T-lymphocytes cloning from rejected kidney. II. Recognition repertoire of alloreactive T-cell clones.

0022 1767/88/14112-4187S02 00/0

Tut JOURNAL ol· IMMUNOLOGY

Copyright © 1988 by The American Association of Immunologibts

Vol 141 4187-4195 No 12 December 15 1988

Prmted in U S Α

Τ LYMPHOCYTE CLONING FROM REJECTED HUMAN KIDNEY ALLOGRAFT

Recognition Repertoire of Alloreactive Τ Cell Clones

1

MARC BONNEVILLE,

2

* JEAN FRAN£OIS MOREAU,* ELS BLOKLAND,

+

JOS POOL,*

JEAN PAUL MOISAN,* ELS GOULMY,* AND JEAN PAUL SOULILLOU»

From the *lnstitut National de la Sante et de la Recherche Medicale (INSERM), Umte 211, U.E.R. de Medecine, Nantes, 44035

Cedex, France; and 'Department of Immunohaematology and Blood Bank, Unwersity Hospital, Leiden, The Netherlands

We analyzed the recognition repertoire of 16

hu-man alloreactive Τ cell clones (ATLC) derived from

cells invading an irreversibly rejected kidney

allo-graft. These clones, which specifically proliferated

against the kidney donor Β lymphoblastoid cell line,

feil into two classes: CD4

+

killers and CD8

+

killers.

Cytotoxic and proliferative activities of the ATLC

were studied by using a panel of allogeneic cells

Sharing HLA specificities with kidney donor cells.

Moreover, mAb recognizing monomorphic parts of

HLA class I and class II molecules were used in

blocking experiments of ATLC cytotoxicity. The

re-sults obtained from proliferative and cytotoxic

as-says were concordant. All ATLC investigated were

directed against HLA molecules, and some clones

were found to recognize HLA-B, -C, -DP, -DQ, or -DR

products. All anti-HLA class I ATLC were CD8

+

,

whereas both CD4

+

and CD8

+

ATLC were committed

against HLA class II specificities. Nine of 16 ATLC

were shown to react against serologically defined

donor HLA determinants. These data indicate the

recognition of HLA determinants in the course of

an in vivo alloimmune response and particularly

emphasize the role of HLA-C and DP loci products

so far ignored in clinical transplantation.

Cellular mechanisms involved in the allograft rejection

process remain highly controversial. Although many cell

types have been implicated, it is still unclear which

pre-dominate and how they act. Τ lymphocytes play a major

role in the development of both mflammatory and cellular

immune responses leading to rejection of an allograft (1,

2). One way to analyze the cellular responses occurring

during this process is to investigate the phenotypic and

functional characteristics of cells invading a rejected

allograft. Early studies with classic markers for lymphoid

subsets (3) and later studies with mAb (4) have

demon-strated convincingly that most of the infiltrating

mono-nuclear cells are Τ lymphocytes. Although the majority

of these graft-invading cells have been shown to exhibit

Received for publication July 7 1988

Accepted for publication August 2, 1988

The costs of publication of this article were defrayed in part by the

payment of page charges This article must therefore be hereby marked

advertisement tn accordance with 18 U S C Section 1734 solely to

tndi-tate this fact

1

This work was in part supported by the Dutch Organization for the

Advancement of Pure Research (Z W 0)

2

Address all correspondence and reprint requests to Dr Μ Bonneville

INSERM U211, Laboratoire d'Immunologie Cltnique, Faculte de Medecine,

1 rue G Veil, 44035 Nantes Cedex, France

a CTL phenotype (e.g., CD4

3

negative, CD8 positive) (4),

both alloimmune helper and cytotoxic Τ lymphocytes

could be evidenced in functional studies (5-7). Several

investigators have developed techniques for culturing Τ

cells from various human allografts (liver, heart, lung,

kidney) in order to delineate functions of intragraft Τ cell

populations during rejection. These studies indicate that

Τ cell responses were mainly directed against HLA class

I and/or class II molecules borne by donor cells (8, 9).

However, in the absence of knowledge about the

frequen-cies of such graft-infiltrating cells and clonal analysis, it

has not been possible so far to assess the level of

anti-HLA response and the specificity.

We previously described a limiting dilution technique

allowing the cloning and expansion of mechanically

har-vested kidney graft-invading lymphocytes (10). High

clon-ing efficiencies (10% of graft-invadclon-ing cells) were

achieved when donor Ag were presented on a kidney

donor Β lymphoblastoid cell line (D-BLCL) (11). Fifty-five

ATLC could be studiea functionally and phenotypically.

We showed that all of these CD2\ CD3

+

, CD4

+

, or CD8

+

ATLC strongly proliferated in the presence of the

irradi-ated D-BLCL and that about 50% of the former CD4

+

ATLC were cytotoxic. None of these clones recognized

recipient BLCL or showed activity against the NK

cell-sensitive K562 cell line (11).

We studied the recognition repertoire c* IG of 55 ATLC

by analyzing their proliferative and cytotoxic activities

against a panel of allogeneic cells covering the HLA

spec-ificities borne by kidney donor cells and the blocking of

these activities by mAb directed against HLA products.

The data suggest that all of the mismatched MHC loci

products can elicit a Τ cell response in the course of an

allograft rejection.

MATERIALS AND METHOL 3

Clinical Status qf the recipient The following case has been

described elsewhere (10, 11) Bnefly, a 46-year-old woman (HLA

typing A2, A3, B7, B51, Cw7, DR2, DRwl4, DRw52; DQwl) with

end-stage renai failure caused by a chronic intersütial nephropathy

received a cadavenc renal allograft (HLA A2, All; B18, B55; Cw3,

Cw7, DRw8, DRwll, DRw52, Dgw3) on December 28, 1983 She

had been pregnant frve times and had received 21 blood

transfu-sions Before grafting, her serum contained antibodies against HLA

A10, B12, B15, and DR7; no autoantibodies were observed. The

cross-match was negative, with donor Τ lymphocytes and positive

with donor Β lymphocytes as determined with currently available

J

Abbreviations used in this paper CD, cluster of differentlation,

D-BLCL, kidney donor-denved Β lymphoblastoid cell line, ATLC, alloreactive

Τ lymphocyte clone, BLCL, Β lymphoblastoid cell line, CML, cell-mediated

[ympholysis, PLT, primed lymphocyte typing, AF, ascitic fiuid

4188 Γ LYMPHOCYfL·, CLONING FROM RLJFC TED HUMAN KIDNCY ALI OGRAFT sora She w i s trt itfd witb tyelosporln Α immedlatefy aftcr grafting

ts prcvfously d< scribed (12| Her urine ouLpul remained bdow 1 liier/24 h and hemodtalysis eould not bc Inten upted after Irans plint ili<Jii Histologie il ( xirnlnaUon showcd extensive cellul sr Infil trallon ( hi kldney was removedon March 27 1984 and Symptoms ol acutt rtjtt tlon supenmposed upon chronic lesion were ( onfirmcd

C ι II liarui bting limlttnq ditution assays and c lonal expanslon

Πκ ni( (find uscd (o obi ιίη grdft inyadingceifs ind thf whole clonlnf* pn i < (im t h ivc bt eil dt si ribi-d previously in det lil (10 II) Hrlel ly f>[ iil inv idlnfi < ι Ils wen plated in RPMI 1640 supplemtnlcd with 10/ h u r n i n s t r u m ιί dcnsilies ranging from 5 to 160 cells/wcttin the limiünf, dilution assay in the presence of 10 irradlitcd (10 000 rad} I> F1I Cl and purt rIL 2 (0 94 ΠΜ) kindty provided by Bloßen (Gentvi Swit/frlind} Once growth frtquencies were takulated et Ils w( re seedtd it 1 teil/will and monoclonal (,ell eultures (p > 97 n) wt rt furlhtr txpanded In II 2 and h u n n n serum (10T v/v) suppk mi nltd nudlum wiüi wtekly addltion of irradi ited stimul itor t d l s I um Uon tl md phtnolypit ch iracterislies of Α ΓΙ C t hosen lor μ mi I st ι idies wert tkfimd by tee hniquts dest nbed e irtier (II)

In Ulis sludy Ifa < loncs weit an ily/td and (heir ph( notvpcs in rtporltd in 1 ible VII

Allaqcm U <,tirmilator/tarqci (.(Ils Phe ΠΙ Α t y p i n ^ (t i r r k t l oul in f hc Deprfrlme iiL of linmunoh lematology ind Blood Hank I (iden rht Ntthirlmd&)of the penpht ral PBL PHA blists md BI CI uscd is stirnnl Uor/largd cells Is given in Table 1 PIIA biistb were ob l tlncdby tulturinglUI f o n l least r> days in the prestmeof 1% (v/ v) I HA Ρ (I)if(O Detroit MI) i n d ι uiture medium Hl Ml 1640+ 10 PCS) (. clis wi κ Ihoroughly w islu d b( fore UM In order lo uile oul k< tin indiirid ι ytotoxk ity Hl C I wtre derived as prtvirusly d( sirlbed (11)

( ( ft Η» dia(< ci ι ijiotaxt Utfawtijs,« MI) A4 h trr(h ist iss i>

(10) w is usid lo mt !surt Ihc < yloloxk < ipa< ity o! tlu ( It n< s In 0 ich cxptrlnitnt Iwo L/Γ ralios were lested In duptit ite Ktstilts arc txpr< ssed (i(h( ι as pt r< ent ige ol spec ifk C r rrk isi or is 1 U / 10 crlls [ffr iphu determinatinnj Spontane ous releise ol tln t ir^el (t lis used nevt r exe (tdeti 2^% of m iximum π k ist indlvidu il wells wert siorcd p>sltiv< whtn the (onnts exettded th( mein (f tht spont im ons C r rcleise ν ikie plus 3 SD

Pjo/i/t ml Ion tissnijs (Pl I) On chy 7 ifler tlu I ist iddili n >1 irr idt Utd donor BLCI clontd ttlls wt re I lytxid ov< r t Ι κ oll Hv ρ lque (ushlon (to remevt dtad (il!&) ind w ishcd Iwlet in 1ΪΡΜΙ lb40 C eil pcllets were rtsuspendetl md idjusled to a densltj ol 10 cilis/ml in II 2 loniaining (ulturt medium (0 94 UM) Irndl ilccJ (5000 rid lor Hl C I md 2000 rid for PB1 /PHA bl ists) stinuil itoi cdls (5 10 cclK/ml) w< rc addtd to 0 I ml of rtspondlnt, r< Ils In triplk i(t in round bottom wclls with 0 1 mi of tufturc imdiuni Cdls wert Inrubikd it 37"C W C O j in <i humidifled itmo&phtrc dui ing

i d iys Prolifcr ition w is assessed by tritialed Ihymldint ine οι p o n

Hon (0 0 ϊ ml ol Ϊ 1/100 dilution of tritlatcd thymidiiK sto< k solulion [IHK 61 20 to ΐθ< i/mmol spttific activity Amcrsham UK]] AlUr in I K h p u l s t teils wt rt harvesttd onto flbciglass disks (Sk itn η Osl< Norw ly) put In 2 ml of semtillation fluld (OC S Amt rsh im) uifl t< iinf ι d in ι btta lujuid •>( intillation countcr 1 osltlvt md ικ (,

iliv( κ KUOJJS weit defined )>y mr m s of Ibi «lusUr m ilytis pr >

gi im dtsirlbed by (airoll et ü (1 ΐ) When rcsulls oblilmd with r>to(oxklty md prolift r ilion lssiys were dlscord in( tht prolikri tlon issriv was rt pi Hid In Ihese instanets teils wert a l k u i d lo prollftriti lon^t r (4 days Insleid ol 3 diys) Rcsulls ir< txprtsscd

is per<< nlafJt. of rel Ü)VL response (RR) in (omparlson to D BLCI ι pm cxp( rimental

- (c pm rcspnnütr ± cpm stjmu) ite r) tpm with donor BI CI

- (t pm respondtr ± rpm I) BI C I ) ftlctklnq of CMI with anti HI Α mAb I h c f o l k w i r i f i m t i I I I Α mAijwtri used in Uu blo< king txpLrlmcnts W6/ Ϊ2 (In AP) (14) ind 41 II 61 1 (in hybridomi supt rnatint) (linmunotu h Marbeille I r i m t ) diref ted agiinst monoinorphk HI Α (iass I strudures wt rt kfndly jjrovidcd by I)r Ollvt (Marseille Γι im e) iill/Q (in ΑΓ) rtt o^ni/<s ι nu noniorphic ipilopeshiredby DR ind DI mt kt uk s (1 5} ΠΜ50 (In ΛΙ·) (lb) Dl 12 (17) ind I 241 (1H) ut rt ittivt with ι DR o^ni/<s ι nu noniorphic ipilopeshiredby DR ind DI mt kt uk s (1 5} ΠΜ50 (In ΛΙ·) (lb) Dl 12 (17) ind I 241 (1H) ut rt ittivt with ι DR b u kbone strudure leulO (19) md 1Λ5 (brth in AF·) rtto^ni/t DQwl w i and nionornorphk DQ tpilopts rcsptdivtiy [17 21 (20) md 1LI5 (21) reeo/;nf/< mononiorphit DI tpitopts mAb 1A1 nd IL15 wlneh h ivc not yd b(<n dtstribid bdongtothi paneloi mti < Uss II mAb utlli/ed in (he 10 Internationil Histotomp Ulbll ty Workshop (Nt.w York NY) ind were obt liiud from the orffani/ing lommittee All mti Ηί Λ < 1 iss !l inAb tx( t ρί 1Λ3 md i I I 5 w< re kindly providtd by Di D Ch trron (Paris I rante) In Initial exp« r( mtnfs tytotoxkity w i s issiytd m Üu presentt of mAb il Ihrtt r o n t t n t r a t k n s (1/500 1/100 md 1/50 fln il dllufions of mAb in AI md 1/400 1/100 ind 1/20 lin d dilutions of mAb in hybndom ι

supernat int) whk h iliowed assessment of the mAb concentration fiivinii opllmil blockinfi of tytotoxicity (i c 1/100 and 1/20 final dilution of mAb in ΑΓ and hybrtdorni supernatant respectlvely) Siniil irly imong thrce F/ I ratlos (20 1 5 1 and 1 1) we chose the first ritio whk h g we the best discrimlnative results in blocking experiments In subsequent experiments the blocking effects of mAb were studitd in tnplitate at Ihese optimal mAb ronctntraüons

ind h / l n i l o s «nly The ptncntißc of Inhibition wts raJruJated

Ktordinii t(> l h < ' iHnwlii^ formula /o Inhibition = (/ spcdlk "• Cr u Itase In tht pre st n< t of mAb)/( / spedfk ^ Cr release without mAb) x 100

Analysts of ATI C repertoire by CML assays Fourteen

of the 16 cytotoxic clones (11) were studied for their lytic activlty against a selected panei of Cr labeled allogeneic PHA blasts and BLCI (listed in fable I) sharing one or more HI Α speeificities with the kidney donor cells Each spe< ifk ity was presented on male ab well as female panel cells Among the Α Π C tesied varlous recognition pat terns were observed (Table II) Some clones clearly rec ogni/ed target cells sharing one well defined HLA class I speufU ity with D BLCL (e g Cw3 for clone 2C5) How ever at thls step of analysis most of them efffciently lyscdtheDR8 and/or DR1 Γ BLCL tested but no precise t haratterl/atfcm of the HLA specificity recognUea could bo made becaust targets gcnerally shared both DR and DQ allt les {in strong linkage disequilibrium with DR) with donor cells Rec ognition of allogeneic cells by some ATLC (2? 7 1D9 1C7 1?2 and 1D7) could not bc correlated with the preseiue of one sero defined HLA allele For fnstantc JD9 retogni/ed (wo of three DR8 cell Jines (BLCL 15 and D BLCI 20 but not BLCL 16) 1C7 1F2 and 1D7 killcd both DR8 DQ blank positive and DR1 1 DQw3 positive targel cells except target cell 8 Moreover c lonc 2Γ7 retogni/cd Α wide ränge of allogeneic eil tar #ets sharing either HLA class 1 or class II speeificities with D BI Ci Despite (he fact that 2C7 clearly lysed D BLCI this clone did not recogni/e any of the panel cells tested I lnally except for 1D9 no differences in ATI C rtactivity weie noted between maie and female target cellb

Τ LYMPHOCYTE CLONING FROM REJECTED HUMAN KIDNEY ALLOGRAFT

₄₁₈₉

HI Α phcnotype qf male (Ml andjei

ΤΛΒΙ L 1

ale (Γ) panel epiis nstd in cijtotoxü and pioliferatiue QSi.i

No 1 2 2 3 4 5 6 7 8 9 10 1 ) 1 2 1? 14 15 )6 17 18 19 20 21 2 2 2 3 24 2 5 26 2 7 2 8 2 9 3 0 i l 32 Ht Λ sp lutnU tipwi Μ F F· Μ Ε· Μ F Μ F Μ F Μ F Μ Γ Μ F Μ Ι Ι Μ Μ Ι Μ Μ F y Ι h Μ h Ρ

l·

cificülcs md rinn Orlf>in PBL PBI ϊ Hl l'lil FBI PBI PBI PBL PBI PBI PBI PBL PBL BI CL BI CL BLC1 BI Cl BLC1 BI C I BLCI BIC1 BI Cl PBI IBI PBI 1 BL I BI 1 BI 1 BI PBI BI CL BI CL B1C1 nh ired with kidn r Β lymphocylcs Λ 2 2 ί 1 ) ; 11 3 2 3 1 30 24 31 1 24 1 1 32 3 33 3 32 25 25 1 3 24 26 24 2 3 2 3 2 1 1 28 30 1 2 2jM 3 24 2 29 2 11 1 1 2 i I I 2 2 4 1 19 2 ey dinor et lls U 44 35 57 51 Η 15 62 J7 18 44 8 18 4J 55 49 55 8 6 0 57 60 7 14 7 12 1 8 T8 7 13 5f> 5 8 7 7 7 •>! 18 55 TT55 / Β 51 55 7 55 57 55 27 35 8 i5 1B27 "7 44 6 2 8 57 51 »re undc C 5 4 2 4 7 Ζ 4 5 7

·)

3 7 3 7 3 6 b 7 1 7 7 7 3 7 6 7 7 1 1 7 3 6 2 4 1 7 2 4 ϊ 4 7 rllnod BI Cl MI Λ UR 4 J 7 2 7 t h Α 6 9 3 6 7 6 11 2 7 2 7 2 3 2 7 11 TT 7 8 1 8 2 2 2 14 8 1 1 6 7 2 1 2 9 Β 1 i 7 14 1 8 1 t Β i i 8 J j 1 1 ί 3 Β 8 19 m d 20 w3 w~ w w w — w w w w w \v w w — w w w u wZ vT w w w w w Η W W W " η 3 w2 w i ^ w 3 w ϊ wl w J w2 \v2 ' \v2 w2 w 1 u 2 w2 w ) v i π d c n v t d Irr n i w4 w5 _ w2 2 u 2 2 u 2 2 w4 wj w5 w2 w4 v.3 w4 w 2 w2 W4 w 4 w2 w2 w3 wl w4 η kidnty reeiptent 111 Λ rf. 1s Ν C yior»A i t a t ( i t i i i / 402 ο ΤΛΒΙ ol AI 1 C t 21 t 11 iqains. ! C 7 Γ alle•>(<< U 2. \L{C 1 U 1 r arqtts 1ί)7 11 1 2( ri Π 7 2DH 2 C 7 Α Conlrols Kidney rtc Ipient Kidnty donor Β PBI Λ2 Dgw ί Λ2 DQw ϊ M l C w7 Λ1 1 ÜQw i B18 Dgw ϊ B18 Cw7 B55 C w Ϊ DQw i B55 Cw) Cw7 DR 1 1 DQw3 Cw3 Cw7 DQw3 Cw3 OQw ί Nont. Noru C BIC1 B18 Ϊ)Κ11 DQw3 B18 DR11 DQwi DRw8 t w7 DRWB

t'cntnl ij,t ol spciiiU lysi Shand Ml Λ sptt Ifldtlcs with kl

imdcr Ihn shdd ol si^

isi i]cu] it(d it onc V/1 rUio (20/1) xydo

is di iiti d In Μ nid Μ

clones recogni/ed BLCL sharing either the DR8 or DR1 1

serotype with donor cells

Proliferatwe activtty qf two clones (2Γ7 and W9)

against α panel of DRw8 cells and HLA DP DQ and

DR typed cell Uncs Among ATLC two clones which

couid not bc assigned as recogni7ing a defined locus (2F7)

or epitope (1D9) were (.ested agaänst another panel of

allogeneic cellb ATLC 2F7 lysed various ailogeneic ceiJs

without any corrclation with the presence ol a particular

HLA Α Β C or DR allele within the panel and was

4190

Τ LYMPHOCYTE CLONING FROM REJECTCD HUMAN KIDNEY ALLOORAFT

ΤΛΒ[ L· Ifi

Prolifi-ratlve acttutty of ATI C in the prt

ct. qf ucirt

rratUateä allogench ce/is (Pßl 1 fo 12 and BLCl 13 to *

Α Controls

Medium

Kidnty df ruir

Η I BL

hl DQwJ

ΑΪ ngwj

All Cw7

Α ! 1 DQw i

1118 DQw t

B18 (

W

7

l\

rr

> < wf DQw i

( w ϊ ( w7 DQw i

Cw Ϊ DQw i

Nonc

( Bl C I

B1H DR11 DQv3

B18 DK1 1 DQw !

ORwH ( w7

DHwH

C w7

270

5 780

9 430

90

15 3 10

110

B4 Η

7TB

220

4 145

16 040

1 800

10 440

780

8 620

310

3 410

540

6 620

810

56 0

70 4

45 2

4Ö~S

68 2

64 Η

5<—ί

mrr

Nl

56 1

4TS

7[ S

NT

7 ) 0

ΝΓ

25 0

4» 7

7X8

NT

28 3

4X9

66 9

H( (ipU nt „_________„___„__ ___

Rcults trt txpnssid i s perunl iffeoi RR (st< Μ md Μ J nid ine m c pm fre^ivc

th irr idl il< d donc r Bl CI I osflive Pl 1 ί lustt rs fsee Μ and Μ ] \η undc rlintd

HI Α spcrii» Ities sliarcd with kldntv donor cclK (no 20) UCSLIUS obl llncd with ri

. UK1BKL17 nid 1H irt tully illnßtiulr from donor Bl C I

Ikkw 10 KR

Ν Γ nuustcd

Sa K< siiiis (/\nnii;->/;,of All C rcpt rloirc b ) Pl I ossdi;'.)

ΙΛΗΙΙ- IV

} roliferativt aeth tttj of ι ι/ίοΐοκιι and noru ytotovh All C ^tlnudatcd ulth all( φ

DRwS PRiult orüQu i cU tt rmmont wltli kut

i tor rtsportdirs (ii)lur<fl JJI

< ipient Hl 11 (ui(o) irr ^ive

r fmrdium) or

, ΡΒ1 1 1 and

MCI *,hartiujlll8 Bu 55 Lu

Midium

Kidney de nor 20

DIB DR11

DQwJ

DRw«( w7

DRw8

Cw7

Cw7

Bw55 Cw7

4

5

6

7

8

ii

5 047

14 787

39 8

J 6 3

994

ί ϊ 521

79 9

Stimulitnrs

Shdrcci Η! Λ bp( (Ifli H

B( low 10 ΙίΚ

rc cullurtd (or 96 Ji

ith kidne

ith irridi ι

ilorsor ( ells

s*i( d ί-ίί κ r J

ind Μ 1

10 reactive target cells were f-ΪΙ Α Β8 Cw7 whereas on

the other 4 reactive cells a Cw7 or C blank could not be

exeluded (ccll 5 B18C? cell 8 B49Cw7 cell 16 B58C

and teil 20 S18Cw7 see Table I) Conversely 5 oi 22

nonreactlve target cells were HLA Cw7 and all of them

were also B7 positive Inasmuch as two subtypes for Cw7

have been described one of which is less frequently seen

In HLA B7 positive individuals (22) it is possiblc thdt

donor and reeipient even though they share C w7 difler

In the Cw7 subtype The Cw7 subtype may thus be as

sociated with the non-B7 serotype and could be recog

ni<;ed by clone 2Γ7 In parallel we assayed ATLO 1 D9

which previously recogm/ed one of the two DRw8+ cell

lines tested (six of seven cell hnes) We confirmed with

this extended panel that 1D9 indeed rtacted with part of

the DR8+ cells (Table V) In addltion thts clone was

further analyzed on the relevant homo?ygous target cells

of the 10' International Histocompatibility Workshop

The results of this study are reported elsewhere (23) and

will be discussed below together with the present ddta

Blocking expcrlments Willi antt HLA mAb Several

m vb recogni?lng a monomorphic part or parts of HLA

class I and class II molecules (see Materials and Methode

Proliferation assays (PLT)) were used in blotking exper

iments Imtially mAb were assayed for their ability ίο

block the proliferation and cytotoxluty of various ATLC

against D BLCL Although Inhibition patterns obtalned

were simllar ior both funetions mAb concentrations

kadlng to a signilkant Inhibition of ATLC proliferation

weie several tirnes higher than those necessary in cyto

toxlc dssays (data noi shown) Insofar as PLT and CML

assays gave cont ordant results only mAb blocking activ

lty in cytotoxic ity cxperiments were assayed Cytotoxicity

of three cloncs (40 2 6 2F7 and 1B4) all CD8

H

was

abrogated bv additlon of anü-HLA class I mAb during the

Τ LYMPHOCYTE CLONING FROM REJECTED HUMAN KIDNEY ALLOGRAFT

₄₁₉₁

Par Π Τ 1 lel analyste 2 Γ 7 11 12 TABLC of 2Γ7 and Cylot !!)9c axlc ty ijtotoxic AI LC I L r i PLT2 Cytotoxl ty Α Controls Medium Kldncy donor Kldney retlpient li PBL 2 2 3 9 120 45 710 40 0 5 400 42 063 20 6001 105 36 1031 700 H K I i l Ν i 67 Η 84 2 68 2 Μ 8 ΪΙ i r 7 i0 8 34 l Clones w u e l e s k d 11 32 and 20 (rinn ir] \ ol Kit loslltvc ΙΓ1 thi

uncler 10 RH R IW cpm ν ilues

.alnst HI Λ DI OQ and DR typtd < tl!s (E,er Tablc [} ind a rt HI Cl Olhcr p n x l ie]K were usid i s 1 Hl Γ< ι Ρϊ Τ ass trs trt ui]d(r)lned(s<( Μ ind Μ )

s DRwS+ccUs (-1 Celli, iß

rt fiiprt ssrd ispcrtentj^c

dcmonstrated that 2Γ7 tndeed recognlzes a HLA class I

molecule (probably a Cw7 rclatcd specificity as men

lioned above) because its cytotoxtcity was unequivocally

blocked by two distinct anti HLA class I mAb (W6/32 and

41 H611)(TableVI)

Cytotoxic activity of the majority of ATLC tested could

be inhibited by adding mAb directed against class II

structures Forexample cytotoxicity of clones 1E3(CD4 )

and 1B5 (CD8 ) was abrogated by addition of mAb B7 21

and PL15 (anti HLA DP) Clone 1B5 was dlso inhibited

by BT2/9 (anti broad HLA class II) whereas 1E3 wab

not which would suggest that these two clonts are r°

active with distfnct HLA DP related epitopes However

LeulO (anti DQ) blocked thc cytotoxic activity of 2C3

suggesting that this clone IS directtd against a DO speci

ficity found in association with the HLA DRw8 serotype

In the Cau( asoid HLA DRw8 is mosl frequently iound in

tssociation wil h DQ blank specti icity litt ause clone 20 Ϊ

was reacUvc with pancl cclls 15 and 16 (DRw8 and DQ

blank+) and because this Τ cell recognized only those

DRwS homozygous cell Unes among the 10 Interna

tional Histocompatibility Workshop panel which also

carry the DQ blank specificity (M Bonneville manuscript

in preparation) it would seem Hkely that it is directed

against DQ blank specificity (Table I) That such

DRw8 DQ blank haplotypes have indeed recognizable de

terminants on DQ rnolecules has beert demonstrated with

mAb (24) Pancl analysis demonstrated that clone 1C7 is

probably dirteted against a determinant iound on DQ

molecule^ expressed by BLCL 14 and 13 as well as by

cells 1? and 16 (Table I) because 1C7 cytotoxteity was

blockeJby t h e L t u l O a n d 1Α3(ιηΜ DQ)mAb Clone 2C7

the

n

'vlty of which was also blocked by the same three

anti OR mAb did not recognize either DRw8 or DRwll

targets except donor cells Two clones (1F5 and 1F2)

were signif icantly blocked by the BT2/9 antibody without

being blocked by any other anti HLA mAb (anti DQ not

tested) Because this antibody recognized an epitope

shared by DP and DR rnolecules no conclusion could be

drawn com erning the pi ecise deflnition of the HLA locus

involved ATLC2D8 as well as clone 1E7 although not

blotked by BM50 mAb was strongly inhibited by both

Dl 12 and L243 mAb Consequently these clones were

probably committed against a DRw8 determinant {shared

between BI CL 15 16 and D BLCL) This was confnmed

by Lestingiheir cytolytic activitv against a panel of homo

/ygous DRw8 cell lines of thc 10 Intern ltional IHsto

compatJbility Workshop

Tinally among the 8 DRw8+ taigets (mcluding D

BLCL) recognized by the 1D9 clone (Table V) two BLCL

could not trigger 1D9 proliferation In addition the three

anti DR monophorphic mAb (BM50 Dl 12 and L243)

routinely used in this study blocked 1D9 proliferation

induced by D BLCL Thcreiore these first lines of data

might suggest that 1D9 clone cells are committed against

a spM of the DRw8 allele However a s mentäoned above

4192

Γ LYMPHOCYTK CLONING FROM R t J E C i T D HUMAN KIDNCY ALI OGRAPT TABI L Vi

Lffcct qfantt HLA mAb on c L//Ü OXIC ai tluity of Ali C

Α Ιί 40 2 ΙΪ7 1Β5 il 5 1134 J|)i) Κ 7 Π 2 Π ί J( ( Π / 21JH 2( / Urco Ν Ι ιί { b tlt 1 Ι Ι I x j r ι C D C D b ( ' C Λ Ι " ( 1) H J. · ϊ Λ ί Ει Λ Β Β Ι! ( Λ ( Λ { Uc >l s p r i , ( s f i d Κ" Μ ϋί π Vi 44 | ' 9 2 71 12 19 11 17 52 55 51 4 5 2 5 57 72 6b b 5 2 5 >5 2 0 4 8 57 57 4 5 1 6 Wf / ti 93 9 Ν7 82 6 ΝΙ 25 1 Νί (J 100 0 Ν ί ΝΓ ΝΓ 20 0 ΝΙ 0 2 8 ΝΙ 1 5 20 0 21 Η 0 Ν Ι 0 19 ( Ν Ι 9 4 ΝΙ iflc ( nclcist ll 20 1 \ /Ι ι» Α ; 4 Ι Ι Ι Γ 1 ] ΝΓ tf ß 6 ΝΙ 6 1 4 Ν i 0 ΝΓ NT 78 9 NT 1 9 6 4 5 0 NT Ν Ι NT 2 7 NT Ρ S 0 Nl Ν Ι ΝΙ Ν 1 Ν Ι Ν ! ml Inhibltio Hl Λ 11 πι j.m Ν Γ i i b NI 0 ! 00 0 95 9 9ίί~5" ~5~9 0 92 h 100 0 NI 0 1 b IM 4 60 b Ν Ι 0 Nl ( j N l 0 8() 0 lÖOl) 0 & r' _ ns fWilf ( x< ι BM D 0 0 4 1 0 io 9 3 0 0 7 7 ο 7 iOJ 0 ! 00 0 0 4 Η 1 2 8 6 9 7 6 4 ( 0 9 1 Ji) 0 1 > 4 2 Ι Η 4 4 »0 6 I0ÖT) r u s Π15 MAb( ii DU 1)1 12 0 0 0 1 2 1 2 NT 0 4 5 ΝΓ 95 6 100 0 2 5 NI 3 4 1 5 0 { 5 i 7 4 5 4 7 0 15 7 iJ 0 4/ 0 4 i 1 57 0 UM nul 1 ι hilii C7) " ! ! 24 i ΝΓ 0 NT 0 15 Ί NI 0 5 1 2 ΝΓ NI 84 0 Nl Nl 1 5 4 S Nl NI 4 7 Ν Γ Ν ! 0 <)5 7 7TÜ) Ν Γ 97 0 Ν Ι Γ< p t r l i 1 < ι ! 0 0 NT 0 Ν Γ 0 NT NT NT NT 0 ΝΓ 67 8 Ν 1 NT Nl Ν Ι 0 Ni 10 9 1 00 ί) 100 0 " Ν 1 Ni ΝΙ ΝΙ Nl m 1Λ3 ΝΙ 0 Nl 1 4 1 0 0 0 3 5 0 ΝΓ 0 ΝΓ 42 0 58 0 4 7 ΝΓ NT 0 ΝΓ Ν Γ 1 00 0 0 0 NT 0 NT crlfnccl Di ιπ -i NT 0 NT 1 2 7 100 0 80 0 ~4~3 0 0 NI 0 ΝΓ 8 4 9 ί 18 1 ΝΓ NT 100 0 NI Nl

o

0 10 5 NI 0 Nl C Ι (Λ Β 1 1 lr 0 ] 5 0 5 1 78 0 95 0 NT NT 3 5 3 0 0 1 5 1 5 0 ΝΓ 8 3 1 5 NT 83 0 0 1 5 NT NT 0 ΝΓ 1 7 C D ί C

sected elscwhcre (23) by usin^other rnAba/id panlt rcIK

as will bc discussed latcr

We previously reportcd on a procedure allowin^f obtam

ment of Γ cell clones dt ived from lymphold (ells inlil

trating an irreversibly rcjected human kidnty allogralt

(11) These experlments were intended to obtain a bettcr

knowledge of the rejeetion process owlng to the pivoial

role of Τ lymphoeyies in this phenomenon Here we

report on Lhe recogmtion repertolrr of 16 of 55 ΛΤΙ Γ

previously described (11) in order to assess the impor

tance of donor HLA rerognition duringailograft rejec tion

For this purpose we carned out proliferative and cytotoxit

assays agalnst a panel of allogeneic teils Sharing sero

defined HLA specificities vviih kidnty donor teils as well

is parallel Inhibition studics ol CMI by well ihar.utcr

i/ed mAb dlrected against monomorphk parts ol Hl Α

class I and II produets Results are summarl/-ed in Table

VII Although we are aware that othtr t link al c ondltlons

might have rcsulted In dlfftrent data the high platlng

efflclency achieved in our clomng System (about lO

i;

c)

(11) strongly suggests that the repeitoire analysis IS in

formative In this view il should bc noted that graft

invading cells were dirertly cloned Immedlatcly ctflcr iso

lation before any bull ilture and in the soll» presence

of rll 2 In this respeel ht in vivo sensittzatioa ol these

cells is noteworthy (ndeed we could imaglne that most

of the clones studled onginated from inillally uncommft

ted cells sensiti?ed in vitro againsl Ag borne by donor

BLCL instead of being derived from cells sensiti/ed in

vivo fhis possibility However is unlikely ior several

reasons In a previous papei we compared the prolifera

tive frequency of graft infiJtrating cells eultured wiih

aulologous BI CI with a pool ol allogeneic PBL in the

picscn<( of PH Α or wlth donor Bt CL (11) Thefrequenty

of(( Ils prollferating with irradiated autologous BLCL was

about 1/2190 lompared lo 1/13 obtained with donor

BIC1 arid 1/250 with allogeneic PBL plus PHA (11) In

addition under these previous condltions the Τ cell

rlones generated from Ihc limlting dilution i.ulture wlth

the pool of irradiated allogeneic PBL were speeifieally

readive agalnst donor BLCL (10) mdicating that in vitro

sensiti/dtion agamst allogeneic decerminants distlnct

Irom thal ol donor cells did not oeeur in this model

Moreover in vitro sensitization of PBL with irradiated

autotogous or allogeneii Β cell hnes led to the generation

of numerous cell colonies exhlbiting NK or NK like cell

at tivitles In contrast none of the Τ cell clones obtained

irom rejecled kidney and tested in this study were abli

lo sifii itkantly lyse lhe NK ceil sensitive K562 targi ι

(10) In addition proliferative frequency of cells agalns

autolo

h

ous BLCL was at least 200 ümes lower than thai

of cells agalnst donor BI CL (15)

LyM ol D BI CL by most Α fLC was slgniflcantly Inhlb

Hed by anti HLA mAb (Table VI) Because ATLC als.

expressed IILA class I and II molecules we were con

cerned by a putative action of these mAb at the effectoi

kvc] Howcver this was not the case for example ii

txpenment C (Table VI) cytotoxicities of stx ATLC e\

pressing slrnilar amounts of HLA-DP Dg and DR mol

e ules (M Boiincvillc et al submltted for publicatioiH

were analy/ed in the presence of antl HLA mAb am ι

varlous Inhibition patterns were observed If a mAb hat

acted at lhe effector level a simllar effect should hav

been expected on cylotoxicity of all ATLC derived from

the samt Indlvldual

Τ LYMPHOCYTE CLONING FROM REJECTED HUMAN KIDNEY ALLOGRArT ₄₁₉₃ AIXC 40 2 6 2L5 1B4 2 F 7 ] D 9 2D8 11-7 2C7 2C5 2C3 1C7 113 ] B 5 ΙΓ'5 ΙΪ 2 I D 7 C D 8 8 8 8 4 4 4 4 4 4 4 4 8 8 4 4 TABLE Recoqnltlon rcpcr I nel ι v&is Bw5"i cells CwJ cells BIS DRwl 1 ctlls > 7/9 DRwH cells DRw8 Bl CL DRw8 BLCI Donor ceils only Donor ceils only DRwSBLtI DQ DRw8/Dg blank and DRwil/DQwJ BLCL DRwl 1 Bl CL DRwll BLCI DRwl 1 BLCL DRw8 DRw11BLCL DRw8 DRwJ 1 Ϊ3! Cl VII toirc of Ai'LC Bio k ΐ-μπιΑΙ HI Λ Ι not tebttd Hl Λ 1 Hl A I Br< ad HI Α Π DR Broid Hl Λ Π DR (Dl 12 oiilv) DR (Dl 12milv) D R ß r o i d H I AH DR (partial blocklnjJ] og DQ DI Ι Π i d HI Α Π DP Brt nd HI Α Π DQ nol U s t t d Brojd HLA Π nol lest« d Spccifl Ifv anti Cw3 dtit BIS mtl Cw7 subt>pe inti DRw8 subtype inll DRw8 intf DRub i n t ! DR prtviU ·> antl DR priviti > -mtl DQ inti DQ inti Di inti DI Ulli ΠΙ Α Π inti HLA II S h ι t d Η Λ ·,ρ( < l U c i t y ol" r t c f fini/cd p u i c l κ Ι Κ w i l l k i d n t \ d ι

the epitopes recognized by each clone piesented in this study rather we have attempted to look at the class I and II MHC produets recogni/ed by this panel of t lones extracted f rom a rejeeted kidney to better understand the role of each loci in in vivo allorecognition

Aecording to the classical dichotomy between MHC class I and class II produets two sets of ATLC were defined All anti HLA class I ATI C were CD8 conflrming the numerous observations obtained with lymphocytes sensiti/ed in vitro (21?] ATI C agalnst all of the mis

matched HLA class I speeificities (except AI 1) betwten kidney reeipient and donor weie found (B18 Cw3 and Hw55) In addition ATLC 2F7 which was blocked by several anti HLA class I mAb and having monoclonahty (as well as that of the Τ < eil clones described in this study) that was demonstrated by DNA hybndlzation wlth Γ cell rearranging gene y and TCR β probes (26) probably rec ognized a Cw7 subtype foi whtch donor and reeipient were mlsmatched The majority of AT I C were directed lgainst HLA class II produets Although Τ eell (lones recognizing HLA class II molecules have been shown to bclong to the CD4 type it has been reported that the same molecules could also elicii CD8 respondei cell > (27) Interestingly two such CD8 Α I"LC were blocked by

inti broad class II (Β Γ2/9) -md/or anti DP (H7 21 , nd PI15)mAb Howcver ATI C reiognizlng DR DQ anrj DP produets were gt neral y CD4

Some of the c lones dt s< nbed gave ret ognltion ρ «t I erns whieh did not fit with determinants predicted by serology before transplantation (clones 2Γ7 and 1D9 for in stance) These known differences in cellular and serol ogical repertolre may have theoretical and practical im portance in transplantation One such example clone 1D9 h a s been further investigated and described eise where (23) Brieily 1D9 1Γ7 and 2D8 lysed only ctlls Hearing DRw8 determinants including hom07ygous DRw8 cell lines and their lytic artivity was abrogated by anti DR mAb (23) However first 1D9 only lysed six of eight DRw8+ cells and in partlcular did not react igalnst BLCL 16 and 31 which nevertheless triggered -iD8 or 1E7 responses Second the antl DR mAb BM50 mhtbited the cytotoxic activity of 1D9 only when anti DR

mAb Dl 12 (and L243) blocked all three clones Third 1E7 differed from 2D8 and 1D9 because its cytotoxicity was resistant to anti broad HLA class II mAb (BT2/9) Moreover a more detailed analysis of the data revealed that 1D9 did not recognize two DRw8+ BLCL among a panel including the Leiden panel used in this study and the 10' International Histocompatibility Workshop panel of nlne additional homozygous DRw8+ cell lines (23) Furthermore olher informative mAb including UK81 (DRw'52) l\DS13(DRw52 3 5 w6 w8) NDS10(DRw52 5 3 wb) MAD88 (DRw8) GSP4 1 (DR) PL8 (DR) and 2D6 (DP DR) were also tested in the blocking assay of 1D9 cyljtoxicity against D BLCI (23) In summary 1D9 recogni/cd a ma|onty of DRw8+ cells (15 of 17 cells) and all D P w 8 - cells (n = 22) were not recognized mAb dl rected against class II monomorphic and polymorphic dete minants coniirmed that 1D9 is committed against DR speeificities RFLP of DRw8+ cells either recognized or not by 1D9 did not reveal differences with DR beta I robes (23) The 1D9 Τ cell might be directed against a DRw8 subtype which would need further investigations at the DNA level Altcrnatively a DRw8 molecule rnight be the lestiiction element for some yet unknown minor Ag

4194

Τ LYMPHOCYTE CLON1NG FROM REJECTLD I UMAN KJDNEY ALLOGRAFT

HLA epitopes recognized by ATLC and mAb were not

close enough to allow antibodies to act by stenc

hfn-drance This hypothesis might also explain the differen

tial blocking activities of the various antl HLA ciass ΙΓ

mAb particularly BT219 BM50 and Dl 12 on the cy

totoxic activity of several anti HLA class II ATLC

In conclusion it can be said that this approach to the

ATLC repertoire combining panel analysls and mAb

blocking experiments, was sufflcient in some cases to

define allelic specificiües (9 of 16 ATLC) In contrast for

the majority of Τ ceJl clones only the locus origin of the

product recognized could be determined The fact that Τ

cell specificity was not fully correlated with serologically

defined Ag ratees several questions First this might

reflcct in some cases a reoognition of minor histocompat

ibllity Ag presented in the context of donor HLA Ag

Regardlcss of the explanation this does not changc the

fInding that donor MHC Ag are recognized by all recipient

Τ cell clones tested (either directly or as a peptide restric

iiori elemcnt;) Turthermore the discordance between se

rologic and cellular specificity rnay have important clln

leal implications Tor lnstance although recipient and

donor seemed to be matched for serologically defined Cw7

specificity they differed for cellularly defined Cw7 sub

type which would aecount for the recognition of this

specificity by one of the cytotoxic clones studied (ATLC

2F7) However we are aware that the preseneeof effector

CTL in a rejeeted graft does not allow them to be definilely

linked with the rejection process because they have been

recent'y shown to be found in well tolerated giafts (28)

Even though nonexhaustive such analysis suggesls that

no MHC gene produets are dominant in eliciting recipient

Τ cell responses and thus clearly demonstrates that a

highly polyclonal Τ cell response has oecurred

Nonethe-less ouröai<i applied to a case of definitive graft rejeetion

in a recipient who was without an immunosuppressive

regimen Jor weeks The immunogenicity of HLA molc

culos borne by allograft cells in other jgrait Situation^

may be somewhat different

Acknowledgments We are indebted to Dr D Charron

(Paris France) and Dr D Olive (Marseille Trance) re

spectively for their generous gift& of anti HLA class II

and ciassi mAb ίο theorgani/ingcommitteeof the 1987

HLA Workshop for providing us with L243 1A3 and

PL15mAb andtoDr R Bontrop Dr G Μ Τ Sthreuder

and Dr J J Van Rood for helpful diseussions

RH PRI-NCI S

1 Platt J L Τ W teoien and Α F Michael }9H2 Inlirsiim) m o n o n u t h ir ti II popul illoiis In r( η Η &r ift n | i < U o n idonlilit tii< η by inonoriondldiitibodki. in fisnUf «.iriJofi«, J ! xp Mi d J"jri 17 2 Hayry Ρ 19Η4 Intragraft c v t n t s In illo^nft tlestruttlon / <nis

pleintadon iß !

t Von Willebrand Ε a n d Hayr y Ρ 1978 ( ompositmn u u ! in vitr> tytotoxkliy aC tcllul tr Intlltr de In rtjeftintf h u m a n kklni y illo gnfls Ctlt Immunol 41 358 1 V o n W i l l e b r a j v i Ε 1 9 8 1 O K 7 4 / 8 n l l o i n t h e n l o o d i n d i n t h t )»r ill d u r l n g ( p i s o c l t s o l h u m t n π η ll a l l o g r i t l rij< (tl< η C < II li a m m o l 77 19(3 IS I 1)1/1 J fVK " JZ r-6 1 <6 / J b ι A s c h e r Ν I R Hoffman D W H a n t s a n d R L S i m m o n s 1 9 8 1 f t U u l i r (v<nLs u i t h l n Ine reju ting illn^rift fran^ptatUatton J1)

191

7 Not ι Α Α Barocci Ο Valente S Carozzi S Burastero Α

Zic Α Cadoni V Dessi and F Celada 1984 In vivo chaneter

\?a of ι donor speUfle cytolylic Τ ce!l liue established from

hu ι lymphwvtcs homlnj» in a icjeclcd kldney allograft Trans

plfj tlon 41 1J5

8 May.- J G Α Α Füller J C Füller Α I Lazarovits L Α Boyle and J Τ Kurnick 1985 Charai terl/alion of in vivo actlvited all« spet ifi< I lymphot ytes propagaled from human rcnal aliograft. biop sks undcrgolng rejcctlon J immunol 134 258

8T Preffer F I R Β Colvin C Ρ Leary L Α Boyle R V Tuazon Α 1 Lazarovits Α Β Cosimi and J Τ Kurnick 1986 Iwocolor flow < ytorot try and functiondl analysis of lymphocytes eultured from humin midi illografts Identlficition of a Leu2+3+ subpopuiatlon

1 Immunol 137 2823

8b Miceb C R S Metygar Μ Chedid F Ward and Ο J Finn 1965 [ ong torrn tulture and rharrfrterl/ation of aliorea<.Uve Τ celJ infJl trales from rena! ncedlc biopsles Η um Immunot 14 295 fit Duquesnoy R J Α Zeevi J Fung C Kaufman Τ Zerbe Β

Orlffith Α Trento R Kormos and R Hardesty 1987 Scquential Infiltration of rhss, I nid i h s s (i bpeclfSc -jJloreaciJve Τ reih in hum tn c irdi ic lllograftf, Transptant Proi 19 2560

81 Zeevi Α J Fung Τ R Zerbe C Kaufman Β S Rabin Β Ρ Inffith R L Hardesty and R J Duquesnoy 1986 AtlospeclflcMy r»f utivifed ί (cIJs gmwci (mm endomyocctrdlai blopsles from heart tnnsp] int pUionts I ranspluntation 41 620

Μ Fung J J Α Zeevi Γ Ε Starzl J Demetris S Iwatsuki and R J Duquesnoy 1986 Pumlion U t hancleri/itton oi inftltrating Τ

lympho<yU^ In Itum^n hrpäih aJioflralts Hum Immunol 16 182

10 Moreau J F Μ Α Peyrat Η Vie Μ Bonneville and J Ρ SoulÜ

Ιου 9β5 1 (olJ (olony formtng frequenov of mononuclt ited cells

cxtndod fr< in 11 |ct teti hum tn kidnty trinsplints Transplantation

Ί9 649

1 1 Moreau J F Μ Bonneville Μ Α Peyrat Α Godard Υ Jacques C Desgranges i n d J Ρ Soulillou 1986 Τ lymphocyteclonlngfrom rijatulhumin kklnty allografts growth ficquenoy ind functional/ plicnotyn» iridlysK J Clin luvest 78 874

12 Hourrnant Μ J Ρ Soulillou and J Guenel 198"5 Comparlsonof Hirn ininiLinosupjJn ^sive nginu ns in kldmy Innsplant ltion

Imtwplant Im 17 Π W

1 1 Carroll Ρ G W C deWolf C R Mehta J Ε Rohan and Ε J Yunis 197) (tiilniid <]uskr malysis of the prtmed tymphocytc tisl Iranspinnt Ϊ tot 11 180c)

Μ Barnstable C J W F Bodmer G Brown G Galfre C Milstein Α F Williams and Α Ziegltr 3 978 Produition ol monodonal

mtibodlt s io gmup Α ( rythrocytcs HI Λ ind oifur hum in surftec mlif,cns New loolsfnrficnttit in ilysls, Cell 14 9

1 ^ Corte G Α Müretta. Μ Ε Cosulich D RamarJi and Α BurgelJe^ 1982 Λ monocknil antiiX 1 iiitlbody stUrtlvcly Inhibits the ßcn trition οΓ (flrUor Tcdi s nudiidiifi ^peeifie cytalytic actliity J

I \p Mtd lr>( l1! I')

16 Tricbel F V Misstnard I ebJond Μ C Conty D J C h a r r o n a n d Ρ Debre 1984 Difft rrrut ι! Inhibition of hum m intimen speclfk Γ (< ii ι ion< pioJilirtiive responso·! by disiincl m u n o r l o m l anli 111 Λ ÜH lnlibcxiKs / Immunol 1 i2 1771

17 Accolla R S Ν Oross S C^rrcl and G Corte 1981 Üistlnci ΙΟΠΪΙΊ of both ilph ι ind bell biibunits am prestnt In the hum in 1 ( HiolfiuLir [>< ol I'iot Nfztl ALOÜ Sii LJ ^ Α 78 4549 18 Lampson L Α and R 1 evy 1980 (wo populillons of la llkc

tnokiult sffi ) Jiiimm JiicU Unt J Immunol 125 293

19 Chen Υ X R L Evans Μ S Pollack L L Lanier J Η Phillips

C R o u s i o Ν L Warner a n d F Μ Brodsky 1984 Ch ir iclerl/ Uion

nid t x p n s M >noi tlic ΠΙ Λ IX uill^< η defined by ιηΜ Ι eu 10 Hum

l'iimunol 10 221

^0 Austin Ρ J Trowsdale C Rudd W Bodmer Μ Teldmann and J Lamb 198^ I und Ion il ixprt^sion of HLA DP gonos trandfuttd into tnoust fibroblists Nature H3G1

j.\ Knowlcs R W 1988 Slrut tur tl polymorphisrns οΓ the »ILA cld&s 11

ilph ι ind b< Ι ι ih Uns surnmary of the ]Ott Workshop to tht gti πι ilysls In Immimobiology o! HLA vol 1 Β Dupont ed Springer V< rl ig Nt w York (in pri s«s)

22 Sasauki Τ J Dausset Κ Tokunaga Η Sato Κ Okodlr and S

Naito 1980 J< inl repi rt Cw7 In Hlstocosnpaliblllty Ttbting 1 I firisiki od Munsk^nrd Coptnhigcn ρ 500

2 i Bonneville Μ J F Moreau Μ L Cheneau F Bonneville Ε

Blockland J Pool Ε Goulmy J D Bignon and J Ρ Soulillou 1988 Ch im te rlzilimi oi a telluhrly defined DRS subiypc In III*· loionipitiblllly fcstfrifi I 1 Terasiki cd Munskgiard Copenh^tn (in pn ssj

24 Bodmer J G L I Kennedy Η Aizawa R L Dawkins V

Lepagt-M C Lepagt-Mtzzilli and Ρ Richiardi 19S4 Hl Α D region monoclon Ί tntibiilUs In Histotompitlbllity Tcstinfi Ρ Ι Teras tki cd Mut) sk(L.nid CcuttnUtfitn Donmark ρ 217

25 Meuer S C S F Srhlossmann and Ε L Reinherz 1982 Clori < in iivsi'i of isuni irr ( ytntOKk Τ ly/nphoiytc T4+ -ind T8+- tffe< tor I (ClK mo^nl/c produt Is of nnjor histocompatlbility reglons f ro

Piatt Acatl S( t U S Λ 79 4 19*>

2h Bonneville Μ J Ρ Moisan J F Moreau 1 Bouyge Μ Μ Hallel

Τ LYMPHOCYTE CLONING FROM REJECTED HUMAN KIDNEY ALLOGRAFT

₄₁₉₅

gtne rearrangements in human alloreactive Τ cell clones extracted

from a rejected kidney Transplant Proc 20 196

27 Flomenberg Ν , Ε Duffy Κ Naito and Β Dupont 198J Two dfstlnct phenotypea of HLA DR specific cytotoxic 1 rcll lines Im

munogenettes 17 317

28 Dalmann Μ J Κ J Wood and Ρ J Morris 1987 Specific