MHC matching and mechanisms of alloactivation in corneal transplantation.

BACKGROUND: In human corneal transplantation the value of matching, particularly for MHC class II, is unclear and controversial. The contribution of the direct pathway to T cell activation is also uncertain. We have determined the relative contribution of class I, II and non-MHC antigens to graft rejection and of the direct and indirect pathways to T cell activation in a rat model mimicking human incompatibilities. METHODS: DA (RT1a) strain recipients received fully mismatched PVG (RT1c) strain grafts or grafts from one of three recombinant strains bearing DA MHC genes on a PVG background. Graft survival was assessed and the specificity of T cells generated in the draining lymph nodes was determined in mixed lymphocyte (MLR) proliferation assays. To assess the contribution of the direct pathway, fully mismatched graft were performed and allospecific proliferation was measured after depletion of recipient APC from the MLR reaction. RESULTS: There was no significant difference in survival of grafts between the four grades of mismatch, which ranged from a full mismatch to non-MHC mismatches alone (median survival 12.5, 11, 13 and 12.5 days respectively). In conformity with clinical results, strong secondary responses were generated against targets matched for MHC with the recipient. Depletion of recipient APC from a fully allogeneic secondary MLR did not fully abrogate donor-specific proliferation. CONCLUSIONS: Class II matching is of no benefit in this model. Strong indirect responses to non-MHC mismatches are sufficient to induce the rapid rejection, but the small numbers of class II+ cells in the donor appear sufficient to generate a direct response.     

Specific suppression of allograft rejection by soluble class I antigen and complexes with monoclonal antibody

In this experiment, we investigated the effect of daily injection or continuous slow infusion of either DA (MHC haplotype, RT1a) serum or soluble DA class 1 MHC antigen or its complexes with monoclonal antibody on rejection of heterotopic heart allograft in the combination of PVG.RT1a (RT1a) donor into PVG (RT1c) recipient. DA serum delayed significantly both the early and late rejection of PVG.RT1a heart grafts in PVG recipients. Removal of soluble class I MHC antigen from DA serum by affinity chromatography on a monoclonal anti-class I antibody column completely abolished the immunosuppressive effect. Continuous infusion of purified soluble class I antigen from DA rat liver, even from day 4 after heart grafting, induced a significant prolongation of graft survival. This effectiveness was donor-specific and amplified by the mixture of monoclonal anti-class 1 (RT1a) antibody with DA serum--this being induced only by using continuous infusion but not by daily injection. The results indicate that soluble class I antigen can act as a specific immunosuppressive agent in allograft rejection and that its effect is amplified by monoclonal anti-class 1 antibody.     

Major histocompatibility complex control of NK-related allogeneic lymphocyte cytotoxicity in rats. The contributions of strong and medial transplantation antigens

Allogeneic lymphocyte cytotoxicity (ALC) describes the elimination of allogeneic lymphocytes in vivo by an NK-related activity. There is evidence that ALC is demonstrable between donor and recipient when these are incompatible at MHC gene loci alone. Since ALC is a property of T cell-deficient nude rats, the role of the MHC in this rejection process needs further study. We have determined the contribution of the MHC to ALC using congenic and recombinant rats. In our analysis we have assumed that ALC involves the recognition of classic alloantigens by clonally distributed effector cells as for other examples of transplant rejection, although this is not yet proved. Strong ALC was measured between congenic rats that differed for MHC genes only. Non-MHC incompatibility alone did not elicit ALC. In the presence of MHC incompatibility the strength of ALC generated in a recipient was dependent on non-MHC genes. The PVG background generated high ALC responses whereas ALC was not measured in the DA rat. However ALC was measured in the congenic PVG-RT1avl (DA) rat. The contributions of classic class I (RT1.A), class II (RT1.B/D), and medial transplantation (RT1.C) regions of the rat MHC were determined by comparing different recombinant donors into the same recipient strain. Single region differences alone in any of these three MHC regions did not elicit full ALC. In two sets of transfers a combination of RT1.B/D and RT1.C region incompatibility was sufficient to generate a full allogeneic response. It can be concluded that the controlling element for allogeneic lymphocyte cytotoxicity is in the RT1.B/D-RT1.C region of the rat MHC.     

Regulatory function of human CD4+ cytolytic T lymphocytes.

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.     

Depletion of recipient CD4+ but not CD8+ T lymphocytes prevents the development of cardiac allograft vasculopathy

BACKGROUND: We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8+ direct allorecognition or by CD4+ indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8+ or CD4+ T cell depletion. METHODS: Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8+ and CD4+ T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4+ or CD8+ T cell depletion. RESULTS: PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8+ T cells. In contrast, chimeric hearts transplanted into permanently CD4+ T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV. CONCLUSIONS: In this specific strain combination, recipient CD8+ T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4+ T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4+ indirect allorecognition and that CD8+ direct allorecognition stimulated by nonprofessional APCs plays a minor role.     

Allograft rejection in athymic nude rats by transferred T cell subsets. II. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to an isolated MHC class I disparity

Athymic PVG-rnu/rnu (RT1c) rats were grafted with skin bearing isolated MHC disparities 7-14 days in advance of cell transfer. The ability of naive CD4+ or CD8+ thoracic duct lymphocytes to induce rejection was assessed by adoptive transfer of one or both T cell subsets into nude recipients bearing congenic PVG.r1 (MHC class I-only disparity, Aa) or PVG.r19 (class I and II-only disparity, Aa B/Da) skin grafts. Recipients of purified CD4+ TDL always rejected r19 allografts, whereas CD8+ TDL were ineffective against this class I + II difference. Neither the injection of CD4+ TDL nor CD8+ TDL alone resulted in destruction of r1 skin grafts. However, rejection of r1 tissue was observed in 63% of cases (19/30) when both CD4+ and CD8+ TDL were present in the nude recipients. Rejection of r1 skin was also induced in some recipients when CD8+ TDL were transferred 8 weeks in advance of CD4+ TDL. In contrast, sequential transfer in the reverse order apparently induced tolerance in the CD4+ population--i.e., surviving r1 skin grafts on 8 week CD4+ T cell-reconstituted nude recipients were not rejected following the subsequent transfer of CD8+ TDL. We conclude that CD4+ T cells were required for rejection of both class I and class II differences. In the presence of a class II difference, CD4+ T cells function autonomously to initiate both the inducer and effector stages of rejection. When the disparity is confined to class I, CD8+ T cells are required (probably at the effector stage) but are dependent on CD4+ T cells for help. There was no evidence of CD4+ effector T cells that could recognize class I directly within the graft.     

Rejection of grafts with no H-2 disparity in TAP1 mutant mice: CD4 T cells are important effector cells and self H-2b class I molecules are target

Our previous results showed that TAP1 mutant mice rejected heart and skin grafts from donors with no H-2 disparity that express normal density of MHC class I molecules at the cell surface. During rejection, CD4 cells were predominant and essentially, no CD8 cells were found infiltrating the grafts. We hypothesized that TAP1 mutant mice, which developed and matured in an MHC class I-deficient environment, may have selected a repertoire of T cells with distinct reactivity to self class I molecules. The rejection of grafts with no H-2 disparity could be mediated by CD4+ T cells reactive to wild type H-2b class I molecules, or derived peptides, in the context of self-APC. Accordingly, we observed that transplanted TAP1 mutant mice presented a significant amplification of the proliferative T cell response to H-2Kb peptides, indicating that the stimulus with the graft was sufficient to induce peripheral expansion of these T cell repertoires. Therefore, the response to H-2Kb molecules could be a relevant pathway of activating T cells and triggering rejection of grafts expressing normal levels of these class I molecules. To test our hypothesis, we investigate the effect of pre-transplantation H-2Kb peptide-immunization on TAP1 mutant, which were then transplanted with C57BL/6 skin grafts (H-2b). Mice were immunized with a pool of five peptides derived from the polymorphic region of Kb alpha chain, before tail skin grafting. To study the role of CD4+ T cells in the rejection of C57BL/6 skin grafts, mice were in vivo depleted with an anti-CD4 monoclonal antibody GK1.5, and transplant evolution was observed. Sensitization of TAP1 mutant mice with H-2Kb peptides accelerated the rejection of skin grafts. Immunized mice rejected grafts with a MST of 13 days, compared to 16 days for the non-immunized mice (P=0.0089). The significant acceleration of graft rejection, induced by immunization with H-2Kb peptides, indicates that these peptides are capable of mobilizing effector T-cells that participate in rejection. These results support our hypothesis that class I molecules may be a target in the rejection of grafts with no MHC disparity. Depletion of CD4 T-cells resulted in a significant delay in rejection compared with the untreated control group. The MST of skin grafts in the controls was 16 days, whereas CD4-depleted recipients rejected skin grafts with a MST of 41 days (P=0.025). Moreover, some animals did not show macroscopic signs of rejection up to > 100 days posttransplantation. The contribution of CD4+ T cells to skin graft rejection, in our model, may reflect the occurrence of the presentation of H-2b peptides during graft rejection, in the context of self-APC. In conclusion, our results demonstrate an important role for H-2b molecules and CD4 T cells in the rejection of C57BL/6 grafts by TAP1 mutant mice. The low expression of MHC-I molecules on TAP1-/- mice may be determinant in the selection of a T cell repertoire strongly reactive to self MHC class I molecules which probably escapes the control of peripheral regulatory mechanisms.     

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.     

Factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model: effect of CTLA4lg treatment on indirect alloactivation induced by immunization with donor MHC I peptides

Abstract We have defined factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model and analyzed the influence of CTLA4Ig treatment on indirect alloactivation induced by donor MHC I peptides in a DA → LEW heart allograft model. Indirect allorecognition of MHC I led to accelerated graft rejection and was accompanied by the induction of anti-peptide antibodies and donor pep-tide-activated T cells. In an attempt to block the B7-induced costimulatory signal of T cell activation, CTLA4Ig was administered to graft recipients in addition to MHC I peptide treatment. CTLA4Ig therapy, however, was not effective in preventing the humoral or cellular anti-donor immune response, nor did it prevent accelerated graft rejection.     

The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts

BACKGROUND: Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS: The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS: We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS: Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.     

Allograft survival following immunization with membrane-bound or soluble peptide MHC class I donor antigens: factors relevant for the induction of rejection by indirect recognition

T cells recognize foreign antigens in the form of peptide fragments resulting from antigen processing by antigen-presenting cells. In contrast to this indirect recognition, MHC molecules of foreign cells can be directly recognized by T cells. Direct recognition has for a long time been considered the only mechanism responsible for transplant rejection. Recent studies have provided evidence of a role of indirect recognition in rejection. In the current series of experiments, we studied the influence of indirect alloactivation, induced either by donor MHC class I peptides or by membrane-bound MHC I molecules, on heart allograft rejection in rats. Recipients were immunized before transplantation with synthetic donor MHC I peptides. The animals developed antibody and T-cell responses. Depending on the rat strain, peptide pretreatment either had no effect on graft survival (DAPVG; untreated controls 8.5±0.6 days, treated rats 9.5±0.6 days) or led to accelerated rejection (DALEW; untreated controls 7.5±0.3 days, treated rats 5.1±0.2 days; P(0.0002). Importantly, sensitization by indirect activation induced acute rejection in a donor-recipient combination (LEW.1ALEW.1WR2) in which neither direct nor indirect recognition led to rejection (untreated controls>400 days, pretreated rats 15±4.2 days). Another group of animals was immunized with allogeneic or congenic erythrocytes carrying the MHC I antigen from which the peptides were derived. Although the immunization elicited a measurable immune response, it did not lead to accelerated rejection. We conclude that sensitization by indirect recognition is able to initiate an acute rejection even in recipients in which neither direct nor indirect recognition is effective, and that this effect is strain-dependent. The form in which the donor antigen is administered is decisive for the induction of rejection by indirect activation.This work was supported by a grant of the Deutsche Forschungsgemeinschaft Bornn     

Alloantibody-associated chronic rejection of MHC class I-disparate heart grafts is modulated by intravenous soluble donor alloantigen

Increasing evidence suggests that there may be a causal relationship between the development of donor-specific alloantibodies and chronic allograft vasculopathy (CAV). PVG.RT1(u) rat heart allografts spontaneously undergo chronic rejection when transplanted into unmodified PVG.R8 congenic recipients that differ only at the classical MHC class I RT1.A locus. Here we show that development of vasculopathy in this experimental model is associated with production of a strong anti-A(u) antibody response. Perioperative intravenous administration of recombinant soluble RT1.A(u) heavy chain that is sequence identical to donor MHC class I, or chimaeric A(u/a) (donor/recipient) protein had a variable effect resulting generally in either sensitisation and accelerated rejection, or abrogation of alloantibody and attenuation of chronic rejection. These findings highlight the potential for soluble donor MHC class I alloantigen given at the time of heart transplantation to influence alloantibody production and graft outcome.     

Multiple pathways in the rejection of skin grafts.

We have analyzed the ability of CD4+ and CD8+ T cells to cause rejection of skin grafts in an Ir gene high responder strain. (DA.RT1u x DA.RT1c)F1 B rats (thymectomized, lethally irradiated, reconstituted with fetal liver cells) were grafted with ear skin of the recombinant strain, DA.RT1rl. The only allogeneic difference was a single class I MHC antigen. The B rats, which do not reject these grafts due to the absence of T cells, were reconstituted at various time intervals after skin grafting with either unsorted lymph node cells (LNCs), CD4+, CD8+ or CD4+ and CD8+ T cells. Unsorted LNCs given any time after graft placement always caused rejection (MST = 15d). CD4+ cells alone never caused rejection (MST greater than 60d, n = 8). CD8+ cells alone caused rejection if given within 3 weeks of graft placement. Thereafter, CD8+ cells alone lost their ability to cause rejection (MST greater than 60d, n = 6). B rats with grafts in place more than 3 weeks, when CD8+ cells alone were ineffective, rejected their skin grafts when given both CD8+ and CD4+ cells. These data suggest that there may be two T cell pathways in skin graft rejection. The first requires only CD8+ cells and causes rejection of a recently placed graft. The second pathway requires both CD4+ and CD8+ cells to reject long-standing grafts in which donor antigen-presenting cells have been putatively depleted and, therefore, may be dependent on host antigen-presenting cells.     

Immunological tolerance-related genes in a spontaneous tolerant model of rat liver transplantation explored by suppression subtractive hybridization

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgapl), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.     

IgM and IgG alloantibody responses to MHC class I and II following rat renal allograft rejection. Effects of transplantectomy and posttransplantation blood transfusion.

Renal allograft rejection in rats and humans is a potent inducer of alloantibody to donor major histocompatibility complex antigens. Alloantibody in such presensitized recipients can cause hyperacute rejection of subsequent renal allografts. In order to characterize alloantibody production in rats presensitized by renal graft rejection, ACI (RT1a) kidneys were transplanted into untreated fully allogeneic PVG (RT1c) recipients and allowed to reject while one native kidney remained in situ for host survival. Serum samples collected at weekly intervals were analyzed by flow cytometry for IgM and IgG antibody binding to ACI lymphoid target cells. The specificity of alloantibody responses was assessed by (1) differential binding to congenic rat strain target cells expressing only donor class I (PVG.R1) versus both donor class I and II (PVG.1A) antigens, (2) differential binding to unseparated donor lymphoid target cells versus lymphoid target cells depleted of class II MHC antigen-expressing cells, and (3) specific blocking of monoclonal antibodies to donor class I (R2/10P, R2/15S) or class II (F17.23.2) epitopes. Alloantibody responses to both donor class I and II MHC antigens were detected. The initial IgM response to donor class I MHC antigens peaked at the time of rejection, followed by a steady decline to relatively low levels by 4 weeks posttransplantation. The IgM response to donor class II MHC antigens was found to be cyclical with apparent peaks at day 7 and 5-6 weeks. The IgG response to donor class I and class II MHC antigens reached maximum by 5-6 weeks before slowly decreasing. IgM and IgG alloantibody specific for class I and class II MHC antigens could be detected through 19 weeks posttransplantation. The effects on circulating alloantibody of two manipulations, posttransplantation donor specific blood transfusion and allograft removal, were examined in this model. The alloantibody responses to class I MHC antigens were not affected by giving DSBT weekly beginning at day 14 after transplantation. However, posttransplantation DSBT eliminated the second peak of IgM alloantibody to class II MHC antigens seen approximately 5-6 weeks posttransplantation and also decreased circulating IgG specific for class II antigens. Transplantectomy at day 5-7 days after transplantation had no apparent effect on circulating IgM or IgG alloantibody through 7 weeks posttransplantation. These data indicate that in a fully allogeneic rat renal allograft model alloantibody responses are elicited to both class I and II MHC donor antigens, but that the kinetics and regulation of the responses to class I differ from those to class II alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. II. Sequential changes in alloreactivity of T cell subsets

A 10-day course of cyclosporine treatment inhibits the capacity of DA rats to reject PVG heart grafts and leads to the development of specific unresponsiveness and indefinite graft survival, which is mediated by a W3/25+ (CD4+) suppressor cell. In this study the sequential changes in the alloreactivity of the CD4+ and CD8+ subsets of CsA-treated DA rats were examined. During the induction phase, 8 and 20 days posttransplant, W3/25+ cells retained normal alloreactivity in that they adoptively restored PVG heart graft rejection in irradiated DA rats. By day 50 they had lost their capacity to restore rejection of PVG grafts but still retained their capacity to effect third party W/F graft rejection. W3/25+ cells from control grafted rats adoptively restored PVG graft rejection even at 75 days posttransplant, suggesting that the loss of alloreactivity of W3/25+ cells in CsA-treated rats was due to the prevention of rejection by CsA, and not a consequence of sensitization to alloantigen. MRCOX8+ cells from CsA-treated rats showed some evidence of sensitization at days 8 and 20 but lost this by day 50. These studies showed that during the induction phase, normal alloreactivity of W3/25+ cells is retained and sensitization of MRCOX8+ cells occurs. Specific loss of reactivity and suppressor potential of W3/25+ cells developed later, when specific unresponsiveness to second donor strain grafts developed in these hosts.     

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4⁺ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8⁺ T cells participate in rejection. To address this, we placed MHC class I-deficient D^bK^b knockout skin grafts onto allogeneic H-2 ^k SCID recipients followed by adoptive transfer of purified H-2 ^k CD8⁺ T cells. The MHC class I-deficient grafts were rejected and only CD8⁺ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8⁺ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8⁺ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.     

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed na?ve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or na?ve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either na?ve or tolerant hosts effected rejection. In contrast neither tolerant nor na?ve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.     

Morphologic studies of acute rat cardiac allograft rejection across an isolated major histocompatibility complex class I (RT1A) disparity

To define the morphologic correlates of acute rat cardiac allograft rejection across an isolated major histocompatibility complex (MHC) class I disparity, rejecting PVG.R1 cardiac allografts transplanted to (PVG x WF)F1 recipients were studied from days 4-8 posttransplantation. Documented ultrastructural tracer techniques as well as immunohistologic and immunoelectron microscopic methods were employed for morphologic analysis. Using intravenously administered horseradish peroxidase as a tracer probe for cell membrane permeability dysfunction, it was shown that severe diffuse loss of integrity of the microvascular endothelium preceded functional rejection, providing strong evidence that the allograft microcirculation is a central target of graft destruction. Also, rejection was associated with localized cardiac myofiber alterations prior to development of significant endothelial changes, indicating that cardiac muscle cells are additional cellular targets of immunologic injury. The ultrastructural features of progressive endothelial and myofiber injury, the predominance of MRC OX8+ lymphocytes and MRC OX6+ macrophages sequestered within the grafts, and the pattern of donor class I expression by allograft endothelium and cardiac myofibers were similar to those observed in rejecting allografts in full MHC-disparate combinations. Since it has been previously shown that MRC OX8+ class I-reactive T cells are absolutely required for rejection in this isolated class I-disparate model, the morphologic data raise the possibility that the OX8+ T lymphocyte subpopulation may also play a highly significant role in rat cardiac allograft rejection across a full MHC disparity.     

Studies on the participation of different T cell subsets in rat liver allograft rejection

In this study, we investigated which subsets of rat T cells (CD8 + vs. CD4 + ) are involved in the rejection of liver allografts by the in vivo administration of monoclonal antibody (OX-8 or OX-38, and W3/25 MAb) into thymectomized recipient Lewis (RTI¹) rats prior to DA (RTI^a) liver transplantation. We also compared the results of allograft survival of liver and heart transplants under the same experimental conditions. In order to deplete either CD8 + T cells or CD4 + T cells from recipient animals, 0.4 ml of OX-8 (ascitic form) or a 0.8 ml cocktail of MAb W3/25 and OX-38 (0.4 ml each) was injected into thymectomized recipient rats, respectively. Untreated Lewis rats consistently rejected donor DA liver grafts between 9 and 11 days (n = 7, 9.8 days ± 1.1 days). In contrast, anti-CD8 MAb pretreatment extended the survival times of DA liver grafts for up to 40 days (n = 5, 26.8 days ± 8.4 days). Furthermore, survival of DA liver grafts was significantly prolonged in Lewis rats that had been pretreated with anti-CD4 MAb (n = 7,35.6 days ± 17.9 days). Two out of seven recipient animals survived for more than 60 days. For heart transplantation, untreated Lewis rats rejected DA heart grafts between 6 and 8 days after operation (n = 6, 6.5 days ± 1.2 days). Anti-CD4 MAb treatment prolonged heart graft survival for more than 60 days in all cases (n = 3, > 60 days). However, there was virtually no effect of anti-CD8 MAb treatment on heart graft survival (n = 4, 7.0 days ± 0.9 days). These results suggested that when whole MHC disparity prevailed between donor and recipient, both subsets of T cells were required for the rejection of liver allografts and that class II reactive T cells predominantly mediated liver graft rejection. Furthermore, CD8 + T cells played a differential role in the rejection of rat liver and heart allograft.