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Alloreactive helper T-lymphocyte precursor frequencies correlate with HLA-DR antigen amino acid residue mismatches
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Ab stracts
IV:6. Allorecognition, Tolerance and Immunosuppression in Transplantation 0876
SELF-RESTRICTED T CELL RECOGNITION OF DONOR HLA-DR PEPTIDES DURING GRAFf REJECTION Liu Zhn oru, Colovai Adriana I. Tugulea Soria. Reed Elaine F. Rose Eric A, Michler Robert E. Cohn David. Hardy MarkA. Renna-Molajooi Elvira, Cortesini Raffaele, Suciu-Foca Nicole College of Physicians & Surgeons of Columbia University. New York. NY USA. To investigate the role of indirect recognition in allograft rejection we have monitored a population of 30 heart and 15 kidney allograft recipients for the capacity of circulating and graft infiltratingT cells to react against allopcptides corresponding to the mismatchedHLA-DR antigens of the donor. Patients were followed for 1-8 months following transplantation, Blood was obtained at two weeksintervals and at the lime of graft biopsy. Lymphocytes from the blood and from biopsy lissues were test ed in LDA for react ivity to synt hetic pept ides corresponding to residue 1-19. 21-39 and 62-80 of donor HLA ORBI' chains. In 22 patients undergoing a total of 35 episodes of acute rejection. T cell reactivity to donor allopeptides was observed in the periphery and in the graft prior and/or at the time when an acute rejection episodeoccurred. 11:0 allopeptide reactive T cells were detected in the periphery or in the graft during periods of quiescence. LDA studiesof lymphocytes infiltrating the graft showed that the frequency of allopeptide-speciflc T cells within the graft was 3O-SO times higber than in the periphery. However, T cell recognition of a1lopeptides is likely (0 have first occurred in the peripherallymphoid tissue rather than in the graft since allopeptide-reactiveT cells wereoftendetected in the circulation 1-4 weeks prior to any histologic evidence of transplant rejection. Reactivity was directed againsta single dominant peptidecorresponding to one rather than (0 both of the mismatched Hl.Avantigens of the donor. Peptide dominancywas determined by the host's HLA-DR restrictive element. The finding that rejection is initiated by T cells which recognize a single dominant allopeptidc suggests the possibility of developing immunosuppressive peptides for specific immunotherapy. Furthermore, the presence of allopcptide-reactive T cells in the peripheryolTers a reliable way for monitoring rejection.
0878 IMPORTANCE OF A SINGLE AMINO ACID SUBSTITUTION OF THE a HELICES OF CLASS I MHC MOLECULES FOR THE INDUCTION OF A PRIMARY ALLOGENE IC RESPONSE Reboul Murielle ' , Noun Ghada " Abastado Jean·Pierr~ . Kourilsky Philippe*. PiaMarika' , 'Mouse Immunogenetics. U93 INSERM. Saint-Louis Hospital. Paris. France *U277INSERM. Pasteur Inslitute, Paris. France
Todelermine whether there is a relation between the localizationof a residue of a MHC class I moleculeand its implicafion inthe induction of a primary allogeneic response. we have tested the ability of various point mutants (a· helices or /l-sheel of the a 1 and a 2 domains) of an H-2 class I molecule to induce a cy1otoxic T cel response in mice carrying the native molecule. Murine L fibroblasts (H.2k hapofype) express ing different Kd mutants were injected into the hind footpads of (BIO.BR x C3H.OH) hybrid mice carrying the class I molecules of the H-i' haplotype together with the Kd molecule. In this combination. an eventual allogeneic response of these mice can only be induced by the singleamino acid difference between the nafive Kd molecule and the point mutant expressed by the fibroblasts_Three days after the injection. the cells from draining lymph nodes were cuhured lor 4 days (in Il ·2·supplemenled medium) in lhe absence of any stimulator cells . The presence of allogeneic CTl was tested in a 4-hr 5' Cr release assay. Under these conditions. on ly 7 out 01 25 mutant Kd molecules tested induced a primary allogeneicresponse. Interestingly all these mutations concern residues located on the ,,-helices. In addition. no correlation was observed between the s ize of the repertoire of peptides selected by these mutants and theircapacity to induce a primary allogeneic response.
0877 A LLOREA CTlV E HELPER T -LYMPHOC YTE PRECURSOR FREQUENCIES CORR EL ATE WITH HL A·DR ANTI GEN AMI NO ACID RESIDUE MISMATCHES Young NT, Roelen DL, Bunce M. Dallman MI. Morris PI, Welsh KI Nuffield Department of Surgery , University of Oxford, Oxford UK Interleukin-2 (IL-2) mediated helper activity is central to the cellular and humor al immune respons e induced by exposure to allogeneic HLA antigens. We have analysed the influence of HLA matching on the in vitro response of primary alloreactive helper T-Iymphocyte precursors (HTLp). Limiting dilution assays were employed to analyse frequencies of IL-2-producing HTLp in van ous liL A-mismatched responder:stimulator comhinations. Mea n IITLp frequencies were significantly (p
0879
TOLERIZING EFFECTS OF PRETRANSPLANTEXPOSURETO DONOR HLA·DR ANTIGEN IN RANDOM TRANSFUS[ONUN[TS FOR KIDNEY RECIPIENTS AM Jackson, C McSherry, K Butters, M Diko, S Almond, AJ Matas, NL Reinsmoen, University of Minoesota, Minneapolis. MN Ourprevious studieshave shown that thein vitroassay of donor antigen-specific hyporeactivity (DASH) is a useful marker for identifying solid organ transplant recipients (kidney, lung.heart or liver) ar low risk of immunologic complications(i.e.• tareacute rejection episodesandchronic rejection). DASH is defined as a significantly decreased post- vs. pretransplant (prctx) donor antigen-specific mixed lymphocyte response to donor cells, while response to third-party cells remains unchanged. We studied whetherpretx transfusions were associated with subsequent donor antigen. specific Tvcellnonrespoasiveoess, identified as DASH. in kidneyrecipientsexposed10 thesameIflA ·DR antigenpretx via the transfusion unit and posttx via the transplanted organ. In this study,24 previously nontransfused patients who received two 150 mI pretx random bloodtransfusions underthe cover of azathioprine are now I )T posttx; theIflA·DRofeachrandom transfusionunit was determined retrospectively using PCR· SSOP. Of the 24 patients. 7 (29%) werehyporesponsive at I }T posttx. Of these 7, 6 were exposed pretx to donor HLA·DR antigen via the transfusion unit vs. 6 of 17 responsive recipients (p=(l.04). Preliminary evidence indicates that, of recipients exposed pretx to donor HLA-DR antigen, those becoming hyporesponsive received fresher transfusion units (mean= It days old) vs. those who remained responsive (mean~24 days old). Neither HLA·DR sharing between the recipient and the transfusion unitsnor the time between tranfusion and tx (mean-e mol influenced thedevelopment of DASH. In conclusion. our results suggest that pretx exposure to donor HLA-DR antigen via fresh transfusion units enhanced the development of DASHfor recipients exposed to the sameHLA-DR antigen posttx via the transplanted organ.
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0881
PREGNANCY CAN INDUCEPRIMING OF CYTOTOXIC T LYMPHOCYTES SPECIflC FOR PATERNAL HLA-ANTIGENS, WIllCH IS ASSOCIATED WITHANTIBODY FOAAIATION GJ. Bouma. P. van Caubergh, F.P.MJ . van Bree, R.M.C Castelli-Visser, ;"1.0 . Witvliet, P.M.W. van der Meer-Prins, J.J. van Rood and F.HJ. Claas, Department of Immunohematology and Blood Bank,LeidenUniversity Hospital. Leiden, The Netherlands Some transplant centers consider paternal HLA-antigens as unacceptable mismatches fnr mothers awaitingkidney transplantation. It is feared that a pregnancy maycausepriming of the maternalimmune response directedtowards paternalIiLAantigens.Should a woman be transplanted with an organfrom a donor who shares those paternalHLA-antigens, tbe risk of graft rejection might be increased, It is known that some women, as a consequence of pregnancy develop antibodies specific for paternal HLA-. ntigens. To investigate whether a pregnancy can also prime the cellular immuneresponseand whether this occurs in all cases. the present study was set up. Frequencies of maternal cytotoxic T lymphocytes directed10 paternal HLA-antigens were evaluated in limitingdilution analysis assays. and compared with those directedto third party HLA-antigens. Differentiationbetween naive and in vivo primed cytotoxic T lymphocytes was made by performing these
MI CR OCIlIM ERI SM FR EQUENCY AND KI NETI CS CAD AVERI C KID NEY ALLOGRAFT RECIPIENTS
I
assays respe ctively in the absence and presen ce of anti-CDS. Na ive cytotoxic T
lymphocyte responses were detectedagainst paternal antigenswhich had never been inherited. and those which had been inherited but had nol inducedantibody formation. In contrast , inheritedpaternal antigens which had induced HLA-specific antibodies in the mother. gaverise to elevatedcytotoxic T lymphocyteprecursor frequencies. as comparedwith the response to thirdparty antigens. Also.the cytotoxic T lymphocytes were found to be more resistant to inhibition by anti-CD8, suggesting that these cells had been primed in vivo. These results suggestthat not all paternal HLA-antigens have to be considered as unacceptable mismatches. Only those individuals. who share a paternalHLA-antigen againstwhich a mother has formedHLA-specific allo-antibodies. should be excluded from organ donation.
IN
Legendre C. Kreis H. Bach JF and Caillat-Zucman S. INSERM U25 and Tran splantation Unit". Hop ital Necker. Paris.France. Whether the development of a donor -type microch imerism is responsible for a state o f long-term donor-specific allograft tolerance is still controversial. Here, we adress the question of the chimerism frequency and time kinetics following cadaveric kidney allografting, and of its functional relevance with graft survival . Four kidney recipient groups were studied for the presence of donor HLA class II alleles and H-Y sequences in peripheral blood and skin : ~ 6 patients studied 3 months after allografting, Jillll!I1.1; 14 patients with normal renal function 2 years after grafting, Jll2l!1L1: 11 patients analysed 10 years after transplantation (8 with normal renal function, 3 undergoing chronic rejection). g!Q!!I!..1; [2 long-term survi ving recipients presenting with normal renal function for more than 20 years. Donor class II genotyping was performed on DNA recovered from frozen graft biopsies. Peripheral microchimerism was detected using sequence-specific amplification (detection threshold 40 pg DNA, i.e 1I4()()() donor/rec ipient cell ratio). Miancbimerism was observed in the skin in the following cases: gm!!J! 1: 2 patients, ~: 2 patients. W!!!I!-3-: 4 patients (2 chronic reject ion, 2 normal graft function). ~ : 4 patients (not different from others by any cri terial .These data indicate that the presence of peripheral microchimerism is not mandatory for the development of prolonged graft acceptance.
Ab stracts
IV:6. Allorecognition, Tolerance and Immunosuppression in Transplantation 0876
SELF-RESTRICTED T CELL RECOGNITION OF DONOR HLA-DR PEPTIDES DURING GRAFf REJECTION Liu Zhn oru, Colovai Adriana I. Tugulea Soria. Reed Elaine F. Rose Eric A, Michler Robert E. Cohn David. Hardy MarkA. Renna-Molajooi Elvira, Cortesini Raffaele, Suciu-Foca Nicole College of Physicians & Surgeons of Columbia University. New York. NY USA. To investigate the role of indirect recognition in allograft rejection we have monitored a population of 30 heart and 15 kidney allograft recipients for the capacity of circulating and graft infiltratingT cells to react against allopcptides corresponding to the mismatchedHLA-DR antigens of the donor. Patients were followed for 1-8 months following transplantation, Blood was obtained at two weeksintervals and at the lime of graft biopsy. Lymphocytes from the blood and from biopsy lissues were test ed in LDA for react ivity to synt hetic pept ides corresponding to residue 1-19. 21-39 and 62-80 of donor HLA ORBI' chains. In 22 patients undergoing a total of 35 episodes of acute rejection. T cell reactivity to donor allopeptides was observed in the periphery and in the graft prior and/or at the time when an acute rejection episodeoccurred. 11:0 allopeptide reactive T cells were detected in the periphery or in the graft during periods of quiescence. LDA studiesof lymphocytes infiltrating the graft showed that the frequency of allopeptide-speciflc T cells within the graft was 3O-SO times higber than in the periphery. However, T cell recognition of a1lopeptides is likely (0 have first occurred in the peripherallymphoid tissue rather than in the graft since allopeptide-reactiveT cells wereoftendetected in the circulation 1-4 weeks prior to any histologic evidence of transplant rejection. Reactivity was directed againsta single dominant peptidecorresponding to one rather than (0 both of the mismatched Hl.Avantigens of the donor. Peptide dominancywas determined by the host's HLA-DR restrictive element. The finding that rejection is initiated by T cells which recognize a single dominant allopeptidc suggests the possibility of developing immunosuppressive peptides for specific immunotherapy. Furthermore, the presence of allopcptide-reactive T cells in the peripheryolTers a reliable way for monitoring rejection.
0878 IMPORTANCE OF A SINGLE AMINO ACID SUBSTITUTION OF THE a HELICES OF CLASS I MHC MOLECULES FOR THE INDUCTION OF A PRIMARY ALLOGENE IC RESPONSE Reboul Murielle ' , Noun Ghada " Abastado Jean·Pierr~ . Kourilsky Philippe*. PiaMarika' , 'Mouse Immunogenetics. U93 INSERM. Saint-Louis Hospital. Paris. France *U277INSERM. Pasteur Inslitute, Paris. France
Todelermine whether there is a relation between the localizationof a residue of a MHC class I moleculeand its implicafion inthe induction of a primary allogeneic response. we have tested the ability of various point mutants (a· helices or /l-sheel of the a 1 and a 2 domains) of an H-2 class I molecule to induce a cy1otoxic T cel response in mice carrying the native molecule. Murine L fibroblasts (H.2k hapofype) express ing different Kd mutants were injected into the hind footpads of (BIO.BR x C3H.OH) hybrid mice carrying the class I molecules of the H-i' haplotype together with the Kd molecule. In this combination. an eventual allogeneic response of these mice can only be induced by the singleamino acid difference between the nafive Kd molecule and the point mutant expressed by the fibroblasts_Three days after the injection. the cells from draining lymph nodes were cuhured lor 4 days (in Il ·2·supplemenled medium) in lhe absence of any stimulator cells . The presence of allogeneic CTl was tested in a 4-hr 5' Cr release assay. Under these conditions. on ly 7 out 01 25 mutant Kd molecules tested induced a primary allogeneicresponse. Interestingly all these mutations concern residues located on the ,,-helices. In addition. no correlation was observed between the s ize of the repertoire of peptides selected by these mutants and theircapacity to induce a primary allogeneic response.
0877 A LLOREA CTlV E HELPER T -LYMPHOC YTE PRECURSOR FREQUENCIES CORR EL ATE WITH HL A·DR ANTI GEN AMI NO ACID RESIDUE MISMATCHES Young NT, Roelen DL, Bunce M. Dallman MI. Morris PI, Welsh KI Nuffield Department of Surgery , University of Oxford, Oxford UK Interleukin-2 (IL-2) mediated helper activity is central to the cellular and humor al immune respons e induced by exposure to allogeneic HLA antigens. We have analysed the influence of HLA matching on the in vitro response of primary alloreactive helper T-Iymphocyte precursors (HTLp). Limiting dilution assays were employed to analyse frequencies of IL-2-producing HTLp in van ous liL A-mismatched responder:stimulator comhinations. Mea n IITLp frequencies were significantly (p
0879
TOLERIZING EFFECTS OF PRETRANSPLANTEXPOSURETO DONOR HLA·DR ANTIGEN IN RANDOM TRANSFUS[ONUN[TS FOR KIDNEY RECIPIENTS AM Jackson, C McSherry, K Butters, M Diko, S Almond, AJ Matas, NL Reinsmoen, University of Minoesota, Minneapolis. MN Ourprevious studieshave shown that thein vitroassay of donor antigen-specific hyporeactivity (DASH) is a useful marker for identifying solid organ transplant recipients (kidney, lung.heart or liver) ar low risk of immunologic complications(i.e.• tareacute rejection episodesandchronic rejection). DASH is defined as a significantly decreased post- vs. pretransplant (prctx) donor antigen-specific mixed lymphocyte response to donor cells, while response to third-party cells remains unchanged. We studied whetherpretx transfusions were associated with subsequent donor antigen. specific Tvcellnonrespoasiveoess, identified as DASH. in kidneyrecipientsexposed10 thesameIflA ·DR antigenpretx via the transfusion unit and posttx via the transplanted organ. In this study,24 previously nontransfused patients who received two 150 mI pretx random bloodtransfusions underthe cover of azathioprine are now I )T posttx; theIflA·DRofeachrandom transfusionunit was determined retrospectively using PCR· SSOP. Of the 24 patients. 7 (29%) werehyporesponsive at I }T posttx. Of these 7, 6 were exposed pretx to donor HLA·DR antigen via the transfusion unit vs. 6 of 17 responsive recipients (p=(l.04). Preliminary evidence indicates that, of recipients exposed pretx to donor HLA-DR antigen, those becoming hyporesponsive received fresher transfusion units (mean= It days old) vs. those who remained responsive (mean~24 days old). Neither HLA·DR sharing between the recipient and the transfusion unitsnor the time between tranfusion and tx (mean-e mol influenced thedevelopment of DASH. In conclusion. our results suggest that pretx exposure to donor HLA-DR antigen via fresh transfusion units enhanced the development of DASHfor recipients exposed to the sameHLA-DR antigen posttx via the transplanted organ.
0880
0881
PREGNANCY CAN INDUCEPRIMING OF CYTOTOXIC T LYMPHOCYTES SPECIflC FOR PATERNAL HLA-ANTIGENS, WIllCH IS ASSOCIATED WITHANTIBODY FOAAIATION GJ. Bouma. P. van Caubergh, F.P.MJ . van Bree, R.M.C Castelli-Visser, ;"1.0 . Witvliet, P.M.W. van der Meer-Prins, J.J. van Rood and F.HJ. Claas, Department of Immunohematology and Blood Bank,LeidenUniversity Hospital. Leiden, The Netherlands Some transplant centers consider paternal HLA-antigens as unacceptable mismatches fnr mothers awaitingkidney transplantation. It is feared that a pregnancy maycausepriming of the maternalimmune response directedtowards paternalIiLAantigens.Should a woman be transplanted with an organfrom a donor who shares those paternalHLA-antigens, tbe risk of graft rejection might be increased, It is known that some women, as a consequence of pregnancy develop antibodies specific for paternal HLA-. ntigens. To investigate whether a pregnancy can also prime the cellular immuneresponseand whether this occurs in all cases. the present study was set up. Frequencies of maternal cytotoxic T lymphocytes directed10 paternal HLA-antigens were evaluated in limitingdilution analysis assays. and compared with those directedto third party HLA-antigens. Differentiationbetween naive and in vivo primed cytotoxic T lymphocytes was made by performing these
MI CR OCIlIM ERI SM FR EQUENCY AND KI NETI CS CAD AVERI C KID NEY ALLOGRAFT RECIPIENTS
I
assays respe ctively in the absence and presen ce of anti-CDS. Na ive cytotoxic T
lymphocyte responses were detectedagainst paternal antigenswhich had never been inherited. and those which had been inherited but had nol inducedantibody formation. In contrast , inheritedpaternal antigens which had induced HLA-specific antibodies in the mother. gaverise to elevatedcytotoxic T lymphocyteprecursor frequencies. as comparedwith the response to thirdparty antigens. Also.the cytotoxic T lymphocytes were found to be more resistant to inhibition by anti-CD8, suggesting that these cells had been primed in vivo. These results suggestthat not all paternal HLA-antigens have to be considered as unacceptable mismatches. Only those individuals. who share a paternalHLA-antigen againstwhich a mother has formedHLA-specific allo-antibodies. should be excluded from organ donation.
IN
Legendre C. Kreis H. Bach JF and Caillat-Zucman S. INSERM U25 and Tran splantation Unit". Hop ital Necker. Paris.France. Whether the development of a donor -type microch imerism is responsible for a state o f long-term donor-specific allograft tolerance is still controversial. Here, we adress the question of the chimerism frequency and time kinetics following cadaveric kidney allografting, and of its functional relevance with graft survival . Four kidney recipient groups were studied for the presence of donor HLA class II alleles and H-Y sequences in peripheral blood and skin : ~ 6 patients studied 3 months after allografting, Jillll!I1.1; 14 patients with normal renal function 2 years after grafting, Jll2l!1L1: 11 patients analysed 10 years after transplantation (8 with normal renal function, 3 undergoing chronic rejection). g!Q!!I!..1; [2 long-term survi ving recipients presenting with normal renal function for more than 20 years. Donor class II genotyping was performed on DNA recovered from frozen graft biopsies. Peripheral microchimerism was detected using sequence-specific amplification (detection threshold 40 pg DNA, i.e 1I4()()() donor/rec ipient cell ratio). Miancbimerism was observed in the skin in the following cases: gm!!J! 1: 2 patients, ~: 2 patients. W!!!I!-3-: 4 patients (2 chronic reject ion, 2 normal graft function). ~ : 4 patients (not different from others by any cri terial .These data indicate that the presence of peripheral microchimerism is not mandatory for the development of prolonged graft acceptance.