Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells.

IAO,

JL

Immunogenenci 16 135-142, 1982 LMUHUilO—

genetics

(_, Springet-Verlag 1982

Differential Recognition of the

Serologically Defined HLA-A2 Antigen

by Allogeneic Cytotoxic Τ Cells

I. Population Studies

Satoshi Horai

, Jan J. van der Poel

*, and Eis Goulmy

1 Department of Anthropology, Faculty of Science, The University of Tokyo, Tokyo, Japan 2 Department of Immunohaematology and Blood Bank, University Hospital, 2333 AA Leiden, the

Netherlands

Abstract. Human alloimmune cytotoxic Τ cells, sensitized selectively against the

HLA-A2 antigen, were tested on a panel of selected target cells. Five HLA-A2

positive outlier cells could be ldentified. These outlier cells were only weakly

lysed by HLA-A2 specific CTLs, although they were serologically

indistinguish-able from the other HLA-A2 positive, strongly lysed target cells. Furthermore, it

was found that the outlier cells were poor cold target inhibitors in contrast to the

other HLA-A2 positive target cells, which showed adequate Inhibition of specific

lysis of HLA-A2 positive target cells. Population studies indicate that the

frequency of such HLA-A2 outlier cells may be approximately 10%.

Introduction

It is generally accepted that gene products of the major histocompatibility complex

(MHC) play an important role in cell mediated lympholysis (CML) both in the

induction phase and m the recognition phase of allogeneic cytotoxic Τ lymphocytes

(CTLs). The antigens recognized by allogeneic CTLs were initially shown to be the

serologically defined HLA-A, -B and -C antigens (Miggiano et al. 1972, Eijsvoogel et

al. 1976, Grunnet et al. 1976), but also the HLA-D region products were proven to

be target antigens (Mawas et al. 1975, Albrechtsen et al. 1979, Feighery and Stastny

1979, Johnsen 1980). Apart from the serologically defined antigens, CTLs were

shown to recognize other determinants (Kristensen et al. 1974, Schapira and

Jeannet 1974, Willumsen and Heron 1974, Sondel et al. 1975). One group of

determinants recognized by allogeneic CTLs comprises the splits or subtypes of

serologically defined antigens (Goulmy et al. 1976, Long et al. 1976, Bradley et al.

1978, Schendel et al. 1978, Kato et al. 1982) for which there is not always as yet a

University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, the Netherlands

serologically dcfincd counterpart Biddison and co-workers (1980a) described an HLA-A2 vananU which by virus immune CTLs and an HLA-A2restnctcd anti-H-Y CTL (Goulmy et al 1982), was shown lo be rccognized differently from most other IILA-A2 positive cells Anothcr possiblc HLA-A2 vanant,detected by an HLA-A2 reslricted anti-H-Y CTL, was reccntly described by Pfeffer and Thorsby (1982)

Wc icport here the rcsults of a systematic study on the occurrence of serologically HLA-A2 positive cells, which are not or only minimally lysed by alloimmune HLA-A2 specific CTLs Approximately 10% of a randomly selected panel of HLA-A2 positive individuals could be identified as couther cells Hirthermore, the outlier cells can be divided into different subscts

Materials and Methods

Ctff dt»wrs CLII donors wcre scleeted from our files of HLA-A B, C and DR lyped, healthy blood dotiors Selecuon was eilher perfoimcd randomly or according to Η LA phenotypes in order to obUun C Π s diiCLted agamsl (he Serologie illy defined Η LA A2 anttgen

CML itihnujut CML was performed acc-ording lo the Luropean Standard techniqucff uiopean CML-gioup rcporl III 1980) In btief mduecr eulturcs (1 e slandard mixed lympliocyte oultures) were estiblishcti for 6 days foilowed by CML lesling (4 h) at four different CfL diluiions against 104 s lC r

labelcd PilA-itimiilatu! (3 days) lymphoblasts

Cold larcftl Inhibition 1 bt CML Inhibition capauly of seleclcd cells was tested by addition of non-^'Ci labcSed (cold) PH Α sümulated ceJls to the specifib combinalion (c g effector ABX agamsl 51Cr labcled

target cctli, U) Α nxed numbt-r of cold targets {105} was added to 104 hot largets at diffetenl CTL/hoV

target CLII latios Control values wcre esl iblislied by idding cold competitorb autologous cither to the responder or the stimul itor eclis

Caliulation of nsults Cytotoxiuty wa calculatcd for each CTL/larget ratio a.ccordmglo the formula (expcrnncntal hpontancous) cpm

— - χ [0Ü = percent relctse (maximitm spontaneous) cpm

The LXpLnmLiilal resuKs fioni diffurLni expctimenls wcre normalized to a peretnt relative cytoloxic response (perccnl RCR) b jsed on tlie 'pecific icsponsc for a given CTL and calciil ited by the foimula

percenl teleasc of cxpenmcntal target

- - - χ 100 = pei cent RCR pcreuil rue \st of sptufic largcl

In all (.xpcnniLiiis described liic pcrcuilRCR wascalculated based on the peicentreleiscobbtivcd Μ Α C Π/luget latio of40 I

Ahsoipiion of ULA Λ2 antisua 1ILA-A2 specifie anliscra were abiorbed using 2 χ 10ri cells per ml of

serum for 10 min at loom temperature UnslimuJated penpheral blood lymphocy'cs Pt-IA stimuidVcd lymphoeyles tnd Γ pstem Bari viru^ transformed cefls were used for absorplions The absorbed antiscra wtre iested on setecteci cells in the National Institutes of Uuilth lechnique

Results

IILA-A2 specific CTLsfatl tolysesomeseroloifitally definedHLA-A2positive tarqet i W/s

Ι η the expenments that initiated Uns study, lt was noted that the percent lysis of an HLA-A2 specific CTL against three HLA-A2 positive target cells was marginal

Differential recogmtion of Η LA A2 Π7 compared with the percent lysis against the specific target cells and most other HLA-A2 positive target cells Since this phenomenon was reproducible, an expanded panel of HLA-A2 positive target cells was subsequently studied using a number of allogeneic CTLs

Four different CTLs were generated against the HLA-A2 antigen between unrelated individuals The HLA-A, -B, -C and -DR genotypes of (he responder and stimulator cells us,ed and the percent lysis of each CTL against autologous and specific target are hsted in Table 1 Cytotoxicity of the CTLs against the corresponding specific target cells langed from 51-78% lysis at CTL to targel ratio 40 1 Marginal cytotoxicity rangmg from 0-4% lysis was observed against the corresponding autologous targets

The results of the panel study in which 58 HLA-A2 positive and 28 HLA-A2 negative target cells have been tested are presented in Figure 1 Α clearcut bimodal distnbution of positive and negative targets was observed As expected, all CTLs lysed most of the HLA-A2 positive target cells strongly (60% RCR or more), while the HLA-A2 negative targels, wilh only a few exceptions, had RCR of 20% or less The three target cells mentioned dbove (designated LV1, LV2 and LV3) were recognized by all HI A-A2 specific CTLs tested, ι e , as outlier cells their RCR was well below 60% Furlhermore.two additional outlier cells (designated LV4 and LV5) were identified, which were consistently lysed less efficiently by two of the CTLs (ι e, CTL1 and 2) However, LV4 and LV5 were not recognized as outlier cells by CTL3 and 4

Inhibition of lysis by outlier cold compelitor cells

The HLA-A2 outlier cells were also tested as cold competitors for cytotoxicity against "normal" HLA-A2 51Cr-labeled target cells As shown for one HLA-A2 specific CTL in Figure 2, none of the outlier cells was capable of inhibiting the specific lysis In this respect they behaved the same as cold competitors, which were

Table 1 HLA genotypes of responder stimulator combinations and percent CML agunst lutologous and specific larget cells

Lffcctor cells CTLI CTL24 CTL3 + CTL4* Responder cel A3 All AI AI AI A2S AI A25 Bw35 B5 B8 Bw44 B8 B44 B8 B44 ls Cw4 Cw5 Cw5 DRI DR4 DRI DRw6 DRI DR3 DRI DR3 Stimulator cells A2 A2 AI A2 AI A2 AI A2 B5 B5 B8 B44 B8 B44 B8 B44 Cw2 Cw4 Cw5 Cw5 DR4 DR7 DR3 DR 3 DR3 DR3 DR3 DR4 Percent lysis* Autologous1 4 0 2 3 Specific 78 67 51 56 * Percent lysis at effector to target n ü o 40 1

Titigei cells from rapondtr ceJl aonor (autologous) and sMmul itor feil donor (specific) CTL2 and 3 were generated against the same stimulator cell white CTL1 and 4 were the same responder cell directed against two different stimulator cells

% RCR UO -ι

r

CTL 1

Fiy 1 1'ua.nt i t l t t i v c cytoloxic rcsponses of III Λ Λ2 spccific C I Ls Opcn urtJcs icpicsuil 1 ILA Λ2 positive t uy-t cclls Closcd urclcs repix seilt HLA A2 ne^ilivc Eny-t cells ü i t oullicr

!l[ Α A2 posihve l u g e t teils I VI t V5 ire nu m

beied 1 ^ 1 4 ind 5 resptxlivcly IILA pheno types of I V] to LV5 I VI IV2 A I Bw6 Λ2 Bw6 Λ2 _ Λ2 Bw6 A2 Iiwfi Λ2 Cw6 Aw29 -Λ26 Cw2 A I Cw4 Aw32 Cwfi I!K Cw7 B7 1)27 Cw6 1!« Bw44 Bw50 DR1 Bw58 DRwf, i m DRw6 Bw15 OR2 Bw50 D R I DR 7 Bw4 -Bw4 DRw9 -„ Bw4 DR7

HLA-A2 negative or (is the autologous cells On the other hand, all strongly lysed

HLA-A2 positive target cells tested werc dble to block specific lysis although the amounl of blocking showcd some vanation

SirolüLjKai cvaluation

Serologitdl analysis using alloimmunc serd with proven specificity for the 1 ILA A2 antigen Wds ptrformcd to confirm the presence of the IILA A2 dntigen on the fivc outlier cells diid on ι andomly chosen strongly lysed target celL As expected, all

cells carncd the serologic.illy defined HLA-A2 specificily As shown in Table 2 positivity in CML and the presence of the serological HLA A2 antigcn werc highly associatcd for all four CTLs (p<000()l by chi-squarc analysis with Ydtes correction)

Diflciential rccogmüon of Η LA Λ2

% Ly 100-,

Fig 2 Cold Lirget mhtbtlton oi<m HLA A2 sptcific CTL CTL 1 wjs testcd on spctific Simulator 1 irgcl cctls (targcl no 7) Cold targct 1 2, 3 4, md 5 conespond with the ouUier cells I VI LV5 wlule cold larget 6 ind 8 irc two normal Η LA A2 positive Uu get cells The perctnl lysis» wilhout adtliiion of coid target ceHs> is indiuitcd by — Η LA phutoiypcs ce)l 6 A2 Awil Iiw"i8 Bw44 Bw4 Cw2 DR2 DRw6 HI Α phenolypes ccll 8 Λ2 A3 Bw62 -Bw6 Cwi OR1 DRw6

lal>lc 2 Correhlion strology and CML IILAA2* CML* CI Ll 2 51 5 28 CML' CTL3 4

* The presence ofthe scrologically defined HLA A2 antigen was lested with the aHoanliscia VR46"U6 and VR49484, whioh arc used as lyping sera in our deparlmcnt

1 Susteptibihty of targU cclls lo lysis by CTLs duected againbi the HLA-A2 untigen

Two sera werc absorbed with three ofthe ouüicr cells and three conti ol cells The

resuhs m Table 3 show Ihat all HLA-A2 positive cells dh>oibed the anti-HLA-A2 activity One absorption was sufficient to remove the anli-HLA-A2 activity

140

1 ablc 3 Absorption of Η LA Λ2 anlist-r ι by outlier <md control eells

Absorbin^ uA\* NüllL 1 VI LV2 I V ! Contro) 1' Control 2 Conlrol 3 Serum VR46M6 - 4-Serum V11494841

4-* fhe ibsorbm^ cells shown in thts t\ble were EBV tr insibrmed cells However no differences in ibsorbm^, c ip tuty were observed when imstimulated lymphocytes or PH Α stimuiated lymphocytes

were used for ibsorplion

I lictiler oilheseium before tbsorplion wis 1 16 Noresiduilreactivity was observed after absorpüon

wilb HLA Λ2 positive cetls

1 Re ictivity of tlic sera w is tcslcd back on the five outher cclls tnd five r indomly chosen olher Η LA A2

positive cclls

1 be HLA phenolypes of oontrol cells arc Ι Λ2 BIS Hwf) Cwi DR4 2 A2 Uwl6 Bw4 DRw6 1 A9 B7 Bw4 DR2

completely No differences in absorbing Ciipacity was observed between un stimulated penpheral blood lymphocytes PHA stimulated lymphocytes, and EBV transformed cells

Discussion

In this study evidence is presented that CTLs directed against the serologically defmed HLA A2 antigen lysed the majonty of HLA A2 positive target ceüs

stiongly Howevt-r in a System itic study compnsing a panel of 58 HLA A2 positive t irget cells five outlier cells were identified which were lysed consistently with Iow efficicney All oullier cells were positive for the HLA A2 antigen as analyzed with well defined alloimmune antisera specific for the A2 antigen When the outliei cells were tested as cold competnors, cytotoxicity against the specific target was hardly blocked lfatall AllotherHLA A2 positive target cells could block the specific lysis Since the CTLs were highly specific for the serologically defmed HLA A2 antigen Ihc Mmplest Interpretation is that the target antigen recognized by the cytotoxic Τ

celUistheHLA A2 antigen ltself Ourdata would then indicate thal around 10y of the seiologically defined HLA A2 antigens are vanant or subtype

Α diffeience was observed in cytotoxic capacity between CTLs 1 and 2 as compared with CTLs 1 and 4 The latter CTLs lysed the outlier cells LV4 and LV5 slrongly (RCR above 60/) To clanfy these differences in cytotoxic capacity scveral possibihties can be considered First cytotoxic responses in bulk eultures aie polyclonal thus several cytotoxic determinants might be recogm?ed by clones pi esent in the bulk population of CTLs tested Since responder and stimulator cells

Differential recognition of HLA A2 141

share HLA-Al, B8 and -B12 antigens, cytotoxic determinants associated with HLA B8 and -B12 (as descnbed by Christiansen et al 1981 and Kato et al 1982) may play a role Interestingly, CTL2, which was generated against the same stimulator cell as CTL3, apparently did not recognize those extra specificities

Second, CTL3 and CTL4 recognize diffeient epitopes of the HLA-A2 antigen as cytotoxic determinant, thereby deßmng a heterogeneity within a heterogeneity Prehminary results show that CTLs directed against the HLA-A2 antigens of the outlier cells indeed subdivide HLA-A2 into at least three bubtypes (J J van der Poel, J Pool and Ε Goulmy, manuscnpt in preparation) Biddison and co-workers

(1980b) documented chemical differences in the heavy polypeptide chain of an HLA-A2 vanant that was detected by virus immune CTL Based on these observations, biochemical studies are in progress to document a molecular basis for the differential recognition of the HLA-A2 antigen using allogeneic CTLs as reagents

Population studies of CTLs between genotypically (Goulmy et al 1976) or phenotypically HLA identical individuals (Schapira and Jeannet 1974, Robinson et al 1978, Kato et al 1982) have demonstrated the existence of cytotoxic de-terminants that are sei ologically indistmguishable Recently, Biddison and co-workers (1981) reported that virus immune CTLs recognized different epitopes of HLA-A 3 antigens in conjunction with different types of mfluenza virus Since five independent HLA A2 outher cells could be documented m this study, the same could hold tiue for the outlier cells in an mfluenza virus restncted System

The present study supportf> the notion that the polymorphism of HLA gene products IS greater than anticipated The picture that arises for a number of HLA gene products resembles the complexity of target determinants descnbed in mouse

Π-2-Κ mutants (Mehef et al 1980, Sherman 1981)

Finally, lt seems of importance to document the influence of the Variation of MHC antigens which is serologically indistmguishable but CTL distinguishable, on the occurrence of kidney graft rejection This may document the bioiogical relevance of vanations in MHC antigens in transplantation biology and infectious diseases, e g viral infections

A<kiu>wlitlqmiiil\ We thank Jos Pool ind Ria Caslelli Visser for then excellent techmcal is>ista«ee ind Ms Γ Η Noordcnjk for piepiring the minuscnpt This work wis in pi t supported by the Dutch Ortini/jtion for Health Reseirch (TNO) the Dutch toundation for Medicil Reseir h (FUNQO) which is subsidi/ed by the Dutch Orgamzation for Ihe Advancement of Pure Research (ZWO) and the J Α Cohen Institute for Radiopathology and Radiation Protection (IRS)

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