Preclinical and clinical studies for transplant tolerance via the mixed chimerism approach

Based upon observations in murine models, we have developed protocols to induce renal allograft tolerance by combined kidney and bone marrow transplantation (CKBMT) in non-human primates (NHP) and in humans. Induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex (MHC)-mismatched primates, with detectable chimerism typically disappearing within 30–60?days. Nevertheless, in MHC mismatched NHP, long-term immunosuppression-free renal allograft survival has been achieved reproducibly, using a non-myeloablative conditioning approach that has also been successfully extended to human kidney transplant recipients. CKBMT has also been applied to the patients with end stage renal disease with hematologic malignancies. Renal allograft tolerance and long-term remission of myeloma have been achieved by transient mixed or persistent full chimerism. This review summarizes the current status of preclinical and clinical studies for renal and non-renal allograft tolerance induction by CKBMT. Improving the consistency of tolerance induction with less morbidity, extending this approach to deceased donor transplantation and inducing tolerance of non-renal transplants, are critical next steps for bringing this strategy to a wider range of clinical applications.     

Recent progress in tolerance induction through mixed chimerism

Organ transplant recipients require life-long treatment with immunosuppressive drugs. Currently available immunosuppression is associated with substantial morbidity and mortality, and is ineffective in inhibiting chronic rejection and graft loss. Therefore, a permanent state of donor-specific tolerance remains a primary goal for transplantation research. The induction of mixed hematopoietic chimerism is an attractive concept in this regard. Hematopoietic chimerism modulates the immunologic repertoire by extending the mechanisms of self-tolerance to donor-specific allotolerance. Despite recent progress in developing nontoxic bone marrow transplantation protocols for rodents, translation to large animals has remained difficult. Here, we outline the concept of tolerance via mixed chimerism, and review recent progress and remaining challenges in bringing this approach to the clinical setting.     

Manipulating the immune system for anti-tumor responses and transplant tolerance via mixed hematopoietic chimerism

Summary: Stem cells (SCs) with varying potentiality have the capacity to repair injured tissues. While promising animal data have been obtained, allogeneic SCs and their progeny are subject to immune-mediated rejection. Here, we review the potential of hematopoietic stem cells (HSCs) to promote immune tolerance to allogeneic and xenogeneic organs and tissues, to reverse autoimmunity, and to be used optimally to cure hematologic malignancies. We also review the mechanisms by which hematopoietic cell transplantation (HCT) can promote anti-tumor responses and establish donor-specific transplantation tolerance. We discuss the barriers to clinical translation of animal studies and describe some recent studies indicating how they can be overcome. The recent achievements of durable mixed chimerism across human leukocyte antigen barriers without graft-versus-host disease and of organ allograft tolerance through combined kidney and bone marrow transplantation suggest that the potential of this approach for use in the treatment of many human diseases may ultimately be realized.     

Immune monitoring of transplant patients in transient mixed chimerism tolerance trials

This review focuses on mechanistic studies performed in recipients of non-myeloablative bone marrow transplant regimens developed at Massachusetts General Hospital in HLA-identical and HLA-mismatched haploidentical combinations, initially as a platform for treatment of hematologic malignancies with immunotherapy in the form of donor leukocyte infusions, and later in combination with donor kidney transplantation for the induction of allograft tolerance. In patients with permanent mixed chimerism, central deletion may be a major mechanism of long-term tolerance. In patients in whom donor chimerism is only transient, the kidney itself plays a significant role in maintaining long-term tolerance. A high throughput sequencing approach to identifying and tracking a significant portion of the alloreactive T cell receptor repertoire has demonstrated biological significance in transplant patients and has been useful in pointing to clonal deletion as a long-term tolerance mechanism in recipients of HLA-mismatched combined kidney and bone marrow transplants with only transient chimerism.     

Monoclonal antibody therapy for the induction of transplantation tolerance

There are three ways in which monoclonal antibodies could be used to facilitate the induction of tolerance to foreign tissues after organ transplantation. First, depleting monoclonal antibodies could be directed against the T cells responsible, thereby reducing their number and acting to non-specifically immunosuppress the patient. This is generally not sufficient to allow tolerance induction in the T cells which repopulate the periphery. Second, depleting monoclonal antibodies could be used to remove donor passenger leukocytes and antigen-presenting cells from the donor organ, which may both reduce immunogenicity and increase the chance of tolerance induction. Third, non-depleting, but functionally blocking, monoclonal antibodies to T cell molecules such as CD4 and CD8 can allow the specific induction of transplantation tolerance in mouse models, an approach which might be applicable to man, not only for organ transplantation, but also in the treatment of autoimmune diseases. These three approaches are, in time, likely to complement each other in clinical practice. Monoclonal antibodies can be tailored to each approach by choosing appropriate specificities and isotypes, and further refinements can be made where necessary by making monovalent or humanised antibodies. The application of each of these approaches to clinical therapy is described.     

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.     

Donor‐specific tolerance induction in organ transplantation via mixed splenocytes chimerism

We have shown previously that donor‐derived splenocytes can replace recipients' bone marrow and induce donor‐specific tolerance (DST). We have also shown the usefulness of the chimeric state for the induction of DST. Further analysis of mixed splenocytes chimera, especially the role of each T cells in mixed splenocytes chimera, is indispensable issue for its clinical use. A chimeric state has been shown to achieve long‐term survival in major histocompatibility complex (MHC)‐mismatched grafts. The donor‐derived splenocytes can replace recipients' bone marrow and induce DST. The long‐term survival of allogeneic skin grafts was achieved without immunosuppressants. In this study we show the role of each T cell type in a splenocyte mixed chimera. This review provides a short summary of our original work, adding some supplemental interpretations. Mixed chimerism is thus considered an attractive approach for the induction of DST without the use of immunosuppressants. In this paper, we summarize some of the findings on mixed splenocyte chimeras and review mixed chimerism in recent organ transplantation.     

Minor histocompatibility antigens: targets for tumour therapy and transplant tolerance

Minor histocompatibility (H) antigens are allogeneic targets of T-cell mediated immune reactivity following allogeneic haematopoietic stem cell transplantation. Depending on the tissue expression profile of the minor H antigens this immune reactivity clinically results in graft-vs-host disease or in graft-vs-leukaemia effects. Targeting haematopoietic-specific minor H antigens by adoptive immunotherapy will evoke leukaemia-specific allo-immune responses, thereby enhancing graft-vs-leukaemia effects. Recently, a novel alternative role for minor H antigens in transplantation has been described; minor H antigen-specific T cells appear to be able to regulate allo-immune responses after solid organ transplantation. This diversity in immune reactivity suggests a broad clinical potential of minor H antigens, both in haematopoietic stem cell transplantation and in solid organ transplantation.     

Gene therapeutic approaches to induction and maintenance of tolerance

Tolerance induction would be an ideal way to treat autoimmune diseases, especially if achievable in primed individuals. Moreover, specific tolerance would preclude the need for immunosuppressive treatment with its side effects. In this review, we will revisit the historical concepts of tolerance, and introduce a novel approach to tolerance via gene therapy. Our model system is based on the tolerogenicity of IgG carriers and B-cell antigen presentation. Results with this model demonstrate that it is simple and effective even in primed recipients, and has proven efficacy in three autoimmune models. We discuss the mechanisms of tolerance with fusion IgG's and analyze how our model of gene therapy approached can be utilized to fit in the future treatment of autoimmune conditions.     

Mixed hematopoietic chimerism and transplantation tolerance

Durable transplantation tolerance can be reliably achieved by inducing engraftment of hematopoietic cells in recipients initially depleted of T-lymphocytes. Engraftment of donor pluripotent hematopoietic stem cells (PPHSC) produces mixed hematopoietic chimeras in which both host and donor cells coexist and are tolerant of each other. The major mechanism of tolerance in these chimeras is central, intrathymic clonal deletion, which is induced and maintained by immigration of both host and donor marrow-derived cells to the host thymus, ensuring the ongoing central deletion of donorand host-reactive cells. In this article, approaches developed in our laboratory to induce stable mixed hematopoietic chimerism and specific central deletional allogeneic and xenogeneic tolerance without toxic or myeloablative host conditioning are reviewed.     

Regulatory T cell therapy for the induction of clinical organ transplantation tolerance

The pursuit of transplantation tolerance is the holygrail in clinical organ transplantation. It has been established that regulatory T cells (Tregs) can confer donor-specific tolerance in mouse models of transplantation. However, this is crucially dependent on the strain combination, the organ transplanted and most importantly, the ratio of Tregs to alloreactive effector T cells. The ex vivo expansion of Tregs is one solution to increase the number of alloantigen specific cells capable of suppressing the alloresponse. Indeed, ex vivo expanded, alloantigen specific murine Tregs are shown to preferentially migrate to, and proliferate in, the graft and draining lymph node. In human transplantation it has been proposed that depletion of the majority of direct pathway alloreactive T cells will be required to tip the balance in favour of regulation. Ex vivo expansion of alloantigen specific, indirect pathway human Tregs, which can cross regulate the residual direct pathway has been established. Rapid expansion of these cells is possible, whilst they retain antigen specificity, suppressive properties and favourable homing markers. Furthermore, considerable progress has been made to define which immunosuppressive drugs favour the expansion and function of Tregs. Currently a series of clinical trials of adoptive Treg therapy in combination with depletion of alloreactive T cells and short term immunosuppression are underway for human transplantation with the aim of minimizing immunosuppressive drugs and completely withdrawal.     

The effect of T lymphocyte depletion on neonatal tolerance induction,graft-vs.-host disease and cellular chimerism

Treatment with a monoclonal anti-Thy-1 antibody and complement completely prevented C57BL spleen cells from causing graft-vs.-host disease following their inoculation into newborn CBA mice. The proportion of mice that became tolerant to C57BL antigens, as measured by skin grafting, was significantly less compared with mice given (CBA × C57BL)F₁ hybrid cells. This was not due to the elimination of T cells, for antibody-treated F₁ cells induced tolerance as readily as complement-treated control F₁ cells. To investigate whether the apparent superiority of F₁ cells over C57BL cells is attributable to differences in the mechanism inducing and maintaining unresponsiveness, two approaches were followed. First, the level of donor cell chimerism in the spleens of tolerant animals was studied. Though no difference between F₁ and C57BL cells was uncovered, the presence of T cells in the donor inocula favored the establishment of chimerism. Second, the involvement of suppressor T cells was examined in adoptive transfer experiments. Splenic suppressor T cells were associated with tolerance regardless of how it was elicited. Preliminary results with F₁ cells show that the tolerogenic property is not confined to any one cell type. It is proposed that the greater tolerogenicity of F₁ cells is brought about by the presence of host-type (self) antigens, which enable the tolerogenic signals to operate without recourse to antigen processing by host cells.     

Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning

Background

For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV⁺) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV⁺ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNγ expression in CD8⁺ and CD8^- T cells, cellular viability, and cellular recovery.

Results

The results indicate that cryopreserved PBMC samples tested for CMV- and HIV- specific interferon-gamma (IFNγ) expression performed equivalent to the respective fresh PBMC processed under both collection conditions. Compared to fresh PBMC, the viability was significantly lower for cryopreserved PBMC derived using Ficoll, although it was never less than 90%. There were no significant differences in the IFNγ response, viability, or recovery between cryopreserved PBMC derived by Ficoll and by CPT.

Conclusion

These data suggest that CPT is an efficient system for the collection and cryopreservation of functionally active HIV⁺ PBMC, as well as a viable alternative to Ficoll gradient separation.     

The practical application of chimerism analyses in allogeneic stem cell transplant recipients: Blood chimerism is equivalent to marrow chimerism

Background

Chimerism defines the amount of donor versus recipient hematopoiesis following allogeneic stem cell transplant (SCT). PCR-based analyses of short tandem repeats (STRs) are commonly used and are accurate and applicable to allogeneic transplant recipients. These analyses are performed on blood and marrow aspirates, but it is unknown if analyses of both are required. We performed a retrospective analysis of 42 consecutive adult allogeneic SCT recipients at our institution to determine if both sample types are needed.

Methods

Chimerism status was determined by multiplex PCR and capillary electrophoresis of STRs. Analyses were performed at 30, 60, and 90 days after SCT on both unfractionated blood and unfractionated marrow aspirate.

Results

PCR analyses of STRs for chimerism performed on unfractionated blood highly correlated with results obtained using unfractionated marrow aspirates at 30, 60, or 90 days following transplant (p < 0.0001 for each time point). Overall and relapse-free survival of patients experiencing full donor chimerism was not statistically different from patients demonstrating mixed chimerism at days 30, 60, and 90 following SCT.

Conclusions

PCR-based chimerism analyses on blood provide similar information as marrow aspirate analyses. These are unique results suggesting that chimerism analyses may be assessed on peripheral blood alone.     

Donor double-negative Treg promote allogeneic mixed chimerism and tolerance

Bone marrow (BM) transplantation is an efficient approach to develop donor-specific tolerance and prevent chronic rejection. Allogeneic BM transplantation is limited by donor T cell-mediated graft-versus-host disease, requirement of cytoreduction and high numbers of BM cells. In addition of these drawbacks, recent studies demonstrate that not only T cells, but also NK cells can mediate BM rejection, and long-term mixed chimerism depends on NK cell tolerance. Thus, NK cell is another potential barrier against engraftment of BM and an important target in efforts to induce transplant tolerance. We have previously identified a novel type of Treg with the phenotype TCRalphabeta+CD3+CD4-CD8- (double-negative, DN). We and others have demonstrated that DN-Treg can effectively suppress anti-donor T cell responses. In this study, we found that donor-derived DN-Treg can suppress NK cell-mediated allogeneic BM graft rejection in both parent-to-F1 and fully MHC-mismatched BM transplantation models. Perforin and FasL in DN-Treg play important roles in the suppression of NK cells. Furthermore, adoptive transfer of DN-Treg can promote a stable mixed chimerism and donor-specific tolerance without inducing graft-versus-host disease. These results demonstrate a potential approach to control innate immune responses and promote allogeneic BM engraftment.