Thomas Wekerle

Allogeneic bone marrow transplantation with co-stimulatory blockade inducesmacrochimerism and tolerance without cytoreductive host treatment

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.

Mechanisms of transplant tolerance induction using costimulatory blockade

The potential use of costimulation-blocking reagents to induce transplantation tolerance has recently created considerable excitement. Recent evidence has begun to delineate the mechanisms by which these powerful effects occur. It has become increasingly clear, firstly, that T cell costimulation is mediated by a delicate network of signaling pathways and, secondly, that interference with these systems can lead to numerous different tolerance mechanisms, including immune regulation, anergy and deletion.

Five-Year Safety and Efficacy of Belatacept in Renal Transplantation

Belatacept is a first-in-class co-stimulation blocker in development for primary maintenance immunosuppression. A Phase II study comparing belatacept with cyclosporine (CsA) for prevention of acute rejection and protection of renal function in kidney transplant recipients demonstrated similar efficacy and significantly higher measured GFR at 1 year for belatacept, but the incidence of posttransplantation lymphoproliferative disorder was higher. Here, we present the results for the extension of this trial, which aimed to assess long-term safety and efficacy of belatacept. Seventy-eight of 102 patients who were receiving belatacept and the 16 of 26 who were receiving CsA completed the long-term extension period. GFR remained stable in patients who were receiving belatacept for 5 years, and the incidences of death/graft loss or acute rejection were low. The frequencies of serious infections were 16% for belatacept and 27% for CsA, and neoplasms occurred in 12% of each group. No patients who were treated with belatacept and one patient who was treated with CsA developed posttransplantation lymphoproliferative disorder during the follow-up period. Serious gastrointestinal disorders occurred more frequently with belatacept (12% belatacept versus 8% CsA), and serious cardiac disorders occurred more frequently with CsA (2% belatacept versus 12% CsA). Pharmacokinetic analyses showed consistent exposure to belatacept over time. CD86 receptor saturation was higher in patients who were receiving belatacept every 4 weeks (74%) compared with every 8 weeks (56%). In conclusion, this study demonstrated high patient persistence with intravenous belatacept, stable renal function, predictable pharmacokinetics, and good safety with belatacept over 5 years.

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.

BACKGROUND Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. METHODS C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. RESULTS Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. CONCLUSIONS One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.

Peripheral Deletion After Bone Marrow Transplantation with Costimulatory Blockade Has Features of Both Activation-Induced Cell Death and Passive Cell Death

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-xL-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.

Treg‐Therapy Allows Mixed Chimerism and Transplantation Tolerance Without Cytoreductive Conditioning

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short‐course costimulation blockade and rapamycin. This combination treatment led to long‐term multilineage chimerism and donor‐specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3‐transduced Tregs, natural Tregs and TGF‐β induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

The Role of Non‐Deletional Tolerance Mechanisms in a Murine Model of Mixed Chimerism with Costimulation Blockade

Peripheral and central clonal deletion are important tolerance mechanisms in models using bone marrow transplantation (BMT) with costimulation blockade (CB). However, since tolerance can be found before peripheral deletion is complete and since elimination of recipient CD4+ cells at the time of BMT prevents tolerance induction, we investigated the potential roles of regulation and anergy in such a murine model. We found that transient elimination of CD25+ cells or neutralization of IL2 immediately after BMT and CB prevented the induction of skin graft tolerance. Cotransfer into SCID mice of CD4+ cells taken from chimeras early after BMT, together with naïve recipient‐type CD4+ cells significantly prolonged donor skin graft survival. In contrast, cotransfer of CD4+ cells harvested from chimeras late after BMT did not prolong donor skin graft survival. Besides, depletion of CD25+ cells in established chimeras several months post‐BMT did not break tolerance. In vivo administration of recombinant IL2 inhibited chimerism and tolerance neither early nor late post‐BMT, arguing against a decisive role for classical anergy. Thus, CD4 cell‐mediated regulation contributes significantly to tolerance induction early after BMT, but appears to have no critical role in the maintenance of tolerance.

Transplantation tolerance through mixed chimerism

The major factors that limit the success of organ transplantation are the host immune response to the foreign graft and the adverse effects of the chronic immunosuppressive therapy required to suppress this immune response. Deliberately establishing tolerance towards the donor tissue by reprogramming the immune system of the recipient thus holds great promise in improving organ transplant survival and eliminating the untoward effects of chronic drug therapy. The transplantation of donor bone marrow into recipients who are appropriately conditioned to allow development of either full or mixed chimerism has long been recognized to effectively induce donor-specific tolerance. Despite the demonstrated effectiveness of this technique, use of the mixed chimerism strategy in regular clinical practice has been hampered by the toxic side effects inherent to conventional bone marrow transplantation protocols. This Review addresses recent advances in preclinical and clinical studies inducing transplantation tolerance through mixed chimerism and discusses both the potential and the challenges of this approach.

Early regulation of CD8 T cell alloreactivity by CD4+CD25– T cells in recipients of anti-CD154 antibody and allogeneic BMT is followed by rapid peripheral deletion of donor-reactive CD8+ T cells, precluding a role for sustained regulation

While acquisition of regulatory function by CD4+CD25– T cells has been reported following antigenic stimulation, “naturally occurring” regulatory CD4+ T cells (Treg) are believed to express CD25. We examined the mechanisms involved in peripheral CD8 T cell tolerance by induction of mixed chimerism using non‐myeloablative conditioning with low‐dose (3 Gy) total body irradiation and anti‐CD154 antibody. Recipient CD4+ T cells were initially required for the induction of CD8 cell tolerance, but were not needed beyond 2 weeks. Depletion of CD25+ Treg prior to bone marrow transplantation and blockade of IL‐2 with neutralizing antibody did not impede tolerance induction. Tolerance was dependent on CTLA4, but not on IFN‐γ. In C57BL/6 mice containing a fraction of 2C TCR transgenic CD8+ T cells, which recognize the MHC class I alloantigen Ld, induction of chimerism with Ld+, but not Ld–, bone marrow cells led to deletion of peripheral 2C+ CD8+ cells within 1 week in peripheral blood and spleen. Complete deletion required the presence of recipient CD4+ T cells. Thus, a novel, rapid form of regulation by CD4+CD25– T cells permits initial CD8 T cell tolerance in this model. Rapid peripheral deletion of donor‐specific CD8 T cells precludes an ongoing requirement for CD4 T cell‐mediated regulation.

Mechanisms involved in the establishment of tolerance through costimulatory blockade and BMT: lack of requirement for CD40L-mediated signaling for tolerance or deletion of donor-reactive CD4+ cells.

We have previously shown that high levels of multilineage 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.