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Blocking the CD28-B7 T lymphocyte costimulatory pathway with the recombinant protein CTLA4Ig induces long term allograft survival in rodents. It has been suggested that this results from selective activation of the Th2 immune pathway. To test this hypothesis, we compared vascularized cardiac allograft survival in wild-type (IL-4 +/+) and homozygous IL-4 gene-knockout (IL-4 -/-) mice. We report in this study that long term survival (>100 days) of fully allogeneic grafts can be induced readily in IL-4 -/- recipients treated with a short course of CTLA4Ig. We also demonstrate that IL-4 -/- mice are deficient in Th2-type cytokine expression following in vitro or in vivo allostimulation. These results suggest that IL-4 production and subsequent generation of a Th2-type immune response are not obligatory for CTLA4Ig-induced long term acceptance of vascularized allografts.

It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen.Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo.In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts.Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.

Resting B (rB) cells are known to be incompetent APCs in vitro, which alone can induce specific unresponsiveness to single minor histocompatibility (miH) Ags and, when combined with CD40 pathway blockade, can induce hyporesponsiveness to MHC molecules in vivo. Here we show that anti-CD40 ligand (CD40L) mAb does not prevent the expression of B7-2 on allogeneic rB cells in vivo but did prolong donor-specific cardiac allograft survival. Moreover, pretreatment with professional APCs combined with anti-CD40L mAb induced hyporesponsiveness to alloantigens in vivo. rB cells from CD40 knockout mice were unable to induce unresponsiveness, while graft prolongation was achieved in CD40L knockout recipients pretreated with wild-type rB cells. These data suggest that CD40-CD40L interactions in the recipient play a critical role in the induction of hyporesponsiveness to alloantigens in vivo and that the effect of the CD40 pathway may be independent of its effect on the B7 costimulatory pathway.

The 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.. 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.Simultaneous 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.Long-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.

Blockade of the B7/CD28 costimulation pathway with the fusion protein, CTLA4-Ig, has been shown to prolong allograft survival in numerous rodent models, suggesting that this pathway is functionally important in the allograft rejection response. This pathway is complex and consists of at least the B7-1, B7-1a, B7-1cyt II, and B7-2 molecules on the antigen-presenting cell and CD28 and CTLA4 molecules on the T cell.The intragraft transcript expression of the B7 molecules and their counterreceptors was defined using reverse transcriptase-polymerase chain reaction in the vascularized mouse cardiac allograft model. In addition, the functional significance of these molecules was investigated both in vitro in the mixed leukocyte response (MLR) and in vivo in the vascularized mouse cardiac allograft model.Intragraft expression of B7-1, B7-1a, B7-1cyt II, B7-2, CD28, and CTLA4 transcripts is up-regulated in allografts when compared with both normal untransplanted hearts and syngeneic transplants at between 5 and 12 days after transplant. Both anti-B7-1 and anti-B7-2 monoclonal antibodies alone inhibited T-cell proliferation in the MLR, however, equivalent maximal inhibition was obtained by a combination of these agents or by CTLA4-Ig. Likewise, in the mouse cardiac allograft model, both anti-B7-1 and anti-B7-2 modestly prolonged graft survival. However, an increased survival was obtained with either a combination of anti-B7-1 and anti-B7-2 or CTLA4-Ig. Blockade of the B7/CD28 pathway in the MLR using T cells from CD28 knockout mice had no effect on the proliferative response. Likewise, blockade of the B7/CD28 pathway did not effect the rate of rejection of cardiac allografts by CD28 knockout recipients.These data suggest that both B7-1 and B7-2 have an important role in allograft rejection in the mouse vascularized cardiac allograft model.

Blocking the CD28-B7 T lymphocyte costimulatory pathway with the recombinant protein CTLA4Ig induces long term allograft survival in rodents. It has been suggested that this results from selective activation of the Th2 immune pathway. To test this hypothesis, we compared vascularized cardiac allograft survival in wild-type (IL-4 +/+) and homozygous IL-4 gene-knockout (IL-4 -/-) mice. We report in this study that long term survival (100 days) of fully allogeneic grafts can be induced readily in IL-4 -/- recipients treated with a short course of CTLA4Ig. We also demonstrate that IL-4 -/- mice are deficient in Th2-type cytokine expression following in vitro or in vivo allostimulation. These results suggest that IL-4 production and subsequent generation of a Th2-type immune response are not obligatory for CTLA4Ig-induced long term acceptance of vascularized allografts.

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.

The rejection of the transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD28. CTLA4-Ig is a fusion protein that has previously been shown to block the CD28-mediated costimulatory signal and inhibit immune responses in vitro and in vivo. In this report we show that treatment of the C3H/He recipient of a BALB/c vascularized cardiac allograft with a 12-day course of CTLA4-Ig produced indefinite graft survival (100 days) in the majority of recipients. In addition, these recipients demonstrated donor-specific transplantation tolerance when tested with donor-specific (BALB/c) and third-party (C57BL/10) skin grafts. These results demonstrate that CTLA4-Ig can induce transplantation tolerance in the adult murine cardiac allograft model.