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We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Five patients with end-stage kidney disease received combined kidney and bone marrow transplants from HLA haploidentical donors following nonmyeloablative conditioning to induce renal allograft tolerance. Immunosuppressive therapy was successfully discontinued in four patients with subsequent follow-up of 3 to more than 6 years. This allograft acceptance was accompanied by specific T-cell unresponsiveness to donor antigens. However, two of these four patients showed evidence of de novo antibodies reactive to donor antigens between 1 and 2 years posttransplant. These humoral responses were characterized by the presence of donor HLA-specific antibodies in the serum with or without the deposition of the complement molecule C4d in the graft. Immunofluorescence staining, ELISA assays and antibody profiling using protein microarrays demonstrated the co-development of auto- and alloantibodies in these two patients. These responses were preceded by elevated serum BAFF levels and coincided with B-cell reconstitution as revealed by a high frequency of transitional B cells in the periphery. To date, these B cell responses have not been associated with evidence of humoral rejection and their clinical significance is still unclear. Overall, our findings showed the development of B-cell allo- and autoimmunity in patients with T-cell tolerance to the donor graft.

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA., (Copyright 2008 Massachusetts Medical Society.)

This is the first study to examine the outcomes in 54 patients with hematologic malignancies who received an HLA-matched related donor bone marrow (BM, n = 42) or GCSF-mobilized peripheral blood stem cells (PBSC, n = 12) following identical nonmyeloablative conditioning with the intention of induction of mixed chimerism (MC) followed by prophylactic donor leukocyte infusion (pDLI) to convert MC to full donor chimerism (FDC) and capture a graft-versus-tumor effect without clinical graft-versus-host disease (GVHD). Neutrophil and platelet recovery were faster and transfusion requirement was less in PBSC recipients (P < 0.05). A total of 48% of BMT recipients achieved FDC with a median conversion time of 84 days, including 13 following pDLI. In contrast, 83% (P = 0.04) in the PBSC group had spontaneous FDC at a median of 14 days, precluding the administration of pDLI. There was no significant difference in the incidences of acute or chronic GVHD, though the rates of chronic GVHD were considerably higher in PBSC group than in the BM group (6/7, 86% vs 10/24, 42%). CD4 and CD8 T-cell recovery was faster in PBSC recipients. In PBSC recipients, a higher number of CD34+ cells was associated with increased rates of severe, grade III-IV acute GVHD.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

Background: We have previously demonstrated that xenogeneic bone marrow engraftment and donor-specific tolerance can be induced in mice receiving anti-CD4, -CD8, -Thy-1.2, and -NK1.1 monoclonal antibodies (mAbs) on Days -6 and -1, 3 Gy total body irradiation (TBI), and 7 Gy thymic irradiation on Day 0, followed by injection of T-cell depleted (TCD) rat bone marrow cells. We have recently demonstrated that anti-CD40L mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance in an allogeneic combination., Methods: We investigated the ability of anti-CD40L mAb to promote mixed xenogeneic chimerism and donor-specific tolerance in B6 mice receiving anti-CD8, -Thy1.2 and -NK1.1 mAbs and 3 Gy TBI followed by TCD bone marrow transplantation (BMT) from F344 rats., Results: Administration of anti-CD4 mAb in this model could be completely replaced by one injection of anti-CD40L mAb. Evidence for deletional tolerance was obtained in mixed chimeras prepared with this anti-CD40L-based regimen. However, anti-NK1.1 and anti-Thy1.2 mAb could not be replaced by anti-CD40L mAb., Conclusions: These results demonstrate that anti-CD40L in combination with xenogeneic BMT can tolerize preexisting peripheral and intrathymic CD4 cells to xenoantigens. However, anti-CD40L does not prevent NK cell and/or gammaDelta cell-mediated rejection of xenogeneic bone marrow.

Anti-CD154 (CD40L) monoclonal antibody (mAb) plus bone marrow transplantation (BMT) in mice receiving CD8 cell-depleting mAb leads to long-term mixed hematopoietic chimerism and systemic donor-specific tolerance through peripheral and central deletional mechanisms. However, CD4(+) T-cell tolerance is demonstrable in vitro and in vivo rapidly following BMT, before deletion of donor-reactive CD4 cells is complete, suggesting the involvement of other mechanisms. We examined these mechanisms in more detail. Spot enzyme-linked immunosorbent (ELISPOT) analysis revealed specific tolerization (within 4 to 15 days) of both T helper 1 (Th1) and Th2 cytokine responses to the donor, with no evidence for cytokine deviation. Tolerant lymphocytes did not significantly down-regulate rejection by naive donor-reactive T cells in adoptive transfer experiments. No evidence for linked suppression was obtained when skin expressing donor alloantigens in association with third-party alloantigens was grafted. T-cell receptor (TCR) transgenic mixing studies revealed that specific peripheral deletion of alloreactive CD4 T cells occurs over the first 4 weeks following BMT with anti-CD154. In contrast to models involving anti-CD154 without BMT, BMT with anti-CD154 leads to the rapid induction of anergy, followed by deletion of pre-existing donor-reactive peripheral CD4(+) T cells; the rapid deletion of these cells obviates the need for a regulatory cell population to suppress CD4 cell-mediated alloreactivity.

Objective: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism., Patients and Methods: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease., Results: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras., Conclusion: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.

Anti-CD40L mAb plus bone marrow transplantation (BMT) and recipient CD8 T-cell depletion permits long-term mixed hematopoietic chimerism and systemic donor-specific tolerance to be achieved across full MHC barriers. Initial tolerance is characterized by peripheral deletion of donor-reactive CD4 cells. In regimens using costimulatory blockade without BMT to achieve allograft survival, cyclosporine inhibited graft survival, suggesting that the combination may not be clinically applicable. We assessed the role of cyclosporine-sensitive mechanisms and the mechanisms of T-cell apoptosis involved in the induction of early peripheral CD4+ T-cell tolerance by BMT with anti-CD40L. Neither a short course of cyclosporine (14 days) nor the absence of FAS-mediated activation-induced cell death (AICD) blocked the induction or maintenance of donor-specific tolerance. IL-2 production was not associated with tolerance induction, consistent with the lack of a role for Fas-mediated AICD. Mice in which passive T-cell death was impaired because of constitutive expression of a Bcl-xL transgene did not develop tolerance with this protocol. These data confirm that deletion of donor-reactive T cells is critical for the induction of mixed chimerism and tolerance. However, the mechanisms involved may differ from those involved in costimulatory blockade regimens that do not include BMT.

We have previously shown that high levels of multiline-age mixed hematopoietic chimerism and systemic T-cell tolerance can be achieved in mice without myeloablation through the use of anti-CD40L and costimulatory blockade alone (plus CTLA4Ig) or with recipient CD8 depletion and allogeneic bone marrow transplantation. Chimeric mice permanently accept donor skin grafts (> 100 days), and rapidly reject third-party grafts. The mechanisms by which costimulatory blockade facilitates the engraftment of allogeneic hematopoietic cells have not been defined. To further understand the in vivo mechanisms by which the administration of anti-CD40L mAb facilitates the engraftment of donor bone marrow and rapidly tolerizes CD4+ T cells, we analyzed the establishment of chimerism and tolerance in CD40L -/- mice. We demonstrate here that anti-CD40L mAb treatment is required only to prevent CD40L/CD40 interactions, and that no signal to the T cell through CD40L is necessary for the induction of CD4+ tolerance. Peripheral deletion of donor-reactive CD4+ T cells occurs rapidly in CD40L -/- mice receiving bone marrow transplantation (BMT), indicating that this deletion in the presence of anti-CD40L is not due to targeting of activated CD4+ cells by the antibody. Complete CD4+ cell tolerance is observed by both skin graft acceptance and in vitro assays before deletion is complete, indicating that additional mechanisms play a role in inducing CD4+ T-cell tolerance as the result of BMT in the presence of CD40/CD40L blockade.

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.

Two major pathways of death of previously activated T cells have been described: activation-induced cell death can be triggered by restimulating activated T cells with high concentrations of Ag, is Fas-dependent, is not influenced by proteins of the Bcl family, and is blocked by cyclosporin A; in contrast, passive cell death is induced by the withdrawal of growth factors and activation stimuli, is Fas-independent, and is blocked by Bcl family proteins. We examined the role of these two forms of cell death in the peripheral deletion of donor-reactive host T cells after allogeneic bone marrow transplantation and costimulatory blockade with anti-CD154 plus CTLA4Ig in two murine models. The substantial decline in donor-reactive CD4 cells seen in wild-type recipients 1 wk after bone marrow transplantation with costimulatory blockade was largely inhibited in Fas-deficient recipients and in Bcl-x(L)-transgenic recipients. We observed these effects both in a model involving low-dose total body irradiation and a conventional dose of bone marrow, and in a radiation-free regimen using high-dose bone marrow transplantation. Furthermore, cyclosporin A did not completely block the deletion of donor-reactive CD4(+) T cells in recipients of bone marrow transplantation with costimulatory blockade. Thus, the deletion of donor-reactive T cells occurring early after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death. Furthermore, these in vivo data demonstrate for the first time the significance of in vitro results indicating that proteins of the Bcl family can prevent Fas-mediated apoptosis under certain circumstances.

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.