Costimulation blockade targeting the CD40-CD154 pathway is an effective approach to induce tolerance to allografts in murine and non-human primate models. However inflammation generated during infection and exposure to pathogen associated molecular patterns abrogates costimulation blockade induced tolerance. Here we have characterized the alloreactive CD8 T cell response during costimulation blockade in the absence or presence of inflammation. Costimulation blockade consisting of donor splenocytes (DST)/anti-CD154 antibody (MR1) stimulated proliferation of alloreactive CD8 T cells but resulted in deletion of the cells by apoptosis. Exposure to LPS during blockade prevented this deletion, but surprisingly reduced proliferation kinetics of the alloreactive CD8 T cells. These data suggest that LPS increases the frequency of alloreactive CD8 T cells by providing survival signals during cell division. RNA-seq analyses revealed differential expression of genes involved in apoptosis (like Fasl, Perp, Birc5, E2F transcription factors), cell cycle (Ki-67, Bub1, Nusap1), CD8 T cell differentiation and function (granzyme B, T-bet, Eomes) and type I interferon induced genes (Ifi44, Ifit1, Usp18) between the treatment groups. Such phenotypic differences will facilitate identification of pathways that can be targeted to prevent the abrogation of transplantation tolerance in situations of inflammation.

Blockade of co-stimulatory signals to T cells is extremely effective for the induction of transplantation tolerance in immunologically naive rodents. However, infections and inflammation compromise the efficacy of co-stimulation blockade regimens for the induction of tolerance, thereby stimulating the rejection of allografts. Previous studies have shown that stimulation of innate immunity abrogates tolerance induction by preventing the deletion of alloreactive CD8+ T cells that normally occurs during co-stimulation blockade. Although inflammation prevents the deletion of alloreactive T cells during co-stimulation blockade, it is not known if this resistance to cell death is the result of a mechanism intrinsic to the T cell. Here, we used syngeneic bone marrow chimeric mice that contain a trace population of T-cell receptor transgenic alloreactive CD8+ T cells to investigate the early apoptotic signature and activation status of alloreactive T cells following exposure to inflammatory signals during co-stimulation blockade with an antibody specific for CD154. Our findings revealed that the presence of bacterial lipopolysaccharide during co-stimulation blockade enhanced the early activation of alloreactive CD8+ T cells, as indicated by the up-regulation of CD25 and CD69, suppressed Fas ligand expression, and prevented apoptotic cell death. However, alloreactive CD8+ T cells from lipopolysaccharide-treated mice remained sensitive to Fas-mediated apoptosis in vitro. These findings suggest that alloreactive T cells rescued from deletion during co-stimulation blockade by inflammation are still sensitive to pro-apoptotic signals and that stimulating these apoptotic pathways during co-stimulation blockade may augment the induction of tolerance.

OBJECTIVE To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell–derived β-cells in the absence or presence of a functional human immune system. RESEARCH DESIGN AND METHODS We backcrossed the Ins2Akita mutation onto the NOD-Rag1null IL2rγnull strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts. RESULTS We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rγnull, Rag1null, and Ins2Akita genes in NOD-Rag1null IL2rγnull Ins2Akita (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rγnull and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts. CONCLUSIONS NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell–derived β-cells in the absence or presence of an alloreactive human immune system.

“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγnull mutation; NOD-scid IL2rγnull, NOD-Rag1null IL2rγnull, and BALB/c-Rag1null IL2rγnull mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.