Eric T. Elwood

Asialo GM1+ CD8+ T cells play a critical role in costimulation blockade–resistant allograft rejection

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.

CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway.

Studies in vivo have documented the importance of CD40-gp39 interactions in the development of T-dependent antibody responses to foreign and auto-antigens. In this report, we demonstrate that allograft rejection is also associated with strong induction of CD40 and gp39 transcripts. When treatment was initiated at the time of transplant, MR1, a mAb specific for gp39, induced markedly prolonged survival of fully disparate murine cardiac allografts in both naive and sensitized hosts. However, when therapy was delayed until postoperative day 5, anti-gp39 failed to prolong graft survival. Allografts from recipients treated with MR1 from the time of transplantation showed decreased expression of transcripts for the macrophage effector molecule, inducible nitric oxide synthase, but essentially unaltered expression of B7 molecules and T cell cytokine transcripts (interleukin [IL]-2, interferon-gamma, IL-10, and IL-4) relative to control allografts. In addition, alloantibody responses in the MR1-treated mice were profoundly inhibited. However, our studies using B cell-deficient mice indicated that the ability of MR1 to prolong allograft survival was not dependent on B cells. These data suggest that blockade of CD40-gp39 interactions may inhibit allograft rejection primarily by interfering with T cell help for effector functions, rather than by interference with T cell activation.

Microchimerism and rejection in clinical transplantation

BACKGROUNDnHaemopoietic microchimerism has been identified in recipients of solid-organ transplants and is thought by some to be critical for the development and maintenance of immunological tolerance. The aim of this study was to correlate prospectively the persistence of donor cells with clinical outcome in recipients of kidney, kidney and pancreas, and liver transplants.nnnMETHODSnPersistence of donor cells in recipient peripheral blood was assessed at 3 days, and at 1, 3, 6, and 12 months after transplantation by a two-stage nested PCR technique to detect donor MHC HLA DR gene specifically. A pretransplant blood sample was collected from each patient to serve as an individual negative control. Seven liver, six kidney and pancreas, and 17 kidney patients were enrolled. 12 of the 17 kidney patients and all of the kidney and pancreas, and liver recipients were suitable for analysis. Exact matches for donors and recipients at the HLA DR loci (n = 1) or inability to obain primer pair specificity among similar HLA DR types (n = 4), meant that we were unable to analyse five patients.nnnFINDINGSnDonor DNA was detected in 20 (80%) of 25, ten (40%) of 25, seven (30%) of 23, five (22%) of 23, and six (32%) of 19 recipients at 3 days, and 1, 3, 6 and 12 months post-transplant, respectively. Within individuals, the detection of donor DNA varied over time; only two patients had detectable donor DNA at all times. Analysis of the whole group of transplant patients showed a similar frequency and severity of rejection episodes in patients with and without microchimerism as defined by detectable donor DR genes.nnnINTERPRETATIONnThese data suggest that a significant percentage of the recipients had persistent donor class II DNA in the peripheral circulation for at least 1 year after transplantation. We showed that a pretransplant blood sample is critical to avoid a false-positive result, and suggest that detectable chimerism may vary over time in individual patients. Therefore, analysis of microchimerism with a single, post-transplant analysis may not help in making clinical decisions for individual patients.

Prolonged acceptance of concordant and discordant xenografts with combined CD40 and CD28 pathway blockade

BACKGROUNDnThe prompt and vigorous immune response to xenogenic tissue remains a significant barrier to clinical xenotransplantation. Simultaneous blockade of the CD28 and CD40 costimulatory pathways has been shown to dramatically inhibit the immune response to alloantigen.nnnMETHODSn. In this study, we investigated the ability of simultaneous blockade of the CD28 and CD40 pathways to inhibit the immune response to xenoantigen in the rat-to-mouse and pig-to-mouse models.nnnRESULTSnSimultaneous blockade of the CD28 and CD40 pathways produced marked inhibition of the cellular response to xenoantigen in vivo and produced long-term acceptance of xenogeneic cardiac and skin grafts (rat-to-mouse), and markedly suppressed an evoked antibody response to xenoantigen. In addition, this strategy significantly prolonged the survival of pig skin on recipient mice.nnnCONCLUSIONSnLong-term hyporesponsiveness to xenoantigen across both a concordant and discordant species barrier, measured by the stringent criterion of skin grafting, can be achieved using a noncytoablative treatment regimen.

CTLA4-Ig plus bone marrow induces long-term allograft survival and donor-specific unresponsiveness in the murine model : Evidence for hematopoietic chimerism

Allograft rejection is dependent on T cell activation, which requires both the engagement of the T cell receptor by antigen in the context of the MHC molecules and costimulatory signals delivered by cell surface molecules such as B7-CD28/CTLA4 pathway. CTLA4-Ig is a fusion protein that blocks this pathway and has previously been shown to prolong both allograft and xenograft survival. The current study demonstrates markedly prolonged murine cardiac allograft survival and specific prolongation of secondary skin grafts using a combination of CTLA4-Ig plus donor bone marrow. A role for hematopoietic chimerism in the establishment of CTLA4-Ig-induced transplantation tolerance was investigated using reverse transcriptase polymerase chain reaction analysis of recipient tissues. Expression of donor-specific MHC class II transcripts in both peripheral and lymphoid tissues was demonstrated at greater than 200 days after transplant. To investigate the functional significance of this observation, heart donors, and donor bone marrow were irradiated before transplantation in CTLA4-Ig-treated recipients. A reduction in allograft survival was associated with irradiation of both the donor heart and the bone marrow. These results suggest that there may be a donor-derived radiosensitive element that enhances allograft survival in this model. Reverse transcriptase polymerase chain reaction analysis of allografts of tolerant and control animals at days 5, 8, and 12 after transplantation failed to demonstrate a dramatic difference in the expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma message. Cytotoxicity effector transcripts were largely intact in CTLA4-Ig + bone marrow-treated recipients as they showed no decrease in intragraft granzyme, perforin, Fas, or Fas ligand transcripts during thr first 8 days after transplant. These results imply that complex mechanisms may be important for the induction and maintenance of transplantation tolerance in the CTLA4-Ig plus bone marrow murine cardiac allograft model.

Fasl is important in costimulation blockade-resistant skin graft rejection

Background. Simultaneous blockade of the CD40 and CD28 costimulatory pathways is effective in prolonging allograft survival in murine and primate models. Recent data suggest that intact apoptotic pathways are crucial for the induction of hyporesponsiveness by costimulation blockade. We have studied the impact of fas/fasL signaling, an important T cell apoptotic pathway, on the effects of costimulation blockade. Methods. Wild type, lpr (fas deficient), and gld (fasL deficient), mice were used as donors and recipients in the murine skin graft model. Allograft survival was compared in untreated and costimulation blockade (500 &mgr;g anti-CD40L and 500 &mgr;g CTLA4-Ig, days 0, 2, 4, 6) treated recipients. In some recipients, CD4+ T cells were depleted using rat anti-murine CD4 (100 &mgr;g day −3, −2, −1, and weekly). Results. gld mice treated with costimulation blockade enjoy a significantly greater increase in skin allograft survival than do wild-type mice. This effect is not replicated using lpr donors or recipients. Experiments in which CD4+ cells were depleted demonstrate that fasL is not necessary for CD8-mediated allograft rejection, and that depletion of CD4+ cells eliminates some of the survival advantage induced by costimulation blockade. Conclusions. FasL is not required for the establishment of costimulation blockade induced hyporesponsiveness, but rather appears to be required for normal costimulation blockade resistant rejection. Fas expression is not critical for costimulation blockade resistant rejection, suggesting that fasL may be interacting with other receptors. Further, it appears that CD4+ cells are important in the maintenance of allograft protection induced by costimulation blockade in this model.