BACKGROUND:Differences in quality and strength of immune responses between individuals are mainly due to polymorphisms in major histocompatibility complex (MHC) molecules. Focusing on MHC class-II, we asked whether the intensity of human anti-pig T-cell responses is influenced by genetic variability in the human HLA-DRB1 and/or the porcine SLA-DRB1 locus. METHODS:ELISpot assays were performed using peripheral blood mononuclear cells (PBMCs) from 62 HLA-DRB1-typed blood donors as responder and the porcine B cell line L23 as stimulator cells. Based on the frequency of IFN-γ-secreting cells, groups of weak, medium, and strong responder individuals were defined. Mixed lymphocyte reaction (MLR) assays were performed to study the stimulatory capacity of porcine PBMCs expressing different SLA-DRB1 alleles. RESULTS:Concerning the MHC class-II configuration of human cells, we found a significant overrepresentation of HLA-DRB1*01 alleles in the medium/strong responder group as compared to individuals showing weak responses to stimulation with L23 cells. Evaluation of the role of MHC class-II variability in porcine stimulators revealed that cells expressing SLA-DRB1*06 alleles triggered strong proliferation in approximately 70% of humans. Comparison of amino acid sequences indicated that strong human anti-pig reactivity may be associated with a high rate of similarity between human and pig HLA/SLA-DRB1 alleles. CONCLUSION:Variability in human and porcine MHC determines the intensity of individual human anti-pig T-cell responses. MHC typing and cross-matching of prospective recipients of xenografts and donor pigs could be relevant to select for donor-recipient combinations with minimal anti-porcine immunity.

RT1.L class I antigens have originally been identified in LEW rats by LEW.1LV3-anti-LEW.1LM1 antisera and have been classified as nonclassical. We report now that LEW.1LV3-anti-LEW.1LM1 antisera react with three different antigens, termed RT1.L1, RT1.L2, and RT1.L3. This was found by serological analysis of a panel of transfectants expressing different class I genes of strain LEW with a LEW.1LV3-anti-LEW.1LM1 antiserum and two monoclonal antibodies (mAbs HT20 and HT21) generated in the same strain combination. The antiserum reacted with all three antigens: the two mAbs with RT1.L1 and RT1.L2, respectively. Sequence analysis showed that the genes encoding RT1.L1, RT1.L2, and RT1.L3 cluster together in a phylogenetic analysis of rat and mouse alpha(1)-alpha(2) sequences and that they share an unusual MHC class I promoter in which Enhancer A and B, as well as the interferon response element (IRE), are missing. Exchange of the promoter in RT1.L2 against the classical RT1.A promoter resulted in high surface expression in appropriate transfectants, indicating that the deviant promoter is responsible for the weak surface expression of the RT1.L2 gene. The very similar promoter structures of RT1.L1 and RT1.L3 are likely to contribute also to the weak expression of these genes. As RT1.L3 maps closely to the deletion in the mutant haplotype lm1, the RT1.L family can be located in the class I region extending from Bat1 to Pou5f1. Different from other allogeneic mAbs detecting known class I molecules encoded by genes of the RT1.C/E region, HT20 and HT21 react with a wide panel of strains carrying different RT1 haplotypes. This suggests that nonclassical class I genes of the RT1.L family are present in most RT1 haplotypes.

Background. In clinical organ transplantation monoclonal antibodies (mAb) to different surface molecules of immunocompetent cells become integral parts of the immunosuppressive therapy. In this study, a mAb against the rat leukocyte common antigen CD45 (RT7) was tested for its immunosuppressive potency after a single perioperative injection. Methods. Binding and depleting properties of the anti-RT7 mAb were investigated by flow cytometry. In the fully major histocompatibility complex–disparate heart and skin transplantation models (LEW [RT1l] → LEW.1W [RT1u]), a single dose of anti-RT7 mAb (10 mg/kg) was administered intravenously (day −1). To characterize the long-term acceptance of heart allografts second set skin transplantation (day 100), mixed lymphocyte reaction studies (day 100) and reverse transcriptase-polymerase chain reaction analysis for intragraft cytokine expression (day 200) were performed. Results. The anti-RT7 mAb bound to nearly all hematopoietic lineage cells, but particularly T and NK cells, and profoundly depleted these cells in circulation and lymphoid tissues. Anti-RT7 mAb-treated rats showed long-term acceptance of heart allografts (>200 days; n=12), whereas untreated recipients rejected allografts by day 8 (n=6). In contrast to hearts, primary skin allograft survival was only moderately prolonged. Animals with stable heart allograft acceptance showed normal in vitro lymphocyte proliferation responses to donor and third party antigen. These recipients also acutely rejected second set donor-strain skin grafts without inducing rejection of persisting heart allografts. Reverse transcriptase-polymerase chain reaction analysis of intragraft cytokines showed up-regulation of Fas-ligand and IL-4 mRNA in long-surviving heart allografts. Conclusions. The findings demonstrate that a single injection of an anti-RT7 mAb in the rat can induce stable long-term acceptance of heart allografts by transient but profound T-cell depletion. Local immunoregulatory mechanisms seem to play a role for maintenance of long-term graft acceptance.

Objective. Organ allografts contain passenger leukocytes that are transferred to the recipient with the transplantation, but their functional relevance to the recipient's immune system is still controversial.Materials and Methods. To clarify the functional capacity of passenger leukocytes, we attempted to enhance their effect in rat heart allograft recipients by selective depletion of recipient leukocytes using a monoclonal antibody (mAb) against a recipient-specific allotype of CD45 (RT7(a)).Results. Although antibody treatment of the recipient alone led to profound lymphopenia and reversible myelosuppression, additional transplantation of an major histocompatibility complex-incompatible heart graft from an RT7(b) donor led to lethal aplastic anemia in the recipients. This lethal effect was completely abrogated by postoperative anti-CD3 treatment of the recipient and was partially abrogated or delayed by depletion of passenger leukocytes through additional anti-RT7(b) antibody treatment of the recipient or gamma -irradiation of the graft,Conclusions. The results suggest a role for both donor and recipient-type T cells for the induction of aplastic anemia in this model. The study shows that, under defined conditions, allogeneic passenger leukocytes in a heart graft can have a profound effect on the recipient's immune system and bone marrow, (C) 2001 International Society for Experimental Hematology. published by Elsevier Science Inc.

With an organ transplant, hematopoietic donor cells are transferred to the recipient. To study the relevance of the resulting microchimerism for allograft acceptance, we analyzed a rat model of cyclosporine-induced tolerance for strongly incompatible heart allografts. Using a monoclonal antibody that detects a donor-specific CD45 allotype (RT7(a)), we selectively depleted donor leukocytes at different times after transplantation (days 0 or 18). Depletion was similarly effective at both times. However, only depletion on day 0 prevented tolerance induction and was associated with severe acute or chronic graft rejection. This indicates that passenger leukocytes have an essential immunomodulatory effect on the induction phase of allograft acceptance.

46 Purpose. It has been a controversial issue whether donor-derived leukocyte microchimerism has functional relevance for allograft acceptance. To clarify this we have tried to eliminate donor-derived hematopoietic microchimerism after transplantation in an allogeneic rat heart transplant model using RT7 (allomorphism of rat common leukocyte antigen) different strain combinations. In this setting donor-derived hematopoietic cells can be selectively eliminated by an antibody against donor-type RT7. Methods. Allografts from male LEW.1W (RT1u. RT7a) donors were transplanted to female LEW.7B (RT11, RT7b) recipients, and the recipients received 15 mg/kg/day cyclosporine (Cy) for 14 days i.m. A monoclonal antibody (mAb) against RT7a was injected to the recipients on days 0 or 18 to eliminate donor-derived hematopoietic cells. The mAb does not affect the graft itself. Microchimerism in peripheral blood was serially checked by PCR analysis for Y-chromosome until day 100. Graft survival and histology of the grafts on day 200 were analyzed. Results. Table Anti-RT7a mAb treatment both on day 0 and on day 18 successfully eliminated PCR detectable microchimerism in the long-term course. One graft was acutely rejected on day 23 after mAb treatment on day 0, all other allografts survived for >200 days. By this time grafts showed severe chronic rejection with massive infiltration by class II positive cells and myointimal proliferation of arteries after mAb treatment on day 0 (group 5), however, while signs of chronic rejection were only mild or minimal in recipients without mAb treatment (group 3) or after mAb treatment on day 18 (group 4). Conclusions. Elimination of microchimerism very early after transplantation (day 0) lead to development of severe chronic rejection in the long-term course, while later elimination (day 18) had no effect. The results suggest a significant role of allogeneic hematopoietic cells only for induction but not for maintenance of allograft acceptance.Table

114 Purpose. The functional relevance of passenger leukocytes in allogeneic organ transplantation is still unclear. To study it more detail we have applied a rat transplant model where we can selectively and transiently eliminate the recipient's lymphocytes without affecting donor lymphocytes. For this purpose rat strains differing in an allomorphic form of the leukocyte common antigen (RT7a/RT7b) and an antibody against one of these antigens (RT7a) have been used. Methods. LEW˙ 1W (RT1u, RT7a) rats were injected with anti-RT7a mAb on day -1. On day 0, these rats received either no or syngeneic (LEW˙1W) or allogeneic (LEW:RT1l, RT7a or LEW˙7B:RT1l, RT7b) heart grafts. In this setting both recipient's leukocytes and leukocytes ofRT7a donors are eliminated by anti-RT7a mAb, while those of RT7b donors are not affected. Some of the recipients with LEW˙7B grafts also received anti-CD3 mAb.Results. After anti-RT7a mAb treatment all rats showed leukopenia most markedly in T cells. Leukopenia recovered over 4-6 weeks in rats that received RT7a allografts (group 3), and these rats survived indefinitely as rats that received no (group 1) or syngeneic grafts (group 2). The rats that received RT7a allografts showed further deteriorated leukopenia after 2 weeks and died of severe anemia and/or bleeding (group 4). This lethal effect was abrogated by anti-CD3 treatment on day 2 (group 5). Autopsy in group 4 showed severe atrophy of the lymphoid tissues. No evidence of graft-versus-host disease was shown. Immunohistology on day 2, but not later, revealed considerable numbers ofRT1l donor cells in the lymphoid organs in group 4, while no donor cells were detected in group 3. Conclusions. The rats with RT7b allografts showed lethal lymphoid aplasia, while the rats with RT7a allografts survived indefinitely. This suggests an essential role of passenger leukocytes from the heart graft for induction of lethal lymphoid aplasia. Abrogation of this lethal effect by anti-CD3 mAb treatment indicates that donor-derived T cells play the most important role. This study shows that under certain conditions even small numbers of passenger leukocytes can have the profound functional relevance.Table

152 Purpose. Common leukocyte antigen is expressed on all hematopoietic cells, and is known to have costimulatory function for T cell activation. We have generated a new monoclonal antibody (mAb) against the rat common leukocyte antigen RT7. This mAb specifically binds an allomorphic region of the RT7 molecule. The purpose of this study was to explore the effects of this mAb ex vivo and in vivo. Methods. Ex vivo effects of this anti-RT7 mAb on non-specific and allospecific lymphocyte proliferation were evaluated. Lymph node cells of LEW 1W (RT1u) rats were stimulated by anti-CD3 mAb or Con-A or by irradiated allogeneic lymph node cells of LEW (RT1l) rats. Anti-RT7 mAb was added to the culture medium at final concentrations of 0, 1, 10, and 100 µg/ml. [3H]TdR uptake was measured on days 3 and 5. In vivo immunosuppressive effect of the mAb was evaluated in an allogeneic rat heart transplant model. Heart grafts of LEW donors were transplanted heterotopically to LEW 1W recipients. Recipients were treated with a single dose of anti-RT7 mAb i.v. on day -1 (n=9). Leukocyte numbers and subpopulations in peripheral blood were serially analyzed, and graft survival was compared to recipients without mAb treatment (n=6). Results. Ex vivo lymphocyte proliferation, both for non-specific (anti-CD3, Con-A) and allospecific stimulation, was significantly enhanced by anti-RT7 mAb in dose-dependent fashion. In the heart transplant model, single injection of anti-RT7 mAb induced indefinite graft acceptance (>200 days × 3, >100 days × 6), while all grafts in non-treated recipients were rejected within 8 days. After injection of anti-RT7 mAb recipients showed transient leukopenia most strikingly of CD3 positive cells that recovered to the normal levels over 4-6 weeks. No other side effects were observed in the long-term course. Conclusions. Our newly generated anti-RT7 mAb induced long term allograft acceptance in a rat heart transplant model in vivo, while enhancing non-specific and allospecific lymphocyte proliferation ex vivo. Although the mechanism of action still has to be elucidated, anti-common leukocyte antigen antibody treatment may be a potent strategy for tolerance induction.

The RT6 alloantigenic system of the rat has originally been defined on T lymphocytes of the peripheral lymphatic organs and has been considered to be selectively expressed on mature peripheral T cells. Studying NK cells and intestinal intraepithelial lymphocytes (IEL), we have now found that both cell types also express RT6 and that the expression patterns found for IEL and NK cells were markedly different from each other and also from the expression pattern previously described for T cells of the peripheral lymphatic organs. In lymph nodes, spleen, and blood both RT6- and RT6+ T cells have been found and the density of RT6 expression on the positive cells has been shown to vary over a broad range. In contrast more than 98% of intestinal IEL stained for RT6 and the RT6 density was about tenfold higher than on strongly positive T cells of the peripheral lymphatic organs. Furthermore, the same high RT6 density was also found on IEL of athymic nude rats althogh these cells, to a large extent, lacked other T cell markers. This probably indicates that RT6 expression is an early event in the maturation of intestinal IEL which can occur already before the expression of T cell-specific membrane molecules. The conclusion that the expression of RT6 may be differently regulated in IEL and other T cell populations was further substantiated by the observation that RT6 was also present on IEL of diabetes-prone BB rats which are known to lack RT6 positive T cells in peripheral lymphatic organs. For NK cells still another pattern of RT6 expression was found. Unlike peripheral T cells and IEL, only a small subset of NK cells in blood and spleen expressed RT6. The percentage of RT6 positive cells was increased by in vitro stimulation of isolated NK cells with high concentrations of recombinant rat IL-2 indicating that RT6 expression may be associated with an activated state in NK cells. Taken together, these findings demonstrate that the expression of RT6 is not restricted to T cells and is differently regulated in normal peripheral T cells, intestinal IEL, and NK cells. Since it has recently been demonstrated that the RT6 gene contains two functional promoter regions with major structural disparity it is very likely that the distinct patterns of RT6 expression in different cell types reflect the differential use of the two promoters. The development of this complex control of RT6 expression in evolution may have been driven by a beneficial effect resulting from the use of the RT6 molecular function by several different lymphocyte populations.