminidase treated sheep red cells.

Statistical analysis. Cultured lymphocytes were defined as reactive towards a given stimulator cell in proliferation assays when the tOtal incorporated sH_ thymidine COunt ± 2 SO was greater than background counts (i.e., counts with cultured lymphocytes with 10% AB serum alone plus irradiated stimulator cells with 10% AB serum alone) ± 2 SO. Analysis of statistical significance was done using the Chi square test.

RESULTS

Patient/Sample Profile

Lymphocytes were grown from 18 biopsies obtained from 14 liver transplant patients. Table 1 summarizes the salient features of these patients including age, sex, primary diagnosis at the time of transplantation, the post-transplant day of the biopsy, and the current status of the allograft. In this paper, each liver biopsy (LB) culture is referred to by a unique number which identifies the patient and the post-transplant day when the biopsy was obtained. In addition, the letters A and B designate the first and second allografts, respectively. For example, LB 1. 7 B was obtained from patient 1 on day 7 post-transplant from the second allograft ~d LB2.16A was obtained from patient 2 on day 16 post-transplant from the first liver transplant. This representative population of transplant patients in­cluded patients with end-stage liver failure due to a variety of causes as well as patients with inborn errors of metabolism and hepatic malignancy (Table 1). Biopsies were obtained from 11 first transplants and five second transplants, including ~o patients (9 and 13), who provided specimens from both first and second allografts. The interval from transplantation to biopsy ranged from 3 days to 330 days, with a median of 14 days post-transplant.

Generation and Expansion of Amvated T Lymphocytes from Liver Biopsies

Biopsies obtained from hepatic allografts were incubated in viero, in the presence of recombinant human 11.,2, but without the addition of irradiated feeder cells. After 2-3 days, an outgrowth of mononuclear cells was seen (FIgUre 1). The cultureS were supplemented at 2-3 day intervals with 11.,2 and transferred into larger wells. Expansion was continued until confluence was obtained. generally within 10-14 days following initial biopsy. Sufficient number of cells (5 x 101-1 X 10') were then obtained for functional assays and phenotypic analysis.

Lymphocytes in Hepatic Allografts 187

FIGURE 1 In vitro propagation of lymphocytes from a liver biopsy culture. This is a 400 X magnification <original magnification) taken with an inverted stage microscope 4 days after expansion in tissue culture medium supplemented with IL-2. The dark area in the left corner is the liver biopsy tissue.

Phenotype analysis of biopsy-grown mononu<:lear cells showed a predomi­nance of CD3 positive T lymphocytes. many of which also expressed DR antigens (an indication of activation [40]). Biopsy cultured lymphocytes were typed phe­notypically berween 2 and 4 weeks following sampling. Table 2 lists representative results which also demonstrate a mixture of CD4 and CDS positive cells in most cultures. Among seven lymphocyte cultures tested, there seemed to be an overall trend towards CD4 predominance. although statistical significance could not be established, possibly because of small sample size. In rwo patients (S and 10), who had undergone a percutaneous liver biopsy, subsequent allograft hepatec­tomy was required for unremitting rejection shortly thereafter. Cells from these rejected livers, extracted by mechanical disruption followed by Ficoll-Hypaque purification and then directly analyzed, showed very similar phenotype profiles co cells grown from the biopsies (Table 2).

Allospecificity of Infiltrating Allograft T Cells in Secondary Proliferation

The alloreacavity of biopsy-grown lymphocytes was determined in secondary proliferation assays (PLT). Table 3 shows the proliferative responses of biopsy­cultured lymphocytes to irradiated spleen cells from the transplant donor and also to exogenous IL-2 in 3-day assays.

All cultured lymphocytes exhibited low spontaneous thymidine incorporation and high proliferative responses to IL-2 suggesting the presence of activated T cells with receptors for 11-2 (Table 3). The majority of expanded lymphocytes also showed strong proliferative responses to original donor lymphocytes. How­ever, five biopsy-grown lymphocyte cultures expressed little or no PLT reactivity towards donor cells (LB2.16A, LB4.12A, LBl1.3A, LB12.3A, and LB14.30A>. When the phenotypes of these cultured T cells were correlated with donor­specific proliferation, no correlation berween a predominance of CDS bearing cells ("suppressor/cytotoxic" subset) and a lack of proliferation could be found.

We next compared the cause of graft dysfunction with the ability of the cultured lymphocytes to proliferate against donor cells. A statistically significant correla-

188

.. ..

...

, i

TABLE 2 Phenotypic analysis of lymphocytes

: . ..;,

Puieol Surface Liver nwnbel' marker biops,.

8 C03 86 CD4 37 CDS 6~ Oil NO

10 C03 94 CD4 3~

CDS 60 Oil ~8

2 C03 94 CD4 62 CDS 2" Oil 6~

C03 93 CD4 ~8

CDS 27 Dil 4~

1" C03 86 CD4 66 CDS 9 Oil 6~

~, bulk CIIIauoed ceIIL 'Bulk ftII'8CIIeCI cells '- .... p.u_, .

J. J. Fuag et al.

91 31 57 NO

NO NO NO NO

NO NO NO NO

NO NO NO NO

tion of low donor induced proliferarion with ischemic hepatic allograft dysfunc­tion was noted, usina the Chi square teSt for 2 X 2 coatigency abies. p < 0.01. Of the five cultures with little PLT reactivity. ischemic iajUI'J was the cause of dysfuactioa in duee.lo me remainder of cultures that demoastnted ItI'ODI donor stimulated proliferation, rejectioa was the cause of snft dysfuoctioo ia all but ODe.

Biopsy-growa lymphocyteS were also teSted for PLT specificity qaiast a panel of uarelaced cells selected co sbare liLA antigens with the oripaal ttaasplant donor. 10 mao, iascaaca. it was possible to determioe the allospecificicy of these aDs. Table" I1IIIUIWiza the appueat PLT specificity for those cultured lym­phocyte lines that proliferaled spiDle donor HLA specificities. Three biopsies hid PLT specilicities limited to class I antigens, seven were class II specific. wbereu three biopsies appeared to concaia cells that recopize both class J and II donor an ..... Ilepraeawive examples of resula of the PLT aaaI,sis on biopsy-growa lymphocytes from three par.ieoa are described below.

P.,iftI; 3. Pacieae 3. who was HLA typed A2",26; B3S,w41; DIU.S; received. lMruansplaatfromadooorwbo typed asA~l,28; B3S,w60; Dllw6,7. On post­tnasplaat day 32, the paieat developed elevaced liver enzymes while uluasound sNdies indicated aormal aUosnft architecture. The hiltolosy of. percutaneOUS

/'

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± 9

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972

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- ~ /'

-_ ... _ .•. _-----

liver biopsy showed subintimal lymphocytic infiltrates in the portal triads con­sistent with mild rejection.

.. The PLT specificity of LB3.32A was associated with DRw6 and DR7 antigens; all six positive stimuJaror ceDs bore either anti&en specificity whereas all of the (our neprive ceDs lacked these class II antigens (Fagure 2).

FIGUllE 2 PLT specificiry of lB3.32A. Tritiated thymidine uptake was determined in ~y proliferation usay •. HLA ..... of the panel cells are listed, chose shared with the original donor are unclerliocd. The beckpouad counl wilh 10% human AB serum was 2097 :: 1608 (1 SD).

3 H-THYMIOINE UPTAKE (<PM)

0 "000 '0,000 11,000 A 8 DR

2,- U.- !.! '.2 7.'" 4.!

'.- 8.17 !.-3.24 7.31 8.7

~ 2.21 t3. , • I.! ... ::z: 2.3 7.82 ' .•

".23 44.11 1.4

2.3 44,82 4.-

1.- !!-.... 2.1

2.28 '''.35 2."

Lymphocytes in Hepatic AllograftS 191

Patient 8. Patient S, was HLA Iyped as A24,25; BS.w62; DR1,4 and had received a liver from a donor with HLA antigens A3.24; B1S.3S; DRS,-. The patient developed elevated liver funaion tests 9 days post-transplant. A percutaneous liver biopsy showed a lymphocytic portal infiltration pattern consistent with moderate to severe rejection. Lymphocytes grown from biopsy LBS.9A showed PLT specificity associated with HLA B1S and B3S, both class I antigens. Seven out of eight positive stimulator cells had either the B1S or B35 antigen while only one of seven negative stimulator cells bore either antigen. In addition, only three of seven stimulator cells cyped as DRS were positive in this PLT assay, indicating weak reactivity to this specificity (Table S).

In spite of aggressive immunosuppression the patient had continued clinical deterioration requiring allograft hepateaomy followed by retransplantation 4 days later. Fragments of the first donor liver (LBS.13A) were incubated under conditions similar to the initial biopsy and the expanded cells were tested .pnst the same panel of cells. Histolosically, the portal infiltrate appeared less extensive but a residium of lymphocyteS and macrophages was noted, consistent with re­solving rejeaion. As shown in Table 5. specificities to which these cells reacted towards were B1S and DRS. AU four B1S positive stimulator cells and all seven DR5 positive panel cells stimulated T cells expanded from this biopsy, whereas five of five B IS and DR5 Deprive panel cells failed to do so. Thus it appears that within the interim. acquisition of DR5 reactivity appeared.

Pa,i,,,, 9. Patient 9. typed as Al,-; B3S,w62; DR5,w6; received a first allograft from an A26.11; B1S.w41; DR2,5 donor. Following initial satisfactory function. elevation of the serum bilirubin 5 days post-transplant necessitated a percutaneous liver biopsy for diagnosis. Histologically. there was no portal infiltrate and no

- Jymphocytes could be grown from the biopsy. Four days later, however. because of increasing liver dysfunction, an allograft hepateaomy was performed. At this time. histologically there was moderate portal tract infiltrates with biliary epi-

TABLE 5 P.LT reactivity of liver biopsies from patient #S

HLA cype 1B 8.9A 1B 8.BA

It. 'A 8 B DR DR (CPM) (CPM)

3 24 18 3) ) 4,00S- 27,n8 29 24 44 3) ) 7 8,022 1&!!

2 31 18 60 4 1,220 2),821 1 2 18 27 4 6 ZA2! 7,n8 2 )0 3) 7 4,600 NO

26 30 49 3) 6 8 z..m NO 26 11 18 41 ) 2 4,879 14,011

2 3 62 3) ) 720 l.llil 2 8 44 ) 3 1.034 4,649

1 30 13 )1 ) 2 123 8.0)0 3 26 14 )1 ) 1 387 6,640 2 3 44 62 4 402 1.))3

11 24 60 2 14) 2,003 2 11 7 44 2 7 3)) 3,008

31 13 60 4 7 NO 1.267 2 24 7 49 2 4 NO 809

'POlitRoe "int....,. are .....xrli .... CRt: __ III....,.. {Of aIcuIMionl'.

/

..

192 J. J. fung et aI.

thelial damage and subendothelial collection of lymphocytes. Immunohistochem­ical staining showed T-cell infiltration with both CD4 and CD8 positive cells. In addition, there was expression of DR antigens on biliary and vascular epithelium, consistent with rejection {26]. Cells grown from this sample (LB9.9A) demon­strated marked proliferation in three of four B 18 positive panel cells. No other donor specificity could be detected in all ten B 18 negative cells which shared other donor-specific antigens (Figure 3).

The second donor was typed as A2,24; B7,49: DR2,4. Again, after initial function, deterioration of liver function occurred. A percutaneous liver biopsy (LB9.11 B) was done 11 days after the second transplant revealed severe choles­tasis with moderate portal lymphocyte infiltration and evidence of bile duct injury. T cells were expanded from this biopsy and as shown in Table 6; allospecificity was determined to be against DR4 and possibly B7. Nine of the nine stimulator cells bearing these antigens induced proliferation of the biopsy cultured T cells in PLT. None of the five DR4 or 87 negative cells induced proliferation in this cell line. We found only slight PLT reactivity ofLB9.11B cells towards cells from the first transplant donor (donor A).

Finally. after 39 days following the second transplant, in spite of~igorous efforts to reverse rejection. another allograft hepatectomy with retransplantation was required for iotractible rejection. as verified by histology. Cells grown from this sample (LB9.39B) were tested in PLT and revealed a similar pattern to cells obtained 4 weeks earlier. although a broader reactivinr pattern was noted from the latter liver sample (Table 6).

Monoclonal Antibody Inhibition Profiles in PL T

A panel of defined monoclonal anti-HLA antibodies were used to inhibit prolif­eration of cultured biopsy lymphocytes towards irradiated donor stimulator cells. As shown in Table 7, the alIoreactivity of the biopsy-cultured cells, as determined by immunogenetic: analysis, was verified by blocking experiments w~th these MoAbs. Additiooof the class I specific MoAb. P A2.6, to cells grown from LB 1. 78

fIGURE 3 PLT specificity of LB9.9A. Legend is the same as for Fisure 2. The back­ground count was 222 :t 23 (1 SO).

3H-THVMlDINE UPTAKE <CPM)

A 8 0

OR 5.000 10.000 15.000 20.000

21.11 !!.~ !.! 2.31 !!.eo ".-1.2 !!.27 ....

2 ... 2. ..... 35 !.7 3.~ 1".51 1.5 2.!.! 7 ..... 2.7

< 3.24 1!.35 5.-..J X !l.24 2".eo 2.-

2.3 ..... e2 2.-1.30 13.51 ~! 2.2" 7 .... !." 2,3 e2,35 !,.-1.2 e ..... 3.5

31.- 13.eO ".7

,/ ./

1'- --

Lymphocytes in Hepatic AUografts 193

TABLE 6 PLT reactivity of liver biopsies from patient #9 (second graft)

HLA cypc ,- -,-LB9.l1B LB 9.39B

A A B 8 Oil DR (CPM) (CPM)

~ 24- 7- 49" :l- 4- ~ NO 25 13 44 4 ~ Qli

2 18 27 6 4 ~ 28.705

2 44 60 5 4 48.165 NO 1 58 55 7 4 ~ NO 2 3 35 5 4 ~ ~

29 31 7 60 7 4 12.,78 21.070 2 29 7 8 4 NO ~ 2 11 7 44 2 7 1'.116 n.!iQ 2 1 7 62 3 7 8.414 lJ2l 2 28 7 44 2 4 NO 1.537 3 24 7 35 6 7 NO 756 2 24 35 62 6 7 NO ~ 2 11 44 51 2 8 NO ~

26' 11' 18' 41' r 5' 1M2 21.(Ml 2 57 6 7 4.185 NO 1 8 57 7 3.405 NO 2 3 62 35 5 2.756 NO

24 29 44 35 5 7 2.000 NO 3 24 18 35 5 4.620 NO

"Se<oad doacw.

'F"anr doacw. Bcx'h inc -' I«ODcI c'- poIiIift ICiaIuIMon _ -'cdiDecI.

(with PLT specificity towards class I donor antigens) resulted in. 51.7% reduction of.dooor-stimulated proliferation. In conuut. none of the class II specific MoAbs sigoificandy inhibited LBl.7B proliferation.

The fine degree of disc:rimioarion of these MoAbs was also evident in the : inhibition profiles of LB7.5A and LB13.28B, both of which demonstrated class

IlrelCtivity by PLT analysis. Cultured ceUs from both biopsies were not inhibited by P A2.6, while sipificant inhibition of prolifenrion was observed with the class II specific MoAhs.

TABLE 7 Monoclonal antibody inhibition profiles of the cultured cells

~

ClaM I CIua II

Biopsy ceUs PAl.6 SGl57 SG465 1.243 Leu 10

LBl.7B 51.7 NO -10.2 6 -6 LB7.5A 8.3 64.1 62.9 33 42.2 LBB.28B -9.3 74.5 43.3 49.1 18.5

..

194 J. J. Fung et aI.

Donor-Specific T-Cell Mediated Cytotoxicity

In several instances, CML assays were used to assess the ability of..expanded biopsy-cultured T cells to lyse donor targets. Using PHA transformed T lympho­blasts and/or E8V-transformed 8 Iymphoblastoid cell lines (derived from original donor spleen cells) as targets, cytotoxcity was determined at a 10: 1 effector/target ratio. As shown in Table 4, several T-cell lines demonstrated significant cytO­toxicity towards donor specificities. In general, those lines showing significant cytotoxicity in this assay correlated to class I antigen induced proliferation in PLT assays.

In patient M.S., LB 13.288, in vitro expanded intragraft T cells demonstrated DR3 allospecificiry in PLT assay. The recipient was HLA typed as Al,24; B35,w55; 0R2.5 while the donor typed as Al.2; B51.8; OR3,7. CML assay showed 21% cytolysis of mY-transformed donor cells which correlated with CML specificity against the DR3 antigen. There was a much lower level of lympholysis against the PHA-transformed donor lymphoblast line (8%) and a significant lympholysis (32%) against another OR3 positive lymphoblastoid cell line (Figure 4). There­fore, while the general rule that class I antigens serve as targets for cell mediated lympholysis, class II antigens can also serve as recognition structures for cell­mediated lympholysis [41,42].

DISCUSSION

We describe methodologies based upon our previous' experiences allowing ex­pansion and characterization of intragraft T-cell populations in hepatic allografts [23]. The rationale behind our approach is based on acquisition of lL-2 receptors

FIGURE" CML specificity of 1.8 13.28B. Percent Iympholysis is determined by [he formula:

If}£ I hi' (experimental - spontaneous) release 7l'} ymp 0 YSIS - • x 100% .

(Tnron-X total - spontaneous) release

HLA amigens of the panel cells arc listed; those shared with the original donor are underlined. Lymphocytes incubarcd with PHA-M for" days prior to testing arc designated as PHA targets. EBV fargees are splenocytes that 'Were originally transformed with EBV.

% CYTOLYSIS

A 0 10 20 30 40 50

B DR

PHA !.~ ~.! :!.!

EBV !.~ !!.!! ~.l

PHA 3.23 !.44 2.5

* 4( EBV ...I 1 - !.35 ~.5 :x: -'

PHA 1.- !.35 ~.5

EBV 23.11 44.51 1.4

EBV 2.11 7.44 2.7

..

, ,

Lymphocytes in Hepatic Allografts 195

during activation [43], and the subsequent requirement ofIL-2 for further prop": agarion {44]. In order to minimize introducing new antigen specificiti~ we used only lecrio-free 11..-2 without feedcrceUs to isolate and expand "the' in61trating cell popularion. In two insances, these ceDs exhibited the same phenotypic makeup of cells obtained in situ.

When the phenotypic makeup of these biopsy bulk cultured ccDs were com­pared to me aUospeciDcity of these cells, there did not appear to be·. consistent pattern lelating the predominance of either C04 or CDS to me HLA class 1 or class II specificity. This may reflect two possibilicies, (i) that CD4 and CD8 positive ceDs are not restricted by HLA class I or class II antigens or (ii) that the cultuled T-cell line lepresena • mixture of ceDs and only • small subpopularion of the expanded ceDs are responsible for aUospecificicy. Generally CD4 aUoractive cells recognize class II anti&ens whereas CDS .uotaetive ceDs rec:ognize class I an­tigens; however, exceptions have recendy been reponed [45-47]. On the other hand, we have preliminary daa., usina limitina dilution analysis of these ceDs, that democmrares dill • proportion of aaivMed T ceDs do nor proliferate to donor HLA antigens. This sugesa dill only • subpopulation of the toW biopsy­cultuled ceDs recognize HLA determinantS.

Kumick and co-workers. usina. similar approach, have reccndy reponed their results chanctaizina apaoded TeeDs from rejectina renal aUosraft from patients on azathioprine and prednisone immunosuppression (22]. Phenotypic analysis of their ceU lines showed • predominance of cells bearing the CDS phenotype, although they also noced that in one patient treated with cyciosporine, that the CD4 and CDS populations were almost equal. The differences in the immuno­suppression regimens may indeed explain the more frequent expression of CD4 populations in our series, and has been noted in other human allograft models [26,48,49].

We have shown, by immUQOSeDCtic analysis. that both class I and class II HLA determinants can sene as recopition anrisens- In several instances, diese PLT findings were vcri6cd by monoclonal anti-HLA antibody inhibicioo profiles. In our preYious studies with aIIoreaaive T -cen clones (32], we have shown that both prolifenrive and cytotoXicicy assays can define allospecificicy to either class 1 or class II antisens and COrlelaled well with monoclonal anti-HLA antibody inhibition profiles. Pmliferaciw: responses to class 1 antigens. where the donor and recipient are DR identical, were noted by Mayer et a1. {22]. In addition, we

; plesent evideoce sugesrlna chat the same HLA antigenic determinant can serve as the recopition suuaure for both proliferarion and cyrolysis. This has been confirmed in Yiuo MIll senenred cloaed T-ceU lines (43].

Analysis of in sim senenred alIospecific: T -ceD lines from the initial and lub­sequent allosnfa during rejection has shown that allorecopirion and infiltration of T ~eDs is specific: and dynamic ill D&CUre. We have shown dw thele may be acquisiaoa of additioaal an ... lpeci6cicies duriD8 unsuccessful ueaanent of rejection. This is in apeemeDC with the obiervaUons on acquisition and loss of donor speci6c:ities in our preWxas repGm ltudyina hem aUosnfts {23]. Follow­ins heparectOaly of die initial snfc containina cells with specificity towards the fint doaor. subsequear aUoarafa appear to have an influx of cells bearing spec­ificity towards the donor antiaens of that respective sraft. Lare in lejection, however, nonspecific: mecb'nisml may aana "irrelevant" ceDs (7].

It is interatina to speculate on the role and ancisen specificicy of culture expanded incngraft T ceDs that do DOl: proliferate to donor HLA antisens nor lyse appropriare targets bearina donor HLA antiaens. 'The finding that non-HLA alloantigens, e.g., tissuMpedfic (50] and vacuIat endcxhelial cell or VEe [50-~2] antigens, can panicipae in allograft rejection, has been detDOnsttated. Unlike

/ /

..

196 J. J. Fung e[ al.

the HLA system, definition of the majority of these non-HLA have been elusive, so that their imponance in allograft rejection is not clearcut. Nevertheless, once appropriate donor cell preparations can be obtained bearing the antigen system in question, application of the techniques described in this paper can begin to shed light on their relative role in allorecognition.

ACKNOW1..EDGMENTS

The authors would lie to thank the PittSburgh Transplant Foundation for their invaluable aid in donor sample procuremcar. We also would like to thank Christina Kaufman, Loretta DiVecchi&. Daniel Graziano, EUeo HamiD. and David Bockstoce for their expert technical help. We thank the following individuals for their generous gifts of monoclonal anu-MHC antibodies. Drs. Sanna Goyen.Jack Silver (Cornell University. New York, NY). and Peter Parham <Stanford University. Palo Alto. CAl.

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....

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LymphocyteS in Hepuic AlIogra(tS 199

..

50. SteilUDuIIer D: T'assue-spcci6c and tissue-resuicted histoCOmpaibiliEy aad&ens. Im­muaol Today 5:234. 1984.

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HIM IIIiMtI i. /mOl: Since submissioo of the manuscript, we have become aware of further evidence to subscaoDale our fiodinp. Dr. James Kumick and co­workers (Boston. NA) have similarly found a preponderance of CD4 positive cells in biopsy srown T cells from liver tnIISplaot puieou 00 cyclosporine (as compared to a preponderance of CDS cells from renal mnsplaot patienu 00 azuhioprille) •

"