Debra A. MacKenzie

GRAIL Is Up-regulated in CD4+ CD25+ T Regulatory Cells and Is Sufficient for Conversion of T Cells to a Regulatory Phenotype

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4+ T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4+ T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4+ CD25+ T regulatory cells (mRNA levels 10-fold higher than naive CD25– T cells). Further investigation demonstrated that CD25+ Foxp3+ antigen-specific T cells were induced after a “tolerizing-administration” of antigen and that GRAIL expression correlated with the CD25+ Foxp3+ antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25+ T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.

Xenogeneic transplantation of porcine islets: an overview.

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.

Role of CD4+ regulatory T cells in hyperbaric oxygen-mediated immune nonresponsiveness.

We have previously shown that hyperbaric oxygen culture (HOC [95% O(2), 5% CO(2), 25 psi]) is an effective pretransplant tissue-modification technique that results in long-term allograft survival and the induction of systemic immune tolerance in a murine model. Here we address the immune modulatory effects of HOC-treatment of human immune responses using the in vitro mixed lymphocyte reaction (MLR). Pretreatment of allogeneic stimulator cells with HOC results in abrogation of cytotoxic T lymphocyte (CTL) activity, proliferative responses, and IFN gamma production in a 7-day MLR. These responses can be restored either by the addition of IFN gamma or IL-2 on day 0, or by blocking the activity of IL-4 and IL-10. The addition of IL-2 on day 4 does not restore allospecific CTL activity. The failure of HOC-treated cells to induce allospecific CTL is not due to the induction of anergy, demonstrated by the failure to restore responses after restimulation with allogeneic cells in the presence of IL-2. Removal of CD4(+) cells prior to restimulation, results in restoration of CTL activity in MLR cultures restimulated with HOC-treated allogeneic cells. These results suggest that HOC-induced immune nonresponsiveness is mediated by the development of CD4(+) regulatory cells in a Th2-type environment.

Acute graft rejection of human fetal pancreas allografts using donor-specific human peripheral blood lymphocytes in the SCID mouse

Transplantation of human fetal pancreas (HFP) is being considered as a potential treatment for insulin-dependent diabetes mellitus (IDDM). Therefore, it is necessary to have an experimental model of HFP-specific allograft rejection in order to understand all the fators that contribute to allograft rejection, and in which to test potential immunomodulatory protocols. The severe combined immunodeficient (SCID) mouse provides such a model. Previously, it has been reported that human allograft rejection can be observed in SCID mice engrafted with human lymphocytes. However, graft rejection is inconsistent and depends on both the number of lymphocytes injected and on the activation state. Here, we describe a model in which SCID mice are injected intraperitoneally with donor-specific lymphocytes generated by an in vitro culture period with irradiated donor splenocytes. Injection of the donor-sensitized PBL results in an acute rejection of HFP allografts (as early as 4 days post-transplant). This model does not require the establishment of chimerism. in the SCID mice, as demonstrated by the lack of detectable human CD45 cells in the peripheral blood of rejecting mice. Allograft rejection was due to human CD4+ and CD8+ cells, as determined by immunohistochemical analysis of graft-infiltrating cells. The advantages of this model include the potential to specifically manipulate either the phenotype of the responding cells or the mechanism in which the responding population is generated. This model can provide a rapid method to test the efficacy of immunomodulatory regimens designed to protect allografts from an acute rejection response.

Decreased immunogenicity of human fetal pancreas allografts following hyperbaric oxygen culture

Human fetal pancreas (HFP) is a potential source of transplantable islets for the treatment of type 1 insulin-dependent diabetes mellitus (IDDM). Pretransplant culture techniques such as long-term culture, high-oxygen culture, UVB irradiation, and low-temperature culture have previously been used to reduce the immunogenicity of tissue for transplantation. In this study, we use hyperbaric oxygen culture (HOC) to modify MHC Class I expression on HFP and to reduce the immunological response of human peripheral blood mononuclear cells (PBMC) to HFP using a sponge matrix allograft model. To study the interaction of naïve PBMC with HOC-treated or untreated HFP allografts, sponges embedded with HFP tissue were implanted into the peritoneal cavity of NOD-SCID mice and injected with 1 x 10(7) freshly isolated human PBMC at the time of transplant. By day 14, human CD45 cells represented less than 2% of the cells recovered from the sponges implanted with HOC-treated HFP. In contrast, human CD45(+) cells represented nearly 15% (P =.0018) of the cells isolated from sponges implanted with conventionally cultured HFP grafts. Approximately 75% of the human CD45(+) cells from conventionally cultured HFP allografts were producing IFNgamma as determined by intracellular cytokine analysis. These data suggest that HOC treatment of HFP abrogates the activation and proliferation of PBMC. Pretransplant HOC treatment of islets is a simple technique that could be used to reduce immunogenicity and increase allograft survival while decreasing the requirement for immunosuppressive drugs.

Sustained Expression of GRAIL during Hematopoiesis Results in Dysregulated Differentiation

Background/Aims: Despite a sophisticated understanding of the hematopoietic developmental program at the transcriptional level, our understanding of the role of E3 ubiquitin ligases remains underdeveloped. The E3 ubiquitin ligase, GRAIL (RNF128), is expressed in the bone marrow, but its role is as yet undefined. In this study, we evaluate the effect of GRAIL expression during hematopoietic differentiation in vitro and in vivo. Methods: Retroviral transduction of hematopoietic multipotent progenitor cells was used for methylcellulose colony assays and bone marrow reconstitution. Results: Enforced expression of GRAIL in colony assays demonstrated skewing of hematopoietic lineage development toward granulocytic/monocytic cells. Bone marrow reconstitution experiments with progenitor cells expressing biologically varied levels of GRAIL demonstrated diminished erythropoiesis and megakaryopoiesis if GRAIL (endogenous or forced) is maintained throughout hematopoiesis. Conclusion: These data highlight a role for GRAIL during hematopoiesis and emphasize the importance of studying posttranslational effects to complement our current understanding of the transcriptional regulation of hematopoiesis.

Removal of CD45+ cells from human fetal pancreas alters immunogenicity in vitro

Human fetal pancreas (HFP) is a potential source of islets for the treatment of diabetes mellitus with the potential for growth and differentiation after transplantation. However, because of the small mass of a given HFP, multiple donors would be required for transplantation, thereby increasing the immunological challenge to the recipient. In this study, we investigate the contribution of hematopoietic cells to the immunogenicity of HFP. Single cell suspensions of HFP were depleted of CD45(+) cells using antibody-conjugated magnetic beads. In vitro mixed lymphocyte islet cultures were established using with CD45-depleted or nondepleted HFP. Depletion of CD45(+) cells resulted in the low levels of IFNgamma production at early time points (day 4), which increased to near normal levels by day 7. The development of donor-specific CTL was not consistently inhibited by CD45 cell depletion. The data suggests that CD45(+) cells within HFP are capable of stimulating immune responses by the direct pathway of antigen presentation, but that the indirect pathway is also involved in the development of CTL. The inhibition of early IFNgamma release, however, may be beneficial for the survival of transplanted islets. Therefore, the combination of CD45 depletion strategies with standard immunosuppressive drug therapies could result in better long-term survival of transplanted islets.

Preparation of Fetal Islets for Transplantation: Importance of Growth Factors

It is estimated that approximately 1.4 million people in the United States suffer from insulin dependent diabetes mellitus (IDDM). Daily insulin therapy and whole organ pancreas transplantation are the only treatments currently available. Results of the diabetes complications and control trial have indicated that while tight control of circulating glucose levels significantly reduced the complications associated with IDDM, it does not prevent them. Insulin replacement therapy is not sufficient to prevent the macrovascular and microvascular complications that make IDDM the third leading cause of death. Pancreas transplant suffers from a shortage of donor organs and requires that the recipient be placed on lifelong immunosuppressive therapy. Adult islet transplantation has met with little success and also suffers from a shortage of donor organs and the need for potent immunosuppression. In contrast, human fetal pancreas (HFP) is readily available, has the potential for further growth and differentiation following transplantation and can be cultured in vitro. This gives HFP the potential for immunologic manipulation such that immunosuppressive therapy may be significantly reduced or eliminated. The growth and differentiation of HFP may be accelerated with short-term culture in growth factors.