Bignon, J.D.

Blokland, E.

Bonneville, F.

Bonneville, M.

Cheneau, M.L.

Goulmy, E.A.J.M.

Moreau, J.-F.

Muller, J.Y.

Pool, J.

Soulillou, J.-P.

Leiden University Scholary Publications

lable 1. DR, DQ and DP β Chains from a Panel of DRw8 ΒCell LinesVvÜD~~Cell Name DR/Dw DQ* DP1 DRß DQß DPß~jßÖ<S6~ΤΑΒ089 w8/DB7 wl 7 ßl ßl ß~4#9067 BTB w8/w8 νΛ_ 4 ßl (32 ß5#9068 BM9 w8/w8 _w4_ 2B1» ßl ß2 ß2#9069 MADURA w8/w8 w4 4 ßl ß2 ß5#9070 LUY w8/8 3 w7 1,4 ßl ß3 ß3,ß5#9071 OLGA w8/8 2 w £ 3 ,4 ' ß2 ß2 ß2,ß4#9072 SPACH w8/8 2 w4 4B"> ß2 ß2 ß4#9100 OLL w8/ w4_ 3 4B> ß2 ß2 ß4+DQ types locally determined, ^DP types by Eckeis( TAB", DRß 1/DQß 1) or DQw3 ("LUY", DRßl/DQß3) The "OLGA" type (DRß2/DQß2) might begenerated by mutaüons of DRßl into DRß2 from "BTB"since both DR β chains carry the DRw8 epitope and aremost hkely evolutionally relatedAnother interesting finding IS that four differentDR/DQ haplotypes perfectly correlated with HLA Dlypes, "TAB" correlated with DB7, "BTB" with Dw8,LUY" with Dw8 3, and "OLGA" with Dw8 2 Inthe pnmary MLR in one combination of these HTCcells, DR molecules are different and may be stimula-tory to each other, while in another combination DQmolecules are different and may be stimulatory Thus,DQ molecules as well as DR molecules appear to beresponsible for the HLA D specificity on the DRw8carrying haplotypeThese DR/DQ haplotypes were found differently withone or two DP molecules isolated by B7/21 The tenta-tive nomenclature was given to these DP molecules aspreviously published (2), where five distinct DP βchains (DPßl-DPß5) were descnbed Four DPß chains(DPß2-DPß5) were identified (Table 1) Α correla-tion between DPß5 and the cellularly defmed DPw4antigen was confirmed However, the other three DP βchains were not correlated with any DP antigens in thisstudyReferences1 Maeda H, Hirata R, Okuyarna M, Thompson A, Tohyama ΗTwo dimensional gel analysis of a second family of class IImolecules by polymorphic HLA DR4, 5, and w9monoclonal antibodies J Immunol 1984,132 24782 MaedaH, Hirata R, JujiT, Katagin Μ Isolation and charactenzation of 9C4 reactive class II molecules In Aizawa Μ(ed) HLA in Asia Oceania 1986 Sapporo, HokkaidoUniversity Press, 1986, 444Author AffiliationsHiroo Maeda, Ranko Hirata, Blood Transfusion Service,Saitama Medical Center, Saitama Medical School, Kawagoe,Saitama, Takeo Juji, Blood Transfusion Service, TokyoUniversity Hospital, Tokyo, JapanIdentification of a Cellularly Defined DRW8 SubtypeΜ Bonneville, J F Moreau, Μ L Cheneau, F Bonneville, Ε Blokland, J Pool, Ε Goulmy,J D Bignon, JY Muller, and J Ρ SouhllouCellular mechanisms involved in the allograft rejectionpiocess remain highly controversial Using monoclonalantibodies. several studies have demonstrated convmcmgly that most oi the infiltrating mononuclear cells areΤ lymphocytes Moreover, several investigators havedeveloped techniques for oulturmg Τ cells out of vanoushuman allografts in order to delmeate functions ofintragraft Τ cell populationsFrom a rejected human kidney graft a limiting dilu-tion technique (1) was used to clonc a large number ofgraft invading cells wi'h clonal efhuency of 1/Π Outof55 clones, 20 were tcsted for 1) prohferative activity inpi mied lymphocyte typing (PLT), 2) cytotoxic activity incell-mediated lympholysis (CML), and 3) cytotoxicactivity mhibited by monoclonal antibodies (MAb) Αlarge panel of well HLA-defined cells (PBL PHA-blasts,and Β lymphoblastoid cell lines-LCL) were used asstimulator or target cells This panel mcluded all DRW8positive cells from Workshop homozygous Β LCL (BM9,TAB 089, MADURA, BTB, OLGA, LUY, S PACHEO,OLL, SPL), seven heterozygous DRW8 positive cellsfrom Blood Bank of Leiden (Netherlands), and cellsfrom recipient BER (DR2 DRW6) and donor DAB(DR5-DRW8) The following MoAbs were used toinhibit cytotoxicity expenments against donor-BLCLW6 32, LEU 10, NDS 10, 1A3, B7/21, 2D6, GSP 4 1NDS 13 UK 8 1, CHE 249 2, MAD 88 (all Workshopreagents), and BT 2 9 (anti class II), Dl 12 (anti DR)VI 15 (anti DR), and BM 50 (anti DR)Clone "1D9" was selected for lts ability to proliferatewith and to kill only but not all cells beanng DRW8phenotype (Table 1) This clone was significantlySpnn tcr Vcrl b NLW York 1989Iminunobioloj.y ol HLAVolume II227Table 1. Prohferative and Cytotoxic Activmes of Clone 1D9Stimulators (PTL)or Targets (CML)Le #15 (BLCL)Le #16 (BLCL)*Le #24 (PHAblasts)Le #26 (PHAblasts)Le #28 (PHAblasts)Le #29 (PHAblasts)Le #31 (BLCL)*LUY (Workshop)(BLCL)BER (retipientBLCL)DAB (donorBLCL)HLADR/DQ7-W8/W21 W8/W1W8-W13/W11-W8/W1W8-W14/W1W8-W13/W13-W8/W2W8/W32-W14/W15-W8/W3Clone 1D9Responder/EffectormPLT(cpm)*210403285]0383110101012113327505518573210020600in CML(% lysis)t25/22-/-////-/-24/12/16/17*Results expressed as mean (tnphcate) cpm of 3H-TdR uptakeof clone cultured 72 hours with stimulating cells, '% specific51 Cr release calculated at two effector/target ratlos (20 1/4 1),* Leiden #16 and #31 were not recognized by 1D9inhibited by broad anti-class II and antl-monomoφhlcDR MoAb, but not by anti-DQ or anti-DP MoAb Mono-clonal antibody MAD 88 (anti-DRW8) elicited againstDRW8 positive cell MADURA, which was lysed byclone "1D9," did not block "1D9" cytotoxicity directedagainst donor cell DAB (DRW8 positive) But this anti-body labeled in lmmunofluorescence the Leiden #16 cell(DRW8 positive), which in lts turn was not lysed by"1D9," clearly suggesting that "1D9" and MoAb MAD88 recognized two different epitopes on the β chain ofthe DRW8 moleculeIn order to identify at the DNA level ihis cellularlydefined DRW8 subtype, a restnction fragment lengthpolymorphism (RFLP) study was carned out to distin-guish a DNA polymorphism of these DRW8 cells accord-mg to their sensitivity to clone "1D9 " Genomic DNAsfrom nine DRW8 positive homozygous Workshop cells,from two recognized heterozygous DRW8 positive cells(Leiden #15 and donor DAB), and from a non-recog-mzed heterozygous DRW8 positive cell (Leiden #16)were digesied with Eco RI, Bam HI, Taq I, and Hind IIIdnd then hybndized with Workshop DR ß, DQ α, andDQ β probes With DR β probe and all enzymes tested,DRW8 haplotype revealed a charactenstic pattern dis-tinct from those of others DR specificities Moreover aspecific fragment of 8 9 Kb was noted with Taq 1 But nopolymorphism was found at the DNA level because thesame RFLP pattern was observed for all DRW8 positivecells includmg the non-recognized Leiden #16 cell Allthe restnction enzyme used to hybndize the DQ α andDQ β probes revealed three different DQ patterns Theseresults were concordant with those previously reportedby serology (2)In summary, clone 1D9 specific for the kidney donorcells IS described It killed neither K562 nor autolo-gous BLCL On a large panel of well HLA-charactenzedcells it recognized a majonty of DRW8 positive cells(15/17 cells) On the other hand, all DRW8 negativecells (N = 22) were not recognized Prohferative andcytotoxic activities were concordant RFLP analysisof DRW8 positive cells either recognized or not by1D9 did not revealed differences with DR β probeHybridization with DQ α and DQ β probes exhibitedthree different patterns without any relationship to 1D9reactivity 1D9 T-cell clone might be directed againsta DRW8 subtype which would need further mvesti-gations (other restnction enzymes and probes) at theDNA level Alternatively, the DRW8 molecule mightbe the restnction element for some yet unknown minoralloantigensAcknowledgments WethankD Gauvinforherexcellenttechnical assistance and Ms V Gallee for preparaiion ofthe manuscnptReferences1 Moreau JF, Bonneville M, Peyrat MA, Godard A, Jacques Y,Desgranges C et al Τ lymphocyte cloning from rejectedhuman kidney dllografts J Chn Invest 1986,78 8742 Betuel H, Gebuhrer L, Schreuder GMT, Laynsse Z, ArnauVillena A, Goldmann SF In Albert ED, et ai (eds) Antigenreport HLA DRW8 in histocompatibihty testing 1984 Berlin, Springer Verlag, ρ 198Author AffiliationsΜ Bonneville Jl· Moreau, JP Souhllou, INSERM U211Nantes, ML Cheneau l· Bonneville JD Bignon JYMuller, Ccnlra Transfusion, Nantes France, £ Blockland JPool F Goulmy, Umversity Hospital, Leiden The Netherlands228

Identification of a cellularly defined DRw8 subtype.

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Identification of a cellularly defined DRw8 subtype.

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