Jennifer O. Manilay

Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen

BACKGROUND Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.

Natural killer cells and their role in graft rejection

Natural killer cells can weakly resist engraftment of allogeneic bone marrow transplants in mice. Functional studies suggest that natural killer cell tolerance can be induced by bone marrow transplantation. Human natural killer cell inhibitory receptor repertoires differ between individuals, depending on their MHC genotype. This supports the concept that the human natural killer cell repertoire, like that of mice, adapts to the MHC molecules presented in its environment. Natural killer cells play a greater role in rejecting xenogeneic than allogeneic bone marrow and have been implicated in the rejection of xenogeneic solid organ transplants. Natural killer cell inhibitory receptors may have a lower likelihood of cross-reacting with xenogeneic than with allogeneic MHC class I molecules; important glycosylation differences between species may affect the propensity of natural killer cells to kill xenogeneic targets.

NK Cell Tolerance in Mixed Allogeneic Chimeras

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c→B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c→ B6 chimeras rejected a low dose of β2-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and β2-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin− BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.

Murine CD133 + CD49f low/+ Cells Derived from ESCs Differentiate into Insulin Producing Cells in vivo

Pancreatic progenitors have been identified and isolated during embryonic development showing their potential to differentiate into insulin producing cells after transplantation. Subsequently, they have been proposed as an alternative to the treatment of Type I diabetes. However, fetal pancreata represent a limited donor supply and we proposed that ESC-derived pancreatic progenitors are another possible option. We isolated committed pancreatic CD133+CD49flow/+ progenitors, expressing neurogenin 3, from insulin producing cells derived from mouse embryonic stem cells in vitro. Here, we demonstrate their potential to differentiate into insulin producing cells in vivo while not forming teratomas. Our results can pave the way for future studies to ascertain the ability of in vitro ESC-derived pancreatic progenitors to be translated for clinical application for Type I diabetes treatments.

Stem Cell Therapies for Type I Diabetes

Diabetes mellitus is a chronic metabolic disease that results in high levels of glucose in the blood. Normally, glucose is transported into cells from the blood for energy, and this transport is initiated in response to the hormone, insulin. In diabetic patients, cellular glucose uptake is defective, in part due to the inability of cells to respond to insulin, or the inability of the body to produce the insulin itself. According to the International Diabetes Foundation, 366 million people worldwide had been diagnosed with diabetes in 2011, and this number is continuing to increase in every country (http://www.idf.org/). It is an especially challenging health problem, as treatments to both control hyperglycemia as well as the debilitating side effects of diabetes, such as injury to the blood vessels and nerves, must be addressed. Amongst the three types of diabetes (Type I autoimmune, gestational and Type II – adult onset), Type II diabetes is the most prevalent, in which hyperglycemia is uncontrolled due to the body’s inability to produce enough insulin or due to the body’s inability to respond appropriately to lower the blood glucose level. In contrast, in patients with Type I diabetes (TID), insulin-producing beta (β) cells are destroyed by the immune system. Studies of TID by many groups have provided extensive insight to the fields of immunological tolerance and pancreatic developmental biology. In this chapter, we will briefly describe TID, provide a synopsis of pancreatic β cell development in mice as compared to humans, review of some of the medical treatments currently available for TID, and discuss current studies that have explored the use of stem cells as alternative possible therapies for TID.