Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2r gamma(null) ) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2r gamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects.

Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non-obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene-1 (Rag1(null)) and interleukin (IL)-2 receptor common gamma chain (IL2r gamma(null)), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17-Prkdc(scid) (NOD-Prkdc(scid)) IL2r gamma(null) mice. We observed that immunodeficient NOD-Rag1(null) IL2r gamma(null) mice tolerated much higher levels of irradiation conditioning than did NOD-Prkdc(scid) IL2r gamma(null) mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation-conditioned adult mice, leading to multi-lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1(null) and scid stocks of immunodeficient NOD mice harbouring the IL2r gamma(null) mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD-Rag1(null) IL2r gamma(null) mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

There is a growing need for effective animal models to carry out experimental studies on human hematopoietic and immune systems without putting individuals at risk. Progress in development of small animal models for the in vivo investigation of human hematopoiesis and immunity has seen three major breakthroughs over the last three decades. First, CB17-Prkdc(scid) (abbreviated CB17-scid) mice were discovered in 1983, and engraftment of these mice with human fetal tissues (SCID-Hu model) and peripheral blood mononuclear cells (Hu-PBL-SCID model) was reported in 1988. Second, NOD-scid mice were developed and their enhanced ability to engraft with human hematolymphoid tissues as compared with CB17-scid mice was reported in 1995. NOD-scid mice have been the "gold standard" for studies of human hematolymphoid engraftment in small animal models over the last 10 years. Third, immunodeficient mice bearing a targeted mutation in the IL-2 receptor common gamma chain (IL2r gamma(null)) were developed independently by four groups between 2002 and 2005, and a major increase in the engraftment and function of human hematolymphoid cells as compared with NOD-scid mice has been reported. These new strains of immunodeficient IL2r gamma(null) mice are now being used for studies in human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine. In this chapter, we discuss the current state of development of these strains of mice, the remaining deficiencies, and how approaches used to increase the engraftment and function of human hematolymphoid cells in CB17-scid mice and in previous models based on NOD-scid mice may enhance human hematolymphoid engraftment and function in NOD-scid IL2r gamma(null) mice.

Regulatory T cells (Treg) are important in peripheral tolerance, but their role in establishing and maintaining hematopoietic mixed chimerism and generating central tolerance is unclear. We now show that costimulation blockade using a donor-specific transfusion and anti-CD154 antibody applied to mice given bone marrow and simultaneously transplanted with skin allografts leads to hematopoietic chimerism and permanent skin allograft survival. Chimeric mice bearing intact skin allografts fail to generate effector/memory T cells against allogeneic targets as shown by the absence of IFNgamma-producing CD44(high)CD8+ T cells and in vivo cytotoxicity. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody prior to costimulation blockade prevents chimerism, shortens skin allograft survival and leads to generation of effector/memory cytotoxic T cells. Depletion of Tregs by injection of anti-CD4 or anti-CD25 antibody two months after transplantation leads to loss of skin allografts even though mice remain chimeric and exhibit little in vivo cytotoxicity. In contrast, chimerism is lost, but skin allografts survive following naïve T-cell injection. We conclude that hematopoietic chimerism and peripheral tolerance may be maintained by different mechanisms in mixed hematopoietic chimeras.

NOD mice develop type 1 autoimmune diabetes and exhibit genetically dominant resistance to transplantation tolerance induction. These two phenotypes are genetically separable. Costimulation blockade fails to prolong skin allograft survival in (NOD x C57BL/6)F1 mice and in NOD-related strains made diabetes-resistant by congenic introduction of protective major histocompatibility complex (MHC) or non-MHC Idd region genes. Here, we tested the hypothesis that the genetic basis for the resistance of NOD mice to skin allograft tolerance also applies to islet allografts. Surprisingly, costimulation blockade induced permanent islet allograft survival in (NOD x C57BL/6)F1 mice but not in NOD mice. After costimulation blockade, islet allograft survival was prolonged in diabetes-resistant NOD.B6 Idd3 mice and shortened in diabetes-free C57BL/6 mice congenic for the NOD Idd3 variant. Islet allograft tolerance could not be induced in diabetes-resistant NOD.B10 Idd5 and NOD.B10 Idd9 mice. The data demonstrate that 1) NOD mice resist islet allograft tolerance induction; 2) unlike skin allografts, resistance to islet allograft tolerance is a genetically recessive trait; 3) an Idd3 region gene(s) is an important determinant of islet allograft tolerance induction; and 4) there may be overlap in the mechanism by which the Idd3 resistance locus improves self-tolerance and the induction of allotolerance.

Background. Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8+ T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. Methods. Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day −7 plus 4 doses of anti-CD154 mAb on days −7, −4, 0, and +4. Deletion of CD8+ alloreactive cells was measured using “synchimeric” CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8+ T cells. Results. Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8+ T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. Conclusions. Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8+ T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.

Abstract: Recent advances in islet transplantation have enabled physicians to cure type 1 autoimmune diabetes, but at the cost of lifelong immunosuppression with its attendant side effects and long‐term health risks. To eliminate the need for immunosuppression, researchers have developed methods for inducing tolerance to transplanted allografts. Tolerance‐based transplantation using costimulation blockade has proven remarkably successful in many animal model systems. The most widely used animal model system for studying islet transplantation in type 1‐like autoimmune diabetes is the NOD mouse. Unfortunately, this strain has proven resistant to costimulation blockade‐based transplantation tolerance protocols that are successful in chemically diabetic mice given islet grafts. It has been assumed that resistance to transplantation tolerance in the NOD mouse is (1) related to autoimmunity directed against its pancreatic β cells, (2) a consequence of that autoimmunity, and (3) under the control of the same genes that control autoimmunity. In this review, we provide arguments to challenge these assumptions. We describe a new animal model and a new conceptual framework based on data indicating that the mechanisms responsible for resistance to transplantation tolerance and β cell autoimmunity are not identical. We believe that the recent discoveries we describe will have important implications for the development of tolerance‐based transplantation therapies and their translation from the laboratory to the clinic.

Curing type 1 diabetes by islet transplantation requires overcoming both allorejection and recurrent autoimmunity. This has been achieved with systemic immunosuppression, but tolerance induction would be preferable. Most islet allotransplant tolerance induction protocols have been tested in nonobese diabetic (NOD) mice, and most have failed. Failure has been attributed to the underlying autoimmunity, assuming that autoimmunity and resistance to transplantation tolerance have a common basis. Out of concern that NOD biology could be misleading in this regard, we tested the hypothesis that autoimmunity and resistance to transplantation tolerance in NOD mice are distinct phenotypes. Unexpectedly, we observed that (NOD × C57BL/6)F1 mice, which have no diabetes, nonetheless resist prolongation of skin allografts by costimulation blockade. Further analyses revealed that the F1 mice shared the dendritic cell maturation defects and abnormal CD4+ T cell responses of the NOD but had lost its defects in macrophage maturation and NK cell activity. We conclude that resistance to allograft tolerance induction in the NOD mouse is not a direct consequence of overt autoimmunity and that autoimmunity and resistance to costimulation blockade-induced transplantation tolerance phenotypes in NOD mice can be dissociated genetically. The outcomes of tolerance induction protocols tested in NOD mice may not accurately predict outcomes in human subjects.

The loss of self-tolerance leading to autoimmune type 1 diabetes in the NOD mouse model involves at least 19 genetic loci. In addition to their genetic defects in self-tolerance, NOD mice resist peripheral transplantation tolerance induced by costimulation blockade using donor-specific transfusion and anti-CD154 antibody. Hypothesizing that these two abnormalities might be related, we investigated whether they could be uncoupled through a genetic approach. Diabetes-resistant NOD and C57BL/6 stocks congenic for various reciprocally introduced Idd loci were assessed for their ability to be tolerized. Surprisingly, in NOD congenic mice that are almost completely protected from diabetes, costimulation blockade failed to prolong skin allograft survival. In reciprocal C57BL/6 congenic mice with NOD-derived Idd loci, skin allograft survival was readily prolonged by costimulation blockade. These data indicate that single or multiple combinations of evaluated Idd loci that dramatically reduce diabetes frequency do not correct resistance to peripheral transplantation tolerance induced by costimulation blockade. We suggest that mechanisms controlling autoimmunity and transplantation tolerance in NOD mice are not completely overlapping and are potentially distinct, or that the genetic threshold for normalizing the transplantation tolerance defect is higher than that for preventing autoimmune diabetes.