Peter Blaha

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.

Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning.

Background. Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. Methods. B6 mice received 200, 100, or 50×106 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. Results. IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200×106 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100×106 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50×106 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200×106 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100×106 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50×106 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. Conclusions. Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50×106 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.

Mechanisms of tolerance induction through the transplantation of donor hematopoietic stem cells: central versus peripheral tolerance.

The transplantation of donor hematopoietic stem cells has been used successfully in numerous experimental settings to induce donor-specific tolerance. After appropriate host conditioning, hematopoietic stem-cell transplantation leads to a lasting state of donor macrochimerism that is associated with a robust form of tolerance. One of the key factors in the success of this approach is its reliance on intrathymic clonal deletion to ensure lifelong tolerization of newly developing T cells. Evidence for ongoing central deletion comes from studies following superantigen-reactive T cells and from experiments using mice transgenic for an alloreactive T-cell receptor. In protocols inducing tolerance through macrochimerism, the preexisting mature T-cell repertoire is controlled by either globally destroying all T cells before the hematopoietic cell transplantation or, in more recent models, by tolerizing it through co-stimulation blockade. The peripheral mechanisms induced by hematopoietic stem-cell transplantation and co-stimulation blockade include both extrathymic clonal deletion and the nondeletional mechanisms anergy, suppression, or both. In addition to these immunologic hurdles, a physiologic engraftment barrier has to be surmounted for the successful induction of mixed chimerism. This can be achieved by cytoreductive host treatment or by the infusion of high numbers of donor hematopoietic cells. A detailed delineation of the mechanisms responsible for tolerance induction after hematopoietic stem-cell transplantation is expected to help in the translation of these experimental protocols to clinical organ transplantation.

Rapid Deletional Peripheral CD8 T Cell Tolerance Induced by Allogeneic Bone Marrow: Role of Donor Class II MHC and B Cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II+ donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c+ dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b+ monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b+ cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.

Tolerance through bone marrow transplantation with costimulation blockade.

The routine induction of tolerance in organ transplant recipients remains an unattained goal. The creation of a state of mixed chimerism through allogeneic bone marrow transplantation leads to robust donor-specific tolerance in several experimental models and this approach has several features making it attractive for clinical development. One of its major drawbacks, however, has been the toxicity of the required host conditioning. The use of costimulation blocking reagents (anti-CD 154 monoclonal antibodies and the fusion protein CTLA4Ig) has led to much less toxic models of mixed chimerism in which global T cell depletion of the host is no longer necessary and which has even allowed the elimination of all cytoreductive treatment when combined with the injection of very high doses of bone marrow cells. In this overview we will briefly discuss general features of tolerance induction through bone marrow transplantation, will then describe recent models using costimulation blockade to induce mixed chimerism and will review the mechanisms of tolerance found with these regimens. Finally we will attempt to identify issues related to the clinical introduction of bone marrow transplantation with costimulation blockade which remain unresolved.

Murine Mobilized Peripheral Blood Stem Cells Have a Lower Capacity than Bone Marrow to Induce Mixed Chimerism and Tolerance

Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T‐cell depletion (TCD). The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony‐stimulating factor (G‐CSF) mobilized PBSC under costimulation blockade (anti‐CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with nonmyeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor BM was also triggered by the cotransplantation of unmanipulated donor T cells isolated from naïve (nonmobilized) donors. Donor‐specific transfusion and transient immunosuppression prevented PBSC‐triggered rejection and mixed chimerism and tolerance were achieved, but graft‐versus‐host disease (GVHD) occurred. The combination of in vivo TCD with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD‐toxicity, and the addition of TCD is required for success.

Breast cancer chemoprevention - a vision not yet realized.

Despite recent advances in the surgical and medical treatment of breast cancer, the number of patients dying from the disease is still high. In addition to improvements of early diagnosis and treatment, the overall mortality of breast cancer could be reduced by means of preventive intervention in both women with particularly normal and with high risk. Preventing the potentially deadly disease is presumably more effective than treatment, for life quality issues as well as for the economic perspective. Chemoprevention though is still a research field with results from large prevention trials being discussed controversially. For women with a defined increased risk for breast cancer, tamoxifen may be a choice for chemoprevention, balancing carefully benefits against risks. With promising results in adjuvant settings, aromatase inhibitors may deliver better prevention treatment options in the future, nevertheless, more research is needed to reliably predict risk on an individual basis in the future.

Molecular signature of mice T lymphocytes following tolerance induction by allogeneic BMT and CD40-CD40L costimulation blockade

Tolerance induction by mixed chimerism and costimulation blockade is a promising approach to avoid immunosuppression, but the molecular basis of tolerant T lymphocytes remains elusive. We investigated the genome‐wide gene expression profile of murine T lymphocytes after tolerance induction by allogeneic bone marrow transplantation (BMT) and costimulatory blockade using the anti‐CD40L antibody MR1. Molecular functions, biological processes, cellular locations, and coregulation of identified genes were determined. A total of 113 unique genes exhibited a significant differential expression between the lymphocytes of MR1‐treated Tolerance (TOL) and untreated recipients Control (CTRL). The majority of genes upregulated in the TOL group are involved in several signal transduction cascades such as members of the MAPKKK cascade (IL6, Tob2, Stk39, and Dusp24). Other genes involved in lymphocyte differentiation and highly expressed in the TOL group are lymphotactin, the estrogen receptors (ERs) and the suppressor of cytokine signaling 7. Common transcription factors such as ER 1 alpha, GATA‐binding protein 1, insulin promoter factor 1, and paired‐related homeobox 2 could be identified in the promoter regions of upregulated genes in the TOL group. These data suggest that T lymphoctes of tolerant mice exhibit a distinct molecular expression profile, which needs to be evaluated in other experimental tolerance models to determine whether it is a universal signature of tolerance.

Influence of immunosuppressive drugs on cell-induced graft tolerance

Purpose of reviewThe transplantation of donor hematopoietic stem cells (HSC) is an attractive strategy for inducing transplantation tolerance. The clinical application of conventional HSC transplantation (HSCT) protocols in organ transplantation, however, has so far been prevented by unacceptable toxicities. In experimental HSC-based tolerance models immunosuppressive drugs have been explored mainly for two reasons: firstly, to find a compatible regimen of conventional immunosuppressive drugs whose transient use would be required as “safety net” when tolerance strategies are first tested in the clinical setting; and secondly, to allow tolerance induction with milder conditioning regimens through the temporary administration of immunosuppression. In this review, we briefly summarize the current experience with immunosuppressive drugs in cell-based tolerance regimens. Recent findingsImmunosuppressive drugs have been routinely used for a long time in clinical HSCT performed for hematological indications. Several cyclosporine (CyA)-based drug regimens have been shown to reduce the incidence and/or severity of graft-versus-host disease (GvHD). Little is known from clinical data, however, about a possible impact of these drugs on engraftment or tolerance. In experimental bone marrow transplantation (BMT) protocols involving global recipient T cell depletion, temporary treatment with cyclosporine (CyA) has been investigated and was found to be not detrimental. CyA can indeed be necessary for tolerance induction in such systems. In contrast, in some, but not all experimental models of BMT involving costimulation blockade (CB), calcineurin-inhibitors displayed a tolerance-abrogating effect. Rapamycin proved beneficial in such models, allowing mixed chimerism and tolerance to be induced with reduced recipient conditioning. SummaryDistinct immunosuppressive drugs can either promote or prevent tolerance after HSC transplantation, depending on the details of the regimen. These effects should be considered when clinical translation of a particular protocol is considered.

Adjuvant bisphosphonates in endocrine-responsive breast cancer: what is their place in therapy?

Recent advances in the treatment of early breast cancer have improved clinical outcomes and prolonged survival, especially in women with endocrine-responsive disease. However, cancer therapies including cytotoxic chemotherapy, ovarian suppression, and aromatase inhibitors can drastically reduce circulating estrogen, increasing bone loss and fracture risk. Because most women with early breast cancer will live for many years, it is important to protect bone health during cancer therapy. Several recent clinical trials combining adjuvant endocrine therapy with bisphosphonates have demonstrated efficacy for preventing cancer treatment-induced bone loss in pre- and postmenopausal women with early breast cancer. The largest body of evidence supporting the use of adjuvant bisphosphonates comes from studies with zoledronic acid; however, studies with risedronate, ibandronate, and denosumab (a biologic agent) have also demonstrated efficacy for preventing bone loss. Adding zoledronic acid to endocrine therapy prevents bone loss and improves bone mineral density (BMD). In addition, preclinical studies suggest that bisphosphonates have direct and indirect antitumor activity, such as inducing tumor cell apoptosis, reducing tumor cell adhesion and invasion, reducing angiogenesis, activating immune responses, and synergy with chemotherapy agents, among others. Clinical trials have demonstrated significantly improved disease-free survival in patients receiving adjuvant endocrine therapy plus zoledronic acid compared with endocrine therapy alone. Ongoing studies will further define the role of adjuvant bisphosphonates in maintaining bone health and improving clinical outcomes. The available evidence suggests that pre- and postmenopausal patients may receive clinical benefit from including bisphosphonates as part of their adjuvant treatment regimen for endocrine-responsive early breast cancer.