Endoscopic Therapy of Bleeding from Radiation Enteritis with Hypertonic Glucose Spray.

Non-variceal and non-ulcerative bleeding in the gastrointestinal (GI) tract, such as radiation enteritis with active and extensive oozing, is common, and management of these conditions can be challenging. We describe the first case in the literature to use hypertonic glucose spray in radiation enteritis-associated diffuse mucosal bleeding.

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen.

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.

Prevention of type 1 diabetes in NOD mice by genetic engineering of hematopoietic stem cells.

Type 1 diabetes is caused by autoimmune destruction of insulin-producing cells in the pancreas. Type 1 diabetes could potentially be treated by islet transplantation; however, the recurrence of autoimmunity leads to the destruction of islet grafts in a relatively short time frame. Therefore, a major goal of diabetes research is the induction of tolerance in diabetic patients to prevent recurrence of diabetes. Diabetes is a polygenic disease, and not all the determinants responsible for disease susceptibility have been identified. However, in both humans and mouse models of this disease, one of the principle determining genetic factors in diabetes incidence is the inheritance of mutant MHC class II alleles that are associated with increased occurrence of disease. We have shown that in the NOD mouse model, the introduction of protective MHC class II alleles through retroviral gene therapy can prevent the onset of autoimmune diabetes. Prevention of diabetes appears to be mediated, at least in part, by the deletion of autoreactive T cells in the presence of protective MHC class II. Here, we outline the procedures involved in the modification of murine hematopoietic cells through retroviral transduction, the reconstitution of recipients with modified bone marrow, and the monitoring of gene therapy recipients after reconstitution.

Induction of transplantation tolerance by combining non-myeloablative conditioning with delivery of alloantigen by T cells.

The observation that bone marrow derived hematopoietic cells are potent inducers of tolerance has generated interest in trying to establish transplantation tolerance by inducing a state of hematopoietic chimerism through allogeneic bone marrow transplantation. However, this approach is associated with serious complications that limit its utility for tolerance induction. Here we describe the development of a novel approach that allows for tolerance induction without the need for an allogeneic bone marrow transplant by combining non-myeloablative host conditioning with delivery of donor alloantigen by adoptively transferred T cells. CBA/Ca mice were administered 2.5 Gy whole body irradiation (WBI). The following day the mice received K(b) disparate T cells from MHC class I transgenic CBK donor mice, as well as rapamycin on days 0-13 and anti-CD40L monoclonal antibody on days 0-5, 8, 11 and 14 relative to T cell transfer. Mice treated using this approach were rendered specifically tolerant to CBK skin allografts through a mechanism involving central and peripheral deletion of alloreactive T cells. These data suggest robust tolerance can be established without the need for bone marrow transplantation using clinically relevant non-myeloablative conditioning combined with antigen delivery by T cells.

Critical role of donor tissue expression of programmed death ligand-1 in regulating cardiac allograft rejection and vasculopathy.

BACKGROUND: Allograft vasculopathy is a major limiting factor in the long-term success of cardiac transplantation. T cells play a critical role in initiation of cardiac allograft rejection and allograft vasculopathy. The negative T-cell costimulatory pathway PD-1:PDL1/PDL2 (programmed death-1:programmed death ligand-1/2) plays an important role in regulating alloimmune responses. We investigated the role of recipient versus donor PD-1 ligands in the pathogenesis of allograft rejection with emphasis on the role of tissue expression in regulating this alloimmune response in vivo. METHODS AND RESULTS: We used established major histocompatibility complex class II- and class I-mismatched models of vascularized cardiac allograft rejection, blocking anti-PDL1 and anti-PDL2 antibodies, and PDL1- and PDL2-deficient mice (as donors or recipients) to study the role of the PD-1:PDL1/PDL2 pathway in chronic rejection. We also used PDL1-deficient and wild-type mice and bone marrow transplantation to generate chimeric animals that express PDL1 exclusively on either hematopoietic or parenchymal cells. PDL1 but not PDL2 blockade significantly accelerated cardiac allograft rejection in the bm12-into-B6 and B6-into-bm12 models. Although wild-type cardiac allografts survived long term, PDL1-/- donor hearts transplanted into wild-type bm12 mice exhibited accelerated rejection and vasculopathy associated with enhanced recipient T-cell alloreactivity. Interestingly, PDL1-/- recipients did not exhibit an accelerated tempo of cardiac allograft rejection. Using chimeric animals as donors, we show that PDL1 expression on cardiac tissue alone significantly prolonged graft survival compared with full PDL1-/- donor grafts in transplanted wild-type recipients. CONCLUSIONS: This is the first report to demonstrate that expression of the negative costimulatory molecule PDL1 on donor cardiac tissue regulates recipient alloimmune responses, allograft rejection, and vasculopathy.

Gene therapy in type 1 diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T1D affects as many as 3 million patients in the United States alone, with 15,000 new cases developing every year (Juvenile Diabetes Research Foundation), and presently there is no cure for T1D. In recent years, there has been a great deal of interest in developing gene therapy approaches to treat T1D. Gene therapy approaches tend to fall into three broad categoriesthose aimed at preventing or curing autoimmunity, those aimed at restoring insulin production through islet transplant or genetically engineered insulin production, and approaches that aim to prevent the morbidity and mortality associated with this complex disease. We review these studies here.

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy.

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.

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.

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells.

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.

Induction of donor-specific tolerance in sublethally irradiated recipients by gene therapy.

Donor-specific transplantation tolerance can be established through the induction of molecular chimerism following reconstitution of lethally irradiated mice with autologous bone marrow expressing retrovirally transduced allogeneic MHC antigens. Here, we set out to define nonmyeloablative host conditioning regimens that would allow for establishment of molecular chimerism and the induction of donor-specific tolerance. Recipient mice received various doses of whole-body irradiation, together with costimulatory blockade using anti-CD154 monoclonal antibody prior to reconstitution with syngeneic bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b). Conditioning consisting of 3 Gy whole-body irradiation and treatment with anti-CD154 was sufficient to induce molecular chimerism resulting in stable multilineage expression of K(b) on hematopoietic cells. T cells from molecular chimeras were unable to lyse allogeneic targets expressing K(b) and contained substantially fewer K(b)-reactive IL-2- and IFN-gamma-producing CD4 T cells than controls receiving mock-transduced bone marrow. Induction of molecular chimerism using nonmyeloablative host conditioning allowed for permanent survival of K(b)-disparate allogeneic skin grafts. These data suggest that nonmyeloablative host conditioning can be used effectively to induce molecular chimerism resulting in transplantation tolerance.

Preventing autoimmune diabetes through gene therapy.

Extract: Type 1 diabetes is an autoimmune disease in which an individual develops T cells that are able to destroy insulin-producing beta cells in the pancreas. Type 1 diabetics require life-long treatment with exogenous insulin for survival. Susceptibility to type 1 diabetes is influenced by both genetic and environmental factors. The first diabetes-susceptibility genes to be identified were the human leukocyte antigen (HLA) genes. Subsequent studies demonstrated an association of these genes with the insulin gene region. High throughput screening of the human genome in families with two or more affected siblings led to the identification of additional chromosomal regions that may contain susceptibility genes for type 1 diabetes. However, linkage between the HLA gene region and susceptibility to disease suggested that the principal genetic component leading to development of diabetes is the inheritance of mutant HLA class II alleles. These are so-called "at-risk" alleles which lack an aspartic acid, a positively amino acid, at position 57 of the major histocompatibility complex (MHC) class II beta chain.

Induction of central tolerance by mature T cells.

Induction of immunological tolerance is highly desirable for the treatment and prevention of autoimmunity, allergy, and organ transplant rejection. Adoptive transfer of MHC class I disparate mature T cells at the time of reconstitution of mice with syngeneic bone marrow resulted in specific tolerance to allogeneic skin grafts that were matched to the T cell donor strain. Mature allogeneic T cells survived long-term in reconstituted hosts and were able to re-enter the thymus. Analysis of T cell development using transgenic mice expressing an alloantigen-reactive TCR revealed that expression of allogeneic MHC class I on adoptively transferred mature T cells mediated negative selection of developing alloreactive T cells in the thymus. Thus, mature allogeneic T cells are able to mediate central deletion of alloreactive cells and induce transplantation tolerance without the requirement for any other alloantigen-expressing cell type.

Prevention of type 1 diabetes by gene therapy.

The autoimmune disease type 1 diabetes in humans and NOD mice is determined by multiple genetic factors, among the strongest of which is the inheritance of diabetes-permissive MHC class II alleles associated with susceptibility to disease. Here we examined whether expression of MHC class II alleles associated with resistance to disease could be used to prevent the occurrence of diabetes. Expression of diabetes-resistant MHC class II I-Abeta chain molecules in NOD mice following retroviral transduction of autologous bone marrow hematopoietic stem cells prevented the development of autoreactive T cells by intrathymic deletion and protected the mice from the development of insulitis and diabetes. These data suggest that type 1 diabetes could be prevented in individuals expressing MHC alleles associated with susceptibility to disease by restoration of protective MHC class II expression through genetic engineering of hematopoietic stem cells.

Induction of T cell tolerance to a protein expressed in the cytoplasm through retroviral-mediated gene transfer.

BACKGROUND: Host immune responses to foreign gene products have been shown to lead to the elimination of genetically modified cells, and are a major barrier to successful therapeutic gene therapy. We have shown that immunological tolerance to retrovirally transduced cell surface proteins can be induced by expressing the gene encoding these products in bone marrow derived cells. Here, we investigate if expression of foreign gene products in bone marrow derived cells can be used to induce tolerance to cytoplasmic proteins. METHODS: Balb/c mice were reconstituted with syngeneic bone marrow cells transduced with retrovirus carrying the gene encoding enhanced green fluorescent protein (eGFP), or mock-transduced bone marrow cells. After reconstitution, mice were immunized with cells expressing eGFP, and T cells were tested for the ability to kill eGFP-expressing targets in in vitro cytotoxic T lymphocyte (CTL) assays. RESULTS: T cells from Balb/c mice reconstituted with mock-transduced bone marrow were able to kill target cells expressing eGFP. In contrast, T cells from mice reconstituted with eGFP-transduced bone marrow were unable to kill targets expressing eGFP. In addition, we observed that T cell responses to eGFP in C57BL/6 mice were minimal even under highly immunogenic conditions. CONCLUSIONS: These data suggest that expression of foreign gene products in bone marrow derived cells is capable of inducing T cell tolerance to proteins expressed exclusively in the cytoplasm.

T cells mediate resistance to genetically modified bone marrow in lethally irradiated recipients.

BACKGROUND: In order for gene therapy to attain clinical relevance, efficient engraftment and long-term survival of cells that express transduced genes of interest must be achieved. In this study, we examined the extent to which host T cells affect engraftment of syngeneic bone marrow cells engineered to express a retrovirally transduced allogeneic major histocompatibility complex class-I gene. METHODS: B10.AKM mice were preconditioned with lethal irradiation or lethal irradiation plus transient CD4 and CD8 T-cell depletion in addition to CD40-CD154 costimulatory blockade and were then reconstituted with syngeneic bone marrow cells transduced with retroviruses that carried the gene that encoded H-2K(b) (K(b)). Expression of K(b) on bone marrow-derived cells was then analyzed, and induction of tolerance to K was evaluated. RESULTS: Mice conditioned using CD4 and CD8 T-cell depletion in addition to CD40-CD154 costimulatory blockade and lethal irradiation showed a significant increase in the frequency of bone marrow-derived cells that expressed K(b) when compared to animals that received lethal irradiation alone. Survival of allogeneic skin grafts that expressed K(b) was significantly prolonged in animals conditioned with anti-CD4, anti-CD8, and co-stimulatory blockade in addition to lethal irradiation (median survival time, 81 days) when compared to mice that received irradiation alone (mean survival time, 31 days; P=0.001). CONCLUSIONS: Radioresistant host T cells significantly affect the ability to induce tolerance by gene therapy by affecting engraftment of transduced cells that expressed allogeneic major histocompatibility complex class-I genes in the absence of host T-cell depletion and costimulatory blockade, even after lethal irradiation. Thus, radioresistant host T cells are a significant barrier to engraftment of transduced bone marrow progenitors and to the induction of tolerance by gene therapy.

Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy.

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K(b) led to long-term expression of K(b) on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K(b)-transduced wild-type bone marrow were tolerant to K(b). In contrast, expression of K(b) in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K(b), demonstrating that expression of K(b) on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.

Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy.

Induction of immunologic tolerance to alloantigens is a major goal in the field of transplantation. Here, we demonstrate that efficient transduction and expression of a retrovirally transduced major histocompatibility complex (MHC) class I gene (H-2K(b)) in bone marrow (BM)-derived cells, resulting in a permanent state of hematopoietic molecular chimerism, induces stable tolerance to the transduced gene product. Reconstitution of lethally irradiated syngeneic recipients with BM transduced with virus encoding H-2K(b) resulted in life-long expression of the retroviral gene product on the surface of BM-derived hematopoietic lineages including Sca-1(+), lineage negative, hematopoietic progenitors. T cells from mice receiving MHC-transduced BM were unable to kill targets expressing H-2K(b) but were able to respond to third-party controls. Mice reconstituted with H-2K(b)-transduced BM exhibited long-term acceptance of H-2K(b) mismatched skin grafts but were able to rapidly reject third-party control grafts. Thus, gene therapy approaches may be used to induce T-cell tolerance.