Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation

Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.     

Tolerance, mixed chimerism and protection against graft-versus-host disease after total lymphoid irradiation

Total lymphoid irradiation (TLI), originally developed as a non-myeloablative treatment for Hodgkin's disease, has been adapted for the induction of immune tolerance to organ allografts in rodents, dogs and non-human primates. Moreover, pretransplantation TLI has been used in prospective studies to demonstrate the feasibility of the induction of tolerance to cadaveric kidney allografts in humans. Two types of tolerance, chimeric and non-chimeric, develop after TLI treatment of hosts depending on whether donor bone marrow cells are transplanted along with the organ allograft. An advantageous feature of TLI for combined marrow and organ transplantation is the protection against graft-versus-host disease (GVHD) and facilitation of chimerism afforded by the predominance of CD4+ NK1.1(+) -like T cells in the irradiated host lymphoid tissues. Recently, a completely post-transplantation TLI regimen has been developed resulting in stable mixed chimerism and tolerance that is enhanced by a brief course of cyclosporine. The post-transplantation protocol is suitable for clinical cadaveric kidney transplantation. This review summarizes the evolution of TLI protocols for eventual application to human clinical transplantation and discusses the mechanisms involved in the induction of mixed chimerism and protection from GVHD.     

造血干细胞嵌合体诱导移植免疫耐受

造血干细胞混合嵌合体是指两个不同基因型个体的骨髓造血干细胞共存的一种状态。在同种异体或异种移植的动物模型中,造血干细胞混合嵌合体巳成功地诱导出针对供者特异性的免疫耐受。现已证实造血干细胞具有否决活性,来自造血干细胞的否决细胞在诱导移植特异性免疫耐受中可能起重要作用。     

Peripheral blood progenitor cell mobilization and leukapheresis in pigs

BACKGROUND AND PURPOSE: The pig is being investigated as an organ donor for humans. Induction of immunologic tolerance to pig tissues in primates would overcome the major immunologic barriers to xenotransplantation. A proven method of inducing tolerance to allografts is by the induction of mixed hematopoietic chimerism by bone marrow transplantation. We are therefore investigating induction of mixed hematopoietic chimerism in the pig-to-baboon model. METHODS: To obtain large numbers of pig hematopoietic cells, leukapheresis was used to collect blood cell products in miniature swine (n = 5) after progenitor cell mobilization by use of a course of hematopoietic growth factors (cytokines), consisting of porcine interleukin 3, porcine stem cell factor, and human granulocyte colony-stimulating factor. RESULTS: Cytokine therapy and leukapheresis were well tolerated. Cytokine therapy increased the total white blood cell count and allowed large numbers of leukocytes (60 x 10(10)) to be obtained by apheresis, of which approximately 0.1% were granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units (CFU-GEMMs), which are considered to be representative of hematopoietic progenitors with multi-lineage potential. CONCLUSIONS: The combination of cytokine therapy and leukapheresis enables hematopoietic progenitor cells to be obtained safely from miniature swine.     

Partial tolerance and immunity after adoptive abrogation of transplantation tolerance in the rat

Lewis rats were rendered hematopoietic and lymphoid cell chimeras by injection of (LBN)F₁ hybrid cells at birth or following treatment with cyclophosphamide in adult life. The establishment of transplantation tolerance was indicated by acceptance of (LBN)F₁ skin grafts and specific unresponsiveness in graft vs. host reaction (GvHR) and mixed lymphocyte interaction (MLI) in vitro. Tolerance was abolished by adoptively transferred Lewis lymphocytes, and the loss of chimerism and recovery of specific reactivity by blood lymphocytes were monitored independently by mixed lymphocyte cultures. Recovery of competence to initiate GvHR by splenic and lymph node cells was monitored by the local renal graft vs. host technique. Both techniques measure essentially the proliferative response of certain lymphocytes to foreign cellular AgB antigens, and both detected a prolonged, but gradually weakening, state of partial tolerance to the AgB factors to which tolerance had originally been induced. During this phase of partial tolerance the former chimera rejects skin and lymph node cell grafts from (LBN)F₁ donors with alacrity, but in some cases accepts (LBN)F₁ kidney grafts. Cytotoxic antibodies appear in the serum soon after allogeneic chimerism is terminated. These results are interpreted to indicate that a state of partial tolerance exists among the cells which proliferate in response to certain AgB antigens in GvHR and MLI in the formerly tolerant chimera, and that a state of transplantation immunity (possibly to other determinants) coexists with this partial tolerance.     

Role of donor-specific regulatory T cells in long-term acceptance of rat hind limb allograft

Background

Vascularized bone marrow transplantation (VBMT) is widely accepted as an efficient means of establishing chimerism and inducing tolerance. However, the mechanism underlying is poorly understood. Recently, regulatory T cells (Tregs) have been shown to play an important role in regulating immune responses to allogeneic antigens. In this study, we explored the role of Tregs in the induction of tolerance in an allogeneic hind limb transplantation model.

Methodology/Principal Findings

Forty-eight Lewis rats were divided into 6 groups. They received isografts and allografts from Brown-Norway hind limbs. Recipients in groups 1 and 2 received isografts and those in the other groups received allografts. The bone components of donor limbs were kept intact in groups 1, 3, and 5 but removed before transplantation into groups 2, 4, and 6. Tapered cyclosporin A (CsA) was administered to recipients in groups 5 and 6 after transplantation. During the 100-day observation period, all isografts survived, but the allografts in groups 3 and 4 were rejected within 8 to 12 days. CsA-treated intact allografts survived rejection-free for more than 100 days, and CsA-treated allografts lacking bone elements were rejected within 2 months. Stable peripheral chimerism and myeloid chimerism were observed in group 5. Declining peripheral chimerism and a lack of myeloid chimerism were observed in group 6. Donor-specific Tregs were exclusively detected in both peripheral blood and in the spleens of long-term recipient rats in group 5, with an increased FoxP3 mRNA expression in the allografts. This was further demonstrated to be responsible for donor-specific hyporeactivity by in vitro one-way mixed lymphocyte reaction (MLR).

Conclusion/Significance

Bone components in the allogeneic hind limbs can induce myeloid chimerism and donor-specific Tregs may be essential to tolerance induction. The bone-removal hind limb model may be a suitable counterpart to the induction of tolerance in the study of limb transplantation.     

Development of either split tolerance or robust tolerance along with humoral tolerance to donor and third-party alloantigens in nonmyeloablative mixed chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.     

Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+ CD25- LAP+ T cells

A major goal of immunotherapy for autoimmune diseases and transplantation is induction of regulatory T cells that mediate immunologic tolerance. The mucosal immune system is unique, as tolerance is preferentially induced after exposure to antigen, and induction of regulatory T cells is a primary mechanism of oral tolerance. Parenteral administration of CD3-specific monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. We found that orally administered CD3-specific antibody is biologically active in the gut and suppresses autoimmune encephalomyelitis both before induction of disease and at the height of disease. Orally administered CD3-specific antibody induces CD4+ CD25- LAP+ regulatory T cells that contain latency-associated peptide (LAP) on their surface and that function in vitro and in vivo through a TGF-beta-dependent mechanism. These findings identify a new immunologic approach that is widely applicable for the treatment of human autoimmune conditions.     

Mixed hematopoietic molecular chimerism results in permanent transgene expression from retrovirally transduced hepatocytes in mice

BACKGROUND: Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. METHODS: Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. RESULTS: In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. CONCLUSIONS: Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.     

Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance.

Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infections, including lymphocytic choriomeningitis virus (LCMV). In this study, we show that LCMV infection prevents prolonged allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance and mixed hematopoietic chimerism, while delay of infection for 3-4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H-2(d) activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative costimulatory pathways subsequent to LCMV-induced dendritic cell maturation.     

Mixed chimerism

Induction of mixed chimerism has the potential to overcome the current limitations of transplantation, namely chronic rejection, complications of immunosuppressive therapy and the need for xenografts to overcome the current shortage of allogeneic organs. Successful achievement of mixed chimerism had been shown to tolerize T cells, B cells and possibly natural killer cells, the lymphocyte subsets that pose major barriers to allogeneic and xenogeneic transplants. Current understanding of the mechanisms involved in tolerization of each cell type is reviewed. Considerable advances have been made in reducing the potential toxicity of conditioning regimens required for the induction of mixed chimerism in rodent models, and translation of these strategies to large animal models and in a patient are important advances toward more widespread clinical application of the mixed chimerism approach for tolerance induction.     

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.     

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.     

Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.     

Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.     

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation

BackgroundUmbilical cord blood (UCB) is an alternative graft source for hematopoietic stem cell transplantation and has been shown to give results comparable to transplantation with other stem cell sources. Donor lymphocyte infusion (DLI) is an effective treatment for relapsed malignancies after hematopoietic stem cell transplantation. However, DLI is not available after UCB transplantation.MethodsIn this study, in vitro–cultured T cells from the UCB graft were explored as an alternative to conventional DLI. The main aim was to study the safety of the cultured UCB T cells used as DLI because such cell preparations have not been used in this context previously. We also assessed potential benefits of the treatment.ResultsThe cultured UCB T cells (UCB DLI) were given to 4 patients with mixed chimerism (n = 2), minimal residual disease (n = 1) and graft failure (n = 1). No adverse reactions were seen at transfusion. Three of the patients did not show any signs of graft-versus-host disease (GVHD) after UCB DLI, but GVHD could not be excluded in the last patient. In the patient with minimal residual disease treated with UCB DLI, the malignant cell clone was detectable shortly before infusion but undetectable at treatment and for 3 months after infusion. In 1 patient with mixed chimerism, the percentage of recipient cells decreased in temporal association with UCB DLI treatment.ConclusionsWe saw no certain adverse effects of treatment with UCB DLI. Events that could indicate possible benefits were seen but with no certain causal association with the treatment.     

Cellular therapies for type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is a disease that results from the selective autoimmune destruction of insulin-producing beta-cells. This disease process lends itself to cellular therapy because of the single cell nature of insulin production. Murine models have provided opportunities for the study of cellular therapies for the treatment of diabetes, including the investigation of islet transplantation, and also the possibility of stem cell therapies and islet regeneration. Studies in islet transplantation have included both allo- and xeno-transplantation and have allowed for the study of new approaches for the reversal of autoimmunity and achieving immune tolerance. Stem cells from hematopoietic sources such as bone marrow and fetal cord blood, as well as from the pancreas, intestine, liver, and spleen promise either new sources of islets or may function as stimulators of islet regeneration. This review will summarize the various cellular interventions investigated as potential treatments of T1DM.     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.