Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade.

Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2b) mice received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2 × 107 bone marrow cells (BMC) from either of three donor strains: Balb/c (H2d) (MHC plus multiple minor histocompatibility antigen (mHAg) mismatched), B10.D2 (H2d) or B10.A (H2a) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities. .

The role of natural killer T cells in costimulation blockade-based mixed chimerism.

Distinct lymphocyte populations have been identified that either promote or impede the establishment of chimerism and tolerance through allogeneic bone marrow transplantation (BMT). Natural killer T (NKT) cells have pleiotropic regulatory properties capable of either augmenting or downmodulating various immune responses. We investigated in this study whether NKT cells affect outcome in mixed chimerism models employing fully mismatched nonmyeloablative BMT with costimulation blockade (CB). The absence of NKT cells had no detectable effect on chimerism or skin graft tolerance after conditioning with 3Gy total body irradiation (TBI), and a limited positive effect with 1Gy TBI. Stimulation of NKT cells with alpha-galactosylceramide (alpha-gal) at the time of BMT prevented chimerism and tolerance. Activation of recipient (as opposed to donor) NKT cells was necessary and sufficient for the alpha-gal effect. The detrimental effect of NKT activation was also observed in the absence of T cells after conditioning with in vivo T-cell depletion (TCD). NKT cells triggered rejection of BM via NK cells as chimerism and tolerance were not abrogated when NKT cells were stimulated in the absence of both NK cells and T cells. Thus, activation of NKT cells at the time of BMT overcomes the effects of CB, inhibiting the establishment of chimerism and tolerance.

Anti-CD154 mAb and rapamycin induce T regulatory cell mediated tolerance in rat-to-mouse islet transplantation.

BACKGROUND: Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0-28 d) or late (100-128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. CONCLUSIONS/SIGNIFICANCES: These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

Toward MSC in solid organ transplantation: 2008 position paper of the MISOT study group.

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance--agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

Is Endocrine Therapy Really Pleasant? Considerations about the Long-Term Use of Antihormonal Therapy and Its Benefit/Side Effect Ratio.

Endocrine therapy has become a key part in the adjuvant treatment of hormone responsive breast cancer. The positive effect on relapse risk reduction is well defined, but therapy is not free from bothersome side effects for which estrogen deprivation accounts to a great extent. Since endocrine therapy is usually prescribed for 5 years or longer to optimally display its protective effect, and because physical strain is missing, good tolerability and safety properties are important, particularly in low-risk patients. While tamoxifen has been the standard adjuvant endocrine treatment with well documented efficiency, it is increasingly replaced by third generation aromatase inhibitors due to their better effectiveness and tolerability. Because tamoxifen holds a risk for life-threatening adverse events such as endometrial cancer, pulmonary embolism, and stroke, its recommended duration of therapy is limited to 5 years, also because extension beyond that time did not produce a measurable advantage. While some side effects are present both with tamoxifen and aromatase inhibitors, differences in side effect profiles are well established. Although side effects of aromatase inhibitor-related therapy usually are mild and common to symptoms of menopause, misconception of the symptoms and their mechanism of action, as well as lack of knowledge about how to handle them, can easily lead to dangerous discontinuation of therapy.

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.

CTLA4Ig promotes the induction of hematopoietic chimerism and tolerance independently of Indoleamine-2,3-dioxygenase.

Bone marrow transplantation (BMT) under costimulation blockade induces mixed chimerism and tolerance in rodent models. Recent data, predominantly from in vitro studies, suggest that in addition to blocking the CD28 costimulation pathway CTLA4Ig also acts through upregulating the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Here we demonstrate that even though CTLA4Ig is critically required for the induction of chimerism and tolerance in a murine model of nonmyeloablative BMT, IDO activity is not. No significant differences were detectable in the kynurenine to tryptophan ratios (indicative of IDO activity) in sera of BMT recipients treated with CTLA4Ig (tolerant group) versus BMT recipients treated without CTLA4Ig (nontolerant group) versus naïve controls. In vivo inhibition of IDO immediately after BMT with CTLA4Ig or several months thereafter did not block achievement of chimerism and tolerance. Thus, IDO does not play a critical role in the induction or maintenance of chimerism and tolerance in a CTLA4Ig-based BMT model.

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy.

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.

Induction of mixed chimerism through transplantation of CD45-congenic mobilized peripheral blood stem cells after nonmyeloablative irradiation.

Clinical translation of the mixed-chimerism approach for inducing transplantation tolerance would be facilitated if mobilized peripheral blood stem cells (mPBSCs) could be used instead of bone marrow cells (BMCs). Because the use of mPBSCs for this purpose has not been investigated in nonmyeloablative murine protocols, we explored the engraftment potential of mPBSCs in a CD45-congenic model as a first step. After 2, 1.5, or 1 Gy of total body irradiation, CD45.1 B6 hosts received unseparated granulocyte colony-stimulating factor-mobilized CD45.2 B6 PBSCs or unseparated CD45.2 B6 BMCs. The same total cell numbers, or aliquots of mPBSCs and BMCs containing similar numbers of c-kit+ cells, were transplanted both with and without a short course of rapamycin-based immunosuppression (IS). Transplantation of mPBSCs induced long-term multilineage macrochimerism, but chimerism levels were significantly lower than among recipients of the same number of BMCs. Transplanting aliquots containing similar numbers of c-kit+ cells reduced the difference between mPBSCs and BMCs, but lower levels of chimerism were nonetheless observed in mPBSC recipients. Chimerism levels correlated more closely with the number of transplanted progenitor cells as determined by colony-forming unit assays. IS did not affect chimerism levels, indicating that the donor CD45 isoform or other minor disparities do not pose a major barrier to engraftment. Our findings indicate that under nonmyeloablative conditions, progenitor cells contained in mPBSCs have an engraftment capacity similar to progenitor cells from BMCs, allowing induction of lasting mixed chimerism with moderate cell numbers. On a cell-per-cell basis, unseparated BMCs have some advantages that may be minimized if the number of progenitor cells is equalized. These results are expected to facilitate the development of mPBSC-based allogeneic tolerance protocols.

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 x 10 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 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 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 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 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 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.

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 naive 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.

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.

The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade.

We recently developed a murine protocol for the induction of allogeneic mixed chimerism and tolerance employing nonmyeloablative total body irradiation (TBI), standard-dose bone marrow transplantation (BMT), and costimulation blockade (cobl) with an anti-CD154 monoclonal antibody (mAb) plus CTLA4Ig. We now evaluated whether a short course (1 month) of immunosuppressive drugs, which would be ethically required in the clinical setting of organ transplantation to prevent graft loss in case tolerance is not achieved, interferes with tolerance induced with this regimen. Our results show that calcineurin inhibitors (cyclosporin A [CyA] or tacrolimus [FK]) inhibit development of long-term chimerism and abrogate tolerance induction in this model. Rapamycin (rapa), methylprednisolone (MP), FTY720, and mycophenolate mofetil (MMF), in contrast, have no negative effect on chimerism or tolerance development. Peripheral deletion of donor-reactive T cells, which usually occurs in the weeks following BMT in this model, is blocked by CyA and FK, but not by the other drugs tested. Furthermore, we found that the additional use of compatible immunosuppressive drugs (rapa plus MMF plus MP) allows the dose of TBI to be reduced, so that mixed chimerism and donor skin-graft acceptance can be achieved with 1 Gy using clinically feasible cell numbers. Thus, this protocol of BMT with costimulation blockade can be safely combined with a clinically tested immunosuppressive regimen to permit success with a lower dose of irradiation. These results should facilitate clinical application of this tolerance strategy.

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.