Why do we reject grafts?

One of the puzzles in clinical and experimental organ and tissue transplantation has been why it is that, with the exception of transplantation between genetically identical twin pairs, transplantation results in a rapidly progressing immune response to the point of tissue necrosis in a matter of a few days, even though the recipient has not been immunized to the donor. Indeed, the numbers of T lymphocytes predetermined to react against grafts are relatively abundant compared to those that recognize microbial antigens. Cellular, molecular, and genetic studies are now revealing the nature of T-cell recognition, and why it is that we cannot freely exchange tissue grafts.

Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies.

The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.

Cellular and humoral mechanisms of vascularized allograft rejection induced by indirect recognition of donor MHC allopeptides.

INTRODUCTION: To investigate the role and mechanisms of indirect allorecognition in allograft rejection, we studied whether priming T cells with donor-derived MHC allopeptides could accelerate rejection in a vascularized allograft model. METHODS: Lewis recipients of fully mismatched Wistar Furth cardiac allografts were immunized before transplantation with donor MHC allopeptides. RESULTS: Animals immunized with immunogenic class II MHC allopeptides rejected their grafts in a significantly accelerated fashion compared with controls. Additional studies demonstrated that a single immunodominant RT1.D (HLA-DR like) allopeptide was responsible for accelerating the rejection process. Histological analysis of rejected allografts revealed marked vascular rejection in the accelerated, although not the control, group as well as severe cellular rejection. Peak production of IgM and IgG donor-specific alloantibodies was detected by flow cytometry 1 week earlier in the sera of the accelerated group compared with the control group. Immunohistological analysis of grafts from the accelerated compared with the control group showed increased endothelial deposition of IgG2b, C3, and fibrin, and up-regulation of class II MHC molecule expression. Increased intragraft expression of interferon-y and the interferon-gamma-induced chemokines, inducible protein-10 and Mig, and infiltration by activated mononuclear cells expressing CXCR3, the receptor for inducible protein-10 and Mig, was also seen. CONCLUSION: These novel data provide evidence of a definitive link between indirect allorecognition of donor-derived MHC class II peptides and the cellular and humoral mechanisms of vascularized allograft rejection.

Inhibition of allorecognition by a human class II MHC-derived peptide through the induction of apoptosis.

The interaction of the T-cell receptor with the major histocomatibility complex (MHC)-peptide complex is central to T-cell activation. Variation in the nature of the peptide bound within the groove of the MHC molecule may result in an altered T-cell response. Because some naturally processed peptides bound within the groove of the class II MHC molecule are derived from the MHC molecules themselves, we studied the inhibitory effects of synthetic class II MHC peptides on alloimmune responses in vitro. Three peptides derived from a highly conserved region of the class II MHC alpha chains inhibited the rat mixed lymphocyte response (MLR) in a dose-dependent manner, with the human HLA-DQA1 peptide also inhibiting the human and mouse MLR. No effect was seen on mitogen-induced T-cell proliferation. HLA-DQA1 inhibited cytolytic T lymphocyte (CTL) generation in a dose-response fashion, with no reduction in preformed CTL killing, suggesting that the inhibitory effect is targeted at CD4(+) T-cell function. Cell-cycle analysis by flow cytometry showed that restimulation of primed T cells in the presence of HLA-DQA1 resulted in increased apoptosis, whereas unstimulated cells were not affected. These data demonstrate that synthetic peptides derived from highly conserved regions of the class II MHC alpha chain can alter CD4(+) T-lymphocyte alloimmune responses in vitro, and this effect is mediated by the induction of apoptosis in activated T cells.

Late blockade of T cell costimulation interrupts progression of experimental chronic allograft rejection.

Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We used the fusion protein CTLA4Ig to block CD28-B7 T cell costimulation late after the onset of initial graft injury. Using the F334 into LEW rat model of chronic renal allograft rejection, transplant recipients were treated with a 10-d course of cyclosporine, and a subgroup received a single injection of CTLA4Ig at 8 wk after transplant. Functionally, CTLA4Ig administration prevented development of progressive proteinuria (14.3+/-4.1 mg/24 h versus 41.0+/-12.0 mg/24 h at 24 wk after transplant, P < 0.05). Histologically, graft mononuclear cell infiltration, glomerular hypertrophy, focal and segmental glomerulosclerosis, and intimal vascular hyperplasia were all attenuated in CTLA4Ig-treated animals. Lastly, reverse transcriptase-PCR and immunohistologic studies showed a significant reduction in the intragraft expression of key products of T cell and macrophage activation, and upregulation of what have recently been termed as "protective" genes, including the bcl family members, Bcl-2 and Bcl-xL, and hemoxygenase. Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection.

Mechanisms of indirect allorecognition: characterization of MHC class II allopeptide-specific T helper cell clones from animals undergoing acute allograft rejection.

BACKGROUND: Recent evidence indicates that T cells primed via the indirect pathway of allorecognition play an important role in allograft rejection, although the effector mechanisms remain unknown. The purpose of this study was to characterize and study the in vivo function of self-restricted MHC allopeptide-specific T-cell clones generated from animals undergoing allograft rejection. METHODS AND RESULTS: We generated self-restricted class II MHC allopeptide-specific T-cell clones from the spleen and kidney of Lewis (LEW; RT1l) rats undergoing acute rejection of MHC-incompatible Wistar Furth (WF; RT1u) renal allografts. RT1.Du/beta20-44 peptide-specific CD4+ T helper 1 clones from the spleen and kidney of rejecting animals expressed a restricted T cell receptor (TCR) Vbeta repertoire: Vbeta4, 8.2, or 9. In comparison, clones generated from RT1.Dubeta20-44 immunized LEW rats all expressed TCR Vbeta9. The amino acid sequence of RT1.Dl (LEW) and RT1.Du (WF) residues 20-44 differ only at positions 30 and 38. T-cell clones expressing TCR Vbeta9 preferentially proliferated to the peptide fragment RT1.Dubeta20-33. T-cell clones expressing TCR Vbeta4 proliferated weakly to peptide fragments RT1.Dubeta20-33 and 31-44, whereas those expressing TCR Vbeta8.2 proliferated preferentially to the peptide fragment 31-44. Adoptive transfer of T-cell clones expressing TCR Vbeta9 or Vbeta8.2, but not Vbeta4, to naive LEW animals elicited significant delayed-type hypersensitivity responses after challenge with the RT1.Dubeta20-44 peptide or allogeneic WF (RT1u) splenocytes. CONCLUSION: This is the first report on the cellular, molecular, and functional characterization of self-restricted MHC allopeptide-specific T-cell clones from animals undergoing acute rejection. Our data provide support for a biologically significant role of indirect allorecognition in allograft rejection.

Indirect allorecognition of major histocompatibility complex allopeptides in human renal transplant recipients with chronic graft dysfunction.

BACKGROUND: It has been suggested that T cells primed by processed donor major histocompatibility complex antigen (the "indirect" pathway of allorecognition) may be responsible for mediating chronic allograft rejection. The purpose of this study was to develop a clinically useful assay to study the occurrence of indirect allorecognition during chronic rejection in humans. METHODS: A panel of 20 mer peptides corresponding to the hypervariable regions of HLA-DRB1*0101, DRB1*1501, and DRB1*0301 were synthesized. Lymphocytes obtained from renal allograft recipients were cocultured with these peptides. Proliferation was assayed by DNA incorporation of [3H]thymidine, and positive proliferation was defined by a statistically significant increase in counts per minute over background with a minimum stimulation index of 2. The precursor frequency of allopeptide reactive T cells was determined by limiting dilution analysis. RESULTS: Lymphocytes from 82% of patients who were mismatched for at least one of the three DR molecules and had chronic allograft dysfunction specifically proliferated to the mismatched allopeptides (n=11). Proliferation was seen in only 6% of control subjects (2/33, P<0.0001). The proliferative response was low grade and was best detected on day 7-8 of culture in vitro. The precursor frequency of peptide-specific T cells was more than 10-fold higher compared with controls (P<0.001). CONCLUSIONS: These data demonstrate for the first time that T cells of patients with chronic graft dysfunction are primed to recognize and respond to specific donor-derived major histocompatibility complex allopeptides. Our results support the hypothesis that T cells primed via the indirect pathway of allorecognition may be important mediators of chronic rejection and provide the rationale to develop specific therapeutic strategies to prevent or interrupt this process.

Role of indirect allorecognition in experimental late acute rejection.

BACKGROUND: Late acute rejection affects up to 28% of renal allograft recipients and remains a major risk factor for late graft loss. As donor-origin antigen-presenting cells are depleted with time, T-cell recognition of donor-derived alloantigenic peptides presented by self antigen-presenting cells (the "indirect pathway" of allorecognition) may play a key role in the initiation of late acute rejection episodes. METHODS: To test this hypothesis, we developed a clinically relevant experimental model in the rat (Wistar-Furth/Lewis) in which allograft recipients received cyclosporine for 1 month after transplantation and were then allowed to reject the graft upon discontinuation of immunosuppression. Lymphocyte proliferation assays to synthetic class II MHC allopeptides of donor origin and also to intact donor (Wistar-Furth) cells were performed at this time. The effector mechanisms studied included delayed-type hypersensitivity (DTH) responses, lymphocyte-mediated cytotoxicity, and alloantibody production. RESULTS: Lymphocytes from recipients undergoing late acute rejection had marked suppression of mixed lymphocyte reaction proliferation to intact donor cells. Significant proliferation to donor-derived 25-mer polymorphic class II MHC allopeptides was elicited, however. In vivo, significant DTH responses were observed to both MHC allopeptides and intact Wistar-Furth cells. Recipient lymphocytes also exhibited significant killing of donor cells, although not third-party cells, and anti-donor alloantibodies were detected by flow cytometry. CONCLUSION: Our results indicate that T cells primed via the indirect pathway are present during acute rejection that occurs after discontinuation of cyclosporine. Mixed lymphocyte reactivity is markedly reduced at this time. Furthermore, there is an association between such allopeptide-primed T cells and the elicitation of specific DTH responses and provision of help to B cells to produce alloantibodies and activation of CD8+ T cells to become effector cytotoxic cells.

Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection.

Blocking CD28-B7 T-cell costimulation by systemic administration of CTLA4Ig, a fusion protein which binds B7 molecules on the surface of antigen-presenting cells, prevents rejection and induces tolerance in experimental acute allograft rejection models. We tested the effect of CTLA4Ig therapy on the process of chronic renal allograft rejection using an established experimental transplantation model. F344 kidneys were transplanted orthotopically into bilaterally nephrectomized LEW recipients. Control animals received low dose cyclosporine for 10 days posttransplantation. Administration of a single injection of CTLA4Ig on day 2 posttransplant alone or in addition to the low dose cyclosporine protocol resulted in improvement of long-term graft survival as compared with controls. More importantly, control recipients which received cyclosporine only developed progressive proteinuria by 8-12 weeks, and morphological evidence of chronic rejection by 16-24 weeks, including widespread transplant arteriosclerosis and focal and segmental glomerulosclerosis, while animals treated with CTLA4Ig alone or in addition to cyclosporine did not. Competitive reverse transcriptase-PCR and immunohistological analysis of allografts at 8, 16, and 24 weeks showed attenuation of lymphocyte and macrophage infiltration and activation in the CTLA4Ig-treated animals, as compared with cyclosporine-alone treated controls. These data confirm that early blockade of the CD28-B7 T-cell costimulatory pathway prevents later development and evolution of chronic renal allograft rejection. Our results indicate that T-cell recognition of alloantigen is a central event in initiating the process of chronic rejection, and that strategies targeted at blocking T-cell costimulation may prove to be a valuable clinical approach to preventing development of the process.

T cell recognition of xeno-MHC peptides during concordant xenograft rejection.

T cell recognition of xenoantigens is likely to play a key role in rejection of xenografts surviving hyperacute and delayed xenograft rejection, but the mechanisms of how this might occur are unknown. We used synthetic rat class II MHC peptides to study the role of the indirect pathway, where processed xenogeneic MHC antigens are presented in the context of self MHC, in a concordant xenograft rejection model in vivo. Mice of four different strains, BALB/c, B1O.A, CBA/ca, and C57BL/6j, were immunized with a mixture of rat class II MHC 25-mer xenopeptides representing the full-length sequence of the beta chain hypervariable domain of either RT1.Du (DR and I-E like) or RT1.Bu (DQ and I-A like) of the Wistar-Furth (WF) (RT1u) rat. Draining lymph node cells were capable of recognizing and proliferating to specific class II xeno-MHC peptides. The immunogenicity of the different peptides varied with the responder mouse strain. Responder T cells were CD4+, and were inhibited by anti-I-A and anti-I-E antibodies. We then examined the proliferative response of T cells from B1O.A primed by WF skin or vascularized cardiac xenografts to the class II MHC xenopeptides, when presented by naive B1O.A splenic antigen-presenting cells. These T cells were capable of proliferating to the same xeno-MHC peptides shown to be immunogenic by immunization. These data confirm the occurrence of self-restricted T cell recognition of xeno-MHC peptides in xenograft rejection, and provide the rationale for further investigating the role of the indirect pathway of recognition in xenotransplantation.

Mechanisms of oral tolerance by MHC peptides.

Recent evidence indicates that MHC peptides play an important role in T-cell recognition of alloantigen. We studied the tolerogenicity of orally administered synthetic MHC allopeptides in the rat model. Initially, we demonstrated that oral administration of synthetic class II MHC allopeptides significantly inhibited the DTH response to the peptides as well as to donor-derived cells. The tolerogenic effect was antigen specific and was induced by immunogenic, but not by nonimmunogenic, allopeptides. Immunohistological studies of DTH skin lesions showed that oral tolerance is associated with a state of "immune deviation" to a predominance of Th2 cell function in the lesions. We recently extended the above observations and examined the tolerogenic effect of orally administered synthetic MHC allopeptides on MLR and CTL generation. We found that oral administration of the class II allopeptides effected significant reduction of MLR proliferation and CTL generation, which was antigen specific. In addition, similar to the DTH results when we compared the tolerogenicity of the immunogenic versus the nonimmunogenic peptides, MLR and CTL suppression was significantly higher with the immunogenic peptides. The gut immune system play an important role in oral tolerance by MHC peptides. Initial experiments showed that intestinal epithelial cells pulsed in vitro with immunogenic MHC allopeptides, or in vivo by oral administration of immunogenic peptides, were capable of presenting these peptides to primed T cells in vitro. Whether such presentation by intestinal epithelial cells or other gut antigen-presenting cells leads to preferential activation of Th2 regulatory cells, which ultimately suppress Th1 alloimmune responses, remains to be determined.

Role of indirect allorecognition in allograft rejection.

It is now clear from animal studies that indirect allorecognition occurs during allograft rejection and that this pathway plays a role in mediating the rejection process. Whether this pathway is the dominant pathway responsible for chronic rejection remains to be established, but this is an intriguing hypothesis that may have major implications for development of novel therapies for this disorder. In addition, there are data to indicate that indirect allorecognition may play a role in maintenance of allograft acceptance, and that provision of appropriate MHC peptides via different routes can result in potent degrees of specific allo-tolerance. Expanded experimentation in animals and in humans is therefore in order; first, to dissect more closely the molecular basis of allorecognition and establish the role of the indirect pathway in acute versus chronic rejection, and second to utilize the immunomodulatory properties of MHC peptides or other novel strategies which targets indirect allorecognition in promoting graft acceptance.

Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo.

Experimental autoimmune encephalomyelitis (EAE), an experimental model for the study of multiple sclerosis, is an autoimmune disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP). MBP-reactive CD4+ T cells, predominantly expressing the V beta 8.2 T cell receptor (TCR), migrate from the peripheral lymphoid organs and initiate the inflammatory response in the brain. We have previously shown that a single intrathymic injection of MBP or its major encephalitogenic peptide (p71-90), but not a nonencephalitogenic peptide (p21-40), induces antigen-specific systemic tolerance and inhibits the induction of EAE in Lewis rats. In this study, we investigated the mechanisms of induction and maintenance of acquired thymic tolerance in this model. First, we investigated which thymic cell is responsible for "induction" of systemic tolerance. Thymic dendritic-enriched cells, isolated by plastic adherence, when incubated in vitro with p71-90 and injected intravenously into Lewis rats, were capable of preventing the development of EAE, but his protection was lost in thymectomized recipients. In addition, intravenous injection of thymic dendritic cells isolated from animals that had been previously injected intrathymically with p71-90 but not p21-40 also prevented the development of EAE. Second, to determine the "effector" mechanisms involved in acquired thymic tolerance, we compared TCR expression in the brains of animals with actively induced EAE with TCR expression in animals that received intrathymic injection of p71-90 or p21-40. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique, we found increased expression of CD4 and V beta 8.2 message in brains of immunized animals compared with those of naive animals. In animals intrathymically injected with p71-90 but not p21-40, CD4 and V beta 8.2 transcript levels were significantly reduced compared with immunized controls. Immunohistologic studies of brain tissue and spleens with specific V beta 8.2 and control V beta 10 monoclonal antibodies confirmed these observations in vivo. These findings, taken together with recent data demonstrating that activated T cells circulate through the thymus, suggest that interaction of thymic dendritic cells with specific TCR of activated peripheral T cells can lead to inactivation of these antigen-specific cells and confirm the role of V beta 8.2-expressing T cells in EAE.

Long-term failure of renal transplants: adding insult to injury.

Although transplantation is the preferred choice for many end-stage renal disease patients, results are far from perfect and the demand for organs exceeds the available supply. After high initial success rates at one year, the subsequent course of randomly HLA matched cadaveric organs is an exponential loss of functioning grafts, with a half-life of seven to eight years. This process is one of progressive sclerosis and fibrosis which may result from the inability of available immunosuppressive agents to control a chronic type of rejection, or it may be the result of early immunological injury with progressive vascular injury occurring as a result of hemodynamically induced injuries, as seen in renal ablation animal models. Matching for HLA antigens has a major impact on this process, with half-lives of 20 years with HLA-A, HLA-B, HLA-DR matched cadaver donors. Various clinical risk factors, including the relative size of the donor kidney, ischemic injuries, and drug toxicities all predispose to a more rapid rate of chronic graft loss. These are likely to be additive to the damaging effects of rejection activity, with a final pathway of glomerulovascular sclerosis initiated when a critically low level of functioning nephrons is reached.

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2.

Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.