Induction Of Donor-specific Tolerance Research Articles

Tolerance and Long-Lasting Peripheral Chimerism After Allogeneic Intestinal Transplantation in MGH Miniature Swine

Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)-inbred miniature swine. Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4/CD25(HIGH+)/Foxp3(+) cells. To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation.

Antagonistic Effect of Toll-Like Receptor Signaling and Bacterial Infections on Transplantation Tolerance

The induction of donor-specific tolerance remains a major goal in the field of transplantation immunology. Therapies that target costimulatory molecules can induce tolerance to heart and pancreatic islet allografts in mouse models, but fail to do so after transplantation of skin or intestinal allografts. We have proposed that organs colonized by commensal bacteria such as skin, lung, and intestine may be resistant to such therapies as a result of bacterial translocation at the time of transplantation, which may promote antigen-presenting cell maturation and the production of proinflammatory cytokines, consequently enhancing responses of alloreactive T cells. Our results indicate that the inability to sense signaling by most toll-like receptors (TLRs), as well as by interleukin-1R and -18R, as a result of genetic ablation of myeloid differentiation factor 88 promotes the acceptance of skin allografts. Conversely, TLR signals and infections by a model bacterium, Listeria monocytogenes (LM), at the time of transplantation can prevent the induction of transplantation tolerance. The effects of the TLR9 agonist CpG are myeloid differentiation factor 88-dependent, whereas the prorejection capacity of LM depends on the intracellular sensing of LM and the production of type I interferon. Therefore, transiently targeting these innate, proinflammatory pathways may have therapeutic value to promote transplantation tolerance.

T regulatory cells in xenotransplantation

The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long-term graft survival. In this regard, the induction of donor-specific tolerance through isolation and expansion of xenoantigen-specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4(+)CD25(+)Foxp3(+) T cells, CD8(+)CD28(-) Treg, double negative CD4(-)CD8(-) T cells, and natural killer Treg. Although only studied in vitro so far, human CD4(+)CD25(+)Foxp3(+) Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8(+)CD28(-) Treg and double negative CD4(-)CD8(-) Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4(+)Valpha14(+) Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4(+)CD25(+)Foxp3(+) Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.

Pankreas- und Inseltransplantation

Pancreas transplantation is a successful and effective procedure resulting in tight glucose control. Due to the postoperative morbidity and the need for immunosuppression pancreas transplantation should be considered in patients with type I diabetes at the time of kidney transplantation. Besides this pancreas transplantation alone can be taken into consideration for patients with very poor metabolic control and quality of life despite optimal medical treatment. Recently, islet transplantation became a less invasive alternative to pancreas transplantation. Due to the lack of long-term follow-up and due to the need of multiple donor grafts for one recipient, islet transplantation should be performed under experimental settings in experienced centers. New developments in protecting transplanted islets and in the induction of donor-specific tolerance could increase the indication to perform the procedure. Therefore alternative sources of beta-cells have to be identified.

Hematopoietic Cell Transplantation for Tolerance Induction: Animal Models to Clinical Trials

The induction of donor-specific immune tolerance is the "holy grail" of transplantation, as it would avoid the toxicities of chronic immunosuppressive therapies while preventing acute and chronic graft rejection. A large number of approaches to tolerance induction have been described in the experimental literature, but only hematopoietic cell transplantation has shown preliminary success for intentional tolerance induction in pilot clinical trials. This review summarizes the conditions that allow progress to be made in moving strategies for tolerance induction from the bench to the bedside and discuss the mechanisms by which tolerance may be achieved through hematopoietic cell transplantation.

Tolerance and Future Directions for Composite Tissue Allograft Transplants: Part II

After studying this article, the participant should be able to: 1. Discuss the definition and basic aspects of tolerance as applied to clinical practice. 2. Understand the role of donor bone marrow cells for tolerance induction in composite tissue allograft. 3. Understand the role of passenger leukocytes in allograft acceptance and rejection. 4. Understand the tolerogenic role of dendritic cells and regulatory cells in composite tissue allograft transplants. 5. Understand modern approaches to transplantation tolerance and their applicability to composite tissue allograft transplants. The introduction of composite tissue allograft transplants into clinical practice has become reality because of well-established microsurgical techniques. However, the success of composite tissue allograft transplants is still dependent on chronic immunosuppression. Clinically applicable protocols for tolerance induction in composite tissue allograft would eliminate the need for lifelong immunosuppression. The major goal of transplantation immunology is to develop immunologic nonresponsiveness (tolerance) to allograft transplants and long-term drug-free survival. A number of protocols have been designed to develop tolerance in experimental systems; however, none of them has been translated to clinical transplantation. In this review, the authors discuss current knowledge and strategies used for tolerance induction and the role of passenger leukocytes for allograft acceptance and rejection. Tolerogenic properties of dendritic cells and immunotherapy with regulatory T cells are introduced as having future potential for tolerance induction in solid organ and composite tissue allograft transplants. The induction of donor-specific tolerance would generate new options for the application of composite tissue allograft transplants and may be considered as a future direction in plastic and reconstructive surgery.

Donor-Specific Tolerance Induction through Expansion of DCregs and Tregs by a Liposomal Formulation of KRN7000 (RGI-2001).

Pharmacological induction of donor-specific tolerance would provide significant benefits in both organ transplantation and bone marrow transplantation settings. We investigated the ability of alpha-galactosylceramide (α-GC) in inducing donor-specific tolerance when given in a liposomal formulation. α-GC is a ligand for CD1d molecules expressed on antigen-presenting cells. Upon presentation by CD1d to invariant natural killer T (iNKT) cells, α-GC induces the rapid release of Th1, Th2, or immune regulatory cytokines and initiation of multiple downstream cellular events such as T cell polarization and expansion of dendritic cell subsets. Previously, using RGI-2001 (a liposomal formulation of a synthetic derivative of α-GC, KRN7000), we have demonstrated that the activation of iNKT cells by RGI-2001 induces expansion of regulatory dendritic cells (DCreg) and subsequent generation of antigen-specific Foxp3+ regulatory T (Treg) cells in the presence of target antigens. In the present study, we examined the effects of RGI-2001 on immune responses against alloantigens using a murine in vivo experimental system. In brief, Balb/c (H-2d) recipients were primed with 5x10e6 C57BL/6 (H-2b) whole spleen cells (WSC) with varying doses of RGI-2001 (0.002 to 20 μg/mouse) given intravenously. Seven days later, WSC from the Balb/c recipients were examined for their cellular composition by FACS analysis. Subtle but reproducible dose-dependent increases were noted in the percentage of the LinnegCD11cintCD45RB+ dendritic cell (DC) population, known to be enriched for regulatory DC (DCreg). Since RGI-2001 induces an increase in the total spleen cells, the absolute DCreg cell numbers in RGI-2001-treated spleen increased in a statistically significant manner as compared with untreated controls. As for the percentages of CD4+Foxp3+ Treg cells, no apparent differences were observed. However, an analysis using the Ki67 cycling cell specific nuclear marker revealed a clear dose-dependent increase in the cycling cell fraction among CD4+Foxp3+ Treg cells. These results together confirmed that RGI-2001 induces expansion of DCregs and Tregs. Next, we investigated the effects of RGI-2001 on immune responses against the donor alloantigens. The WSC of the recipient mice were restimulated in vitro with the mitomycin C-treated, T cell depleted donor (C57BL/6) WSC, and the levels of proliferation were measured by MTT colorimetric assay (one-way MLR). It was found that RGI-2001 treatment reproducibly and significantly suppressed proliferation of host WSC in response to donor alloantigens. A dose of 2μg/mouse of RGI-2001 induced in average ~30% reduction in host WSC proliferation. IL-2 production was also reduced to ~50%, further indicating the suppressive effects of RGI-2001. Notably, it was confirmed that suppressive effect of RGI- 2001 was restricted to the responses towards donor specific alloantigens, as no suppression in proliferation nor IL-2 production was noted when a third party (C3H) WSC was used as the stimulators. Collectively, the results suggest that RGI-2001, when administered together with allogeneic donor cells, can induce donor specific tolerance by expanding DCregs and inducing antigen-specific Tregs. RGI-2001 may have a potential to be a novel therapy to prevent organ rejection as well as GvHD in bone marrow transplantation.

Long-Term Donor-Specific Tolerance in Rat Cardiac Allografts by Intrabone Marrow Injection of Donor Bone Marrow Cells

Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens. A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gyx2 or 4.0 Gyx2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation. Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gyx2 or within 3 months after the treatment with irradiation of 4.0 Gyx2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gyx2, but the chimerism was lost within 1 month after the treatment. These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

Splenocytes Can Replace Chimeric Cells and Maintain Allograft Tolerance

The induction of donor-specific tolerance (DST) has recently attracted widespread attention as a new approach to facilitate engraftment without using immunosuppressants. One way in which to induce DST is to establish a chimeric state that allows donor-derived cells to exist within a recipient. This study aims to investigate whether splenocytes can be used to maintain chimerism and to prolong graft survival. Mixed bone marrow (BM) was established in this chimeric model by lethally irradiating C3H mice on day 0, and transplanting BM from C3H and B6D2F1 mice into them. Skin grafts from C57BL/6 mice were transplanted on day 30. On day 60, splenocytes from C3H (group A), B6D2F1 (group B) and B6C3F1 (group C) mice were administered to the chimeric mice. The class I major histocompatibility complex (MHC) type, the percentage of chimeric cells in the peripheral blood, and the survival of skin grafts were assessed. After splenocyte infusion, BM-derived chimeric cells were eliminated from the periphery in group A (86.2+/-5.9% to 0.04+/-0.03%, P=0.0008), B6D2F1-derived cells increased in quantity and established an allochimera in group B (83.7+/-7.2% to 99.6+/-0.2%, P=0.021), and in group C the B6C3F1-derived cells significantly increased to a level of 77.8% at 180 days after infusion (P=0.014), thereby maintaining the new chimerism. Skin grafts in groups B and C survived for at least 200 days (P=0.0003 and P=0.0001, respectively). Chimerism arising from cells with a partial MHC match to the graft allows the maintenance of specific immunotolerance and graft survival.

Allogenic Mixed Chimerism Induced by Nonlymphoablative Regimen Including Donor BMT with Low‐Dose TBI and Anti‐CD40L Cured Proliferative Glomerulonephritis in Lupus Mice

Allogeneic mixed chimerism achieved by low-dose total body irradiation (TBI) and anti-CD40L monoclonal antibody (mAb) with donor bone marrow transplantation (BMT) and host T cell depletion overcomes both allo- and autoimmunity. We investigated whether a similar regimen without T cell depletion cured diffuse proliferative glomerulonephritis. Male BXSB mice (H-2b) were injected with 20 x 10(6) BALB/c (H-2d) BM cells. When indicated, 3 Gy TBI on day -1 and anti-CD40LmAb (2 mg) on day 0 of BMT was given. Skin grafting was performed 1 day after BMT. BXSB mice were divided into four groups--I: BMT with TBI and anti-CD40LmAb; II: TBI; III: TBI and anti-CD40LmAb; and IV: no treatment. Chimerism in peripheral blood was analyzed. The kidney was examined histologically. TBI with anti-CD40LmAb and BMT allowed induction of multilineage mixed chimerism and donor-specific tolerance to skin grafts without graft-versus-host disease (GVHD). There was significant decrease in glomerular PAS-positive material deposition score, glomerular cell numbers, IgG, and C3 deposition in chimeric mice. All chimeric mice survived. Allogeneic mixed chimerism induced by a less toxic, nonlymphoablative regimen achieved allograft tolerance and cured glomerulonephritis in BXSB lupus mice.

New Approaches to the Prevention of Organ Allograft Rejection and Tolerance Induction

The therapeutic use of organ allograft transplantation is dependent on the discovery and clinical application of immunologic strategies to blunt the immune response and prevent graft rejection. It was the discovery of powerful immunotherapeutics such as cyclosporine A and rapamycin that has allowed for the widespread use of organ transplantation to treat organ failure. However, despite the attainment of impressive survival rates 1 year after organ transplantation, a significant number of organ allografts are lost to immune-mediated chronic rejection. Furthermore, significant morbidity and mortality can be associated with the use of currently available immunosuppressive regimens. Thus, the development of novel approaches to prevent of organ allograft rejection remains extremely important. Here we discuss two promising and novel avenues of research. First, the discovery and characterization of naturally occurring immune inhibitory signals have led to recent research aimed at exploiting these pathways to induce peripheral tolerance to alloantigen. Furthermore, we discuss new approaches to the induction of donor-specific tolerance by induction of molecular chimerism and the transfer of alloantigen-expressing mature T cells.

Mixed Chimerism Approach to Induction of Transplant Tolerance: A Review of the Massachusetts General Hospital Experience

URRENTLY, long-term immunosuppression is required for successful solid organ transplantation in humans. However, immunosuppressive regimens are associated with multiple complications, including infection, malignancies, and renal failure due to drug toxicity. In addition, although short-term allograft survival has increased significantly in the current era of immunosuppression, there has been little improvement in the rate of graft loss after the first posttransplantation year. Approximately 3% to 5% of all kidney transplants are lost in each subsequent year and chronic rejection accounts for almost half of the graft losses. 1 Many in the transplantation field believe that induction of donor-specific tolerance is the only way to control late alloimmune responses and to reduce the morbidity of immunosuppression. It has been shown that individuals with complete hematopoietic chimerism after myeloablative therapy and bone marrow transplantation will later be tolerant to solid organ transplants from the same donor. 2 However, the morbidity and mortality that result from the conditioning regimen is such that this method of tolerance induction is not appropriate for routine solid organ transplantation. Ildstad and Sachs 3 reported in 1984 that mice reconstituted with a mixture of donor and recipient strain bone marrow after ablative treatment developed mixed chimerism, where hematopoietic cells of both donor and recipient origin coexist in the recipient. These animals maintained their mixed chimerism and became tolerant to skin grafts from the donor strain. Later studies by Sharabi and Sachs 4 used nonmyeloablative protocols to achieve the same result. More recently, these studies were extended to large animal models. 5 Renal allograft tolerance through mixed chimerism has been reported in swine 6 and nonhuman primates. 7 In the monkey studies, combined hematopoietic stem cell and kidney transplants from the same donor resulted in mixed chimerism, but only for a few weeks. However, despite the loss of mixed chimerism, long-term tolerance of the kidney allograft was maintained. The low toxicity, lack of graft versus host disease (GvHD), and longterm tolerance that developed in these animals allowed similar protocols to be used in humans. The first trial of inducing renal allograft tolerance using mixed chimerism started in 1998 and involved HLA-matched bone marrow and kidney transplantation for multiple myeloma with renal failure secondary to the myeloma. 8,9 The protocol was easily justifiable for this patient population because patients with multiple myeloma are not candidates for conventional kidney transplants, and patients with end-stage renal disease (ESRD) are not candidates for conventional bone marrow transplantation (BMT). In addition, nonmyeloablative HLA-matched BMT was associated with a potent graft versus tumor effect, so this was the only treatment option available for these patients that could potentially treat their myeloma while restoring renal function. The conditioning regimen was derived from the nonhuman primate studies as well as experience with nonmyeloablative transplants for patients with hematologic malignancies. 10 Total body irradiation used in the animal studies was replaced with cyclophosphamide to provide more cytoreductive capability for the malignancy. Because the patients had renal failure, cyclophosphamide was limited to 2 doses at 60 mg/kg. Patients underwent thymic irradiation (7.0 Gy) on day 1 and antithymocyte globulin (ATG; 15 mg/kg) was given on days 1, 1, 3, and 5. Cyclosporine (CyA) was also started on day 1, with plans to discontinue it at 2 to 3 months posttransplantation. A kidney and bone marrow transplantation was performed from the same HLA- matched sibling donor on day 0. Donor leukocyte transfusions were given posttransplantation in an attempt to convert mixed chimerism to full donor chimerism to enhance the antitumor response without associated GvHD. Six such transplantations have been performed so far and

Adoptive therapy with allogeneic CD4+CD25+ Foxp3+ Treg cells induces transplantation tolerance (102.10)

Abstract Alloantigen primed Treg cells offer the potential for non-toxic suppression of transplant rejection reactions. If they are not rejected by the recipient, MHC-mismatched Treg cells might also establish transplant tolerance to the MHC molecules that the Treg cells were selected on during their development. In this regard, we have shown that the adoptive transfer of allogeneic Treg cells into neonatal IL-2Rβ−/−mice, which lack Treg cells and develop rapid lethal autoimmunity, lead to long-term engraftment by the donor Treg cells, effective prevention of this autoimmunity, and simultaneous long-term tolerance to the skin grafts expressing alloantigens shared by the Treg cells. This tolerance depended upon active suppression by Treg cells, not deletion or anergy. Mice tolerant to allogeneic C57BL/6 skin grafts were also tolerant to MHC class II-deficient C567BL/6 skin grafts, but rejected bm3 and bm12 skin grafts. These latter findings demonstrate that Treg cells are not required to directly recognize alloantigens within the skin grafts and that a single neo-antigen was sufficient to break tolerance. Collectively, these data demonstrate induction of dominant donor-specific transplantation tolerance by allogeneic Treg cells provided that the MHC antigens of both the Treg cells and transplant are matched. (Supported by the NIH)

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.

Allopeptide‐pulsed dendritic cells and composite tissue allograft survival

Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.

Generation and Expansion of Human CD4+CD25+ Regulatory T Cells with Indirect Allospecificity: Potential Reagents to Promote Donor-Specific Transplantation Tolerance

Harnessing naturally arising CD4+ CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number. CD4+ CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+ A2- individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs). Here we show that CD4+ CD25+ Tregs specific for an HLA A2 (103-120) peptide can be selected from the peripheral blood CD4+ CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+ CD25+Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+ CD25+ Tregs. These data may pave the way for clinical studies using CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

Efficacy and Limitations of Natural Killer Cell Depletion in Cyclophosphamide-Induced Tolerance

We previously developed a cyclophosphamide (CP)-induced tolerance protocol, consisting of an intravenous injection of 1 x 10(8) donor spleen cells (SC) given on day 0 and an intraperitoneal injection of 200 mg/kg CP given on day 2. In the present study, we modified this protocol with natural killer cell (NK) depletion in recipient mice, and evaluated the efficacy of tolerance induction. We used B10.D2 (H-2d; IE+) and B10 (H-2b; IE-) mice as both donors and recipients. The recipient mice were treated with donor SC, CP, and donor bone marrow cells (BMCs) with or without NK depletion. A higher level of mixed chimerism was achieved in the NK-depleted recipients. Survival of both the skin and heart donor grafts was significantly prolonged in the NK-depleted recipients. Donor reactive Vbeta11+ T cells were found at the same level as in untreated control mice. Pretreatment with recipient NK cell depletion was effective in inducing higher levels of donor mixed chimerism; however, permanent engraftment of donor bone marrow was not achieved. Survival of donor grafts was remarkably prolonged in the NK cell-depleted group, but transplantation tolerance could not be induced. Our results suggest that NK cell depletion in CP-induced tolerance conditioning has some effect on the induction of donor-specific tolerance.

Undifferentiated Murine Embryonic Stem Cells Cannot Induce Portal Tolerance but May Possess Immune Privilege Secondary to Reduced Major Histocompatibility Complex Antigen Expression

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.

Distinctive role of donor strain immature dendritic cells in the creation of allograft tolerance

Dendritic cells (DCs) are pivotal antigen-presenting cells and serve a unique role in initiating immunity. To test the hypothesis that pre-immunization of recipient with certain DC subsets of donor origin can influence graft outcome, we have studied the effects of immunization with allogeneic CD4(+)CD8(-)CD11c(+) dendritic cell (CD4(+)DC) and CD4(-)CD8(+)CD11c(+) dendritic cell (CD8(+)DC) on the allograft response. Although both immature CD4(+)DC and CD8(+)DC subsets from DBA/2 were able to prime naive allogeneic C57BL/6 (B6) T cells in mixed lymphocyte reaction (MLR), CD8(+)DC exerted more vigorous alloimmune responses than CD4(+)DC did. Also, CD4(+)DC-driven allogeneic T cell response was attenuated more significantly by anti-CD154 mAb than CD8(+)DC-driven response. Consistent with the MLR results, combined pre-treatment with CD4(+)DC, but not CD8(+)DC, plus anti-CD154 mAb produced donor strain-specific long-term graft survival and induced tolerance while treatment with CD8(+)DC plus anti-CD154 mAb created minimal prolongation of allograft survival in a pancreas islet transplant model (DBA/2-->B6). The beneficial effects exerted by CD4(+)DC and anti-CD154 mAb pre-treatment were correlated with T(h)1 to T(h)2 immune deviation and with the amplified donor-specific suppressive capacity by recipient CD4(+)CD25(+) T cells. These findings highlight the capacity of CD4(+)DC to modulate alloimmune responses, and suggest therapeutic approaches for the induction of donor-specific tolerance.