Full Major Histocompatibility Complex Barriers Research Articles

Induction of mixed chimerism using combinatory cell-based immune modulation with mesenchymal stem cells and regulatory T cells for solid-organ transplant tolerance.

Establishment of mixed chimerism is an ideal approach to induce donor-specific tolerance while expanding its potential in various clinical settings. Despite the developments in partial conditioning regimens, improvements are still needed in reducing toxicity and bone marrow transplantation-related complications. Recently, cell-based therapies, including mesenchymal stem cells (MSCs), have been incorporated in establishing noncytoreductive mixed chimerism protocols; however, its efficacy is only partial and shows reversed immunosuppressive properties. This study demonstrates a novel approach to induce mixed chimerism and tolerance through combinatory cell-based immune modulation (CCIM) of MSCs and regulatory T cells (Tregs). We hypothesize that the interaction between these cells may lead to greater inhibition of host immune responses. Compared with single cell therapy, CCIM induced a higher engraftment rate and robust donor-specific tolerance to skin allografts across full major histocompatibility complex barriers. These regulatory effects were associated with inhibition of natural killer cell cytotoxic activity, CD4(+)IL-17(+) cells, memory B cells, plasma cells, and immunoglobulin production levels along with increased frequencies of CD4(+)Foxp3(+) cells, IL-10-producing mature B cells, and myeloid-derived suppressor cells. Furthermore, CCIM was able to regulate mortality in a graft-versus-host disease model through reciprocal regulation of Treg/Th17. Taken together, we suggest CCIM as a clinically applicable strategy for facilitating the induction of mixed chimerism and permanent tolerance.

Induction of Cardiac Allograft Tolerance Across a Full MHC Barrier in Miniature Swine by Donor Kidney Cotransplantation

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice

AbstractInduction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes—a new approach to translate immunological tolerance into clinically applicable protocols.

T-Regulatory Cells—What Relationship With Immunosuppressive Agents?

The immune system maintains self-tolerance through the interplay of several mechanisms. One of the key mechanisms is the suppression of effector cells through regulatory T cells (Tregs). Recent progress has allowed the identification, analysis, and experimental manipulation of various subpopulations of Tregs. Several major issues regarding Tregs have emerged in relation to organ transplantation, including the questions of whether immunosuppressive drugs influence Tregs and whether Tregs can be used therapeutically to achieve graft acceptance or even tolerance. Although none of the available immunosuppressive drugs have been specifically designed to influence Tregs, they nevertheless do so through various pathways. Notably, current evidence suggests that rapamycin has a favorable effect on Tregs in organ transplant recipients, whereas calcineurin inhibitors (CNI) have markedly negative effects on Tregs. The idea of inducing tolerance through the therapeutic administration of Tregs has received much attention in recent years. To obtain sufficient numbers of recipient-derived Tregs, various strategies are being investigated to expand sorted Treg populations. Rapamycin has been found to significantly promote the in vitro expansion of murine and human Tregs; it may thereby help to generate clinically relevant quantities of Tregs. However, no data have yet been published that would demonstrate that the therapeutic use of Tregs induces transplantation tolerance across full major histocompatibility complex barriers in immunocompetent mice displaying a polyclonal T-cell repertoire.

Posttransplant lymphoproliferative disease after allogeneic transplantation of the spleen in miniature swine.

Spleen transplantation (SpTx) was performed in miniature swine across full major histocompatibility complex barriers to study the tolerogenic effect of the spleen. This study describes the development of posttransplant lymphoproliferative disease (PTLD) after allogeneic SpTx. Recipient pigs underwent whole body irradiation (100 cGy), thymic irradiation (700 cGy), and native splenectomy (day 0), and received a 45-day course of intravenous cyclosporine (trough level 400-800 ng/ml). After SpTx, two of seven pigs developed PTLD (1 donor-type, 1 host-type). These two pigs had greater T cell depletion and higher trough levels of cyclosporine. Early changes that occurred prior to the development of clinical features of PTLD were increased porcine lymphotropic herpesvirus-1 viral loads in blood and tissues, and increased numbers of leukocytes, B cells, and total serum IgM. PTLD can occur after allogeneic SpTx in swine. This model may be useful in studies of the pathogenesis of PTLD.

Permanent acceptance of both cardiac and skin allografts using a mild conditioning regimen for the induction of stable mixed chimerism in mice

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.

Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion

AbstractIn utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism and donor-specific tolerance without cytoreductive conditioning or immunosuppression. The primary limitation to the clinical application of IUHSCTx has been minimal donor cell engraftment, well below therapeutic levels for most target diseases. Donor lymphocyte infusion (DLI) has been used in postnatal circumstances of mixed chimerism as targeted immunotherapy to achieve a graft-versus-hematopoietic effect and to increase levels of donor cell engraftment. In this report we demonstrate in the murine model that a combined approach of IUHSCTx followed by postnatal DLI can convert low-level, mixed hematopoietic chimerism to complete donor chimerism across full major histocompatibility complex barriers with minimal risk for graft-versus-host disease (GVHD). Time-dated embryonic day 14 (E14) to E15 Balb/c (H-2Kd, CD45.2) fetuses underwent intraperitoneal injection of 5 × 106T-cell–depleted B6 (H-2Kb, CD45.2) bone marrow cells. Chimeric recipients then received transplants at either 4 or 8 weeks of age with 1 of 3 doses (5, 15, or 30 × 106cells) of donor congenic splenocytes (B6-Ly5.2/Cr, H-2Kb, CD45.1). The response to DLI was dose dependent, with conversion to complete donor peripheral blood chimerism in 100% of animals that received high-dose (30 × 106 cells) DLI. Only 1 of 56 animals receiving this dose succumbed to GVHD. This study directly supports the potential therapeutic strategy of prenatal tolerance induction to facilitate nontoxic postnatal cellular therapy and organ transplantation, and it has broad implications for the potential treatment of prenatally diagnosed genetic disorders.

Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.

Inbred miniature swine provide a large animal model in which the effects of selective major histocompatibility complex (MHC) matching can be reproducibly studied. We have previously demonstrated that although a 12-day course of cyclosporine uniformly induces tolerance to class I-mismatched renal allografts, it does not induce tolerance across full MHC barriers. In this study, we assessed whether and at what dose tacrolimus might permit allografts to induce tolerance across different MHC barriers. Recipients of MHC disparate renal allografts were treated with a 12-day course of tacrolimus by continuous intravenous infusion. Groups were divided as follows: (1) class I-mismatched kidneys with 0.3 mg/kg/day tacrolimus (n=3); (2) fully MHC-mismatched kidneys with 0.3 mg/kg/day tacrolimus (n=2); and (3) fully MHC-mismatched kidneys with 0.12-0.16 mg/kg/day tacrolimus (n=4). In groups 1 and 2, recipients with tacrolimus levels of 45-80 ng/ml accepted renal allografts long-term with stable renal function. Donor-specific hyporesponsiveness was demonstrated by cell-mediated lymphocytotoxicity and mixed lymphocyte response, and subsequent donor-matched grafts were also accepted, without further immunosuppression (n=4), confirming systemic tolerance. In group 3, recipients that achieved tacrolimus levels of 35 ng/ml (n=2) accepted their grafts without chronic changes, whereas recipients with levels of 20-26 ng/ml (n=2) developed chronic allograft glomerulopathy, suggesting 35 ng/ml as the threshold blood level for tolerance induction. In vitro assays demonstrated that peripheral blood lymphocytes from tolerant animals produced inhibitory cytokines, suggesting the involvement of regulatory mechanisms. To our knowledge, this study represents the first demonstration of the induction of transplant tolerance across a two-haplotype full MHC barrier with a short course of immunosuppression in a large animal model. These studies may also have clinical applicability, because the time course required to induce tolerance was sufficiently short that the high drug levels required might be expected to be tolerated clinically with only transient toxicity.

An alternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers

Previously, we developed a well-tolerated single-day protocol for induction of stable multilineage chimerism and permanent donor-specific tolerance across major histocompatibility complex (MHC) barriers, with preservation of the host's normal immune responses. In our murine model, recipient mice were treated with a single dose and anti-CD3, anti-CD4, low dose total body irradiation (TBI; 3–6 Gy) and allogenic bone marrow cells. An alternative cytoreductive strategy that is well-recognized in bone marrow transplantation, but has not been evaluated extensively in organ allograft recipients, involves the use of a combined chemotherapuetic drug treatment. The present data show that conditioning with low dose TBI, in a MHC-disparate donor-recipient combination, can be successfully substituted by a combined single low-dose dimethyl myleran (DMM)/cyclophosphamide (CY) therapy, resulting in both stable, mixed chimerism and specific skin graft tolerance.