Erin E. Hillhouse

A comprehensive review of the phenotype and function of antigen-specific immunoregulatory double negative T cells

Double negative T cells that lack the expression of both CD4 and CD8 T cell co-receptors exhibit a most unique antigen-specific immunoregulatory potential first described over a decade ago. Due to their immunoregulatory function, this rare T cell population has been studied in both mice and humans for their contribution to peripheral tolerance and disease prevention. Consequently, double negative cells are gaining interest as a potential cellular therapeutic. Herein, we review the phenotype and function of double negative T cells with emphasis on their capacity to induce antigen-specific immune tolerance. While the phenotypic and functional similarities between double negative T cells identified in mouse and humans are highlighted, we also call attention to the need for a specific marker of double negative T cells, which will facilitate future studies in humans. Altogether, due to their unique properties, double negative T cells present a promising therapeutic potential in the context of various disease settings.

Interleukin-10 limits the expansion of immunoregulatory CD4 − CD8 − T cells in autoimmune-prone non-obese diabetic mice

Regulatory T cells appear to show great potential for use in cellular therapy. In particular, CD4−CD8− (double negative (DN)) T cells, which compose 1–3% of the total number of T lymphocytes, exhibit prominent antigen‐specific immune tolerance properties and confer immune tolerance in models of allografts and xenografts. We have recently shown that autoimmune‐diabetes‐prone mice carry fewer DN T cells and that this phenotype contributes to autoimmune‐prone diabetes susceptibility, suggesting that increasing DN T‐cell number in autoimmune‐prone individuals may be of therapeutic interest. To achieve this goal, we must first determine whether the remaining DN T cells in autoimmune‐prone mice are functional. In addition, we must identify the parameters that regulate the numbers of DN T cells. Herein, we evaluate the immunoregulatory properties of DN T cells in the autoimmune‐prone non‐obese diabetic (NOD) genetic background. Using 3A9 TCR transgenic mice, we show that DN T cells from both diabetes‐resistant B10.Br and genetically autoimmune‐prone NOD.H2k mice show an equivalent immunoregulatory potential on a per cell basis. However, upon stimulation, there is a 10‐fold increase in the number of 3A9 TCR transgenic DN T cells that produce interleukin10 (IL‐10) from NOD.H2k mice in comparison with B10.Br mice. We further showed that IL‐10 facilitates DN T‐cell apoptosis and thus may regulate the number of DN T cells. Taken together, our results show that, although reduced in number, DN T cells from mice carrying an autoimmune‐prone genetic background exhibit a potent cytotoxic potential and that DN T‐cell expansion is regulated, at least in part, by IL‐10.

Implication of the CD47 pathway in autoimmune diabetes.

CD47 and signal regulatory protein (SIRP) interactions have been proposed to take part in autoimmune disease susceptibility. Importantly, a recent genome-wide association study for type 1 diabetes susceptibility highlighted the association of the 20p13 region comprising the SIRP cluster, where some of the SIRP proteins encode functional ligands to CD47. Using a TCR transgenic mouse model at the brink of autoimmune disease, we demonstrate that CD47-deficiency is sufficient to break the immune tolerance and provoke the onset of autoimmune diabetes. Interestingly, CD47-deficient mice show a severe reduction in the number of mature CD4(-)CD8(-) T cells, and passive transfer of these CD4(-)CD8(-) T cells is sufficient to restore immune tolerance and prevent diabetes progression. Together, these findings constitute an in vivo demonstration that CD47 is involved in diabetes susceptibility and controls the homeostatic regulation of CD4(-)CD8(-) T cells.

Immunoregulatory CD4-CD8- T cells as a potential therapeutic tool for transplantation, autoimmunity, and cancer

A central objective in organ transplantation and the treatment or prevention of autoimmune disease is the achievement of antigen-specific immune tolerance. An additional challenge in bone marrow transplantation for the treatment of hematological malignancies is the prevention of graft-vs-host disease (GVHD) while maintaining graft-vs-tumor activity. Interestingly, CD4-CD8- (double negative, DN) T cells, which exhibit a unique antigen-specific immunoregulatory potential, appear to exhibit all of the properties to respond to these challenges. Herein, we review the therapeutic potential of immunoregulatory DN T cells in various immunopathological settings, including graft tolerance, GVHD, cancer, and autoimmunity.

Nearby Construction Impedes the Progression to Overt Autoimmune Diabetes in NOD Mice

Construction nearby animal houses has sporadically been reported to affect various aspects of animal health. Most of the reports have focussed on the impact on stress hormone levels and the hypersensitivity of animals relative to humans. There has also been an anecdotal report on the impact of construction on autoimmune diabetes in NOD mice. Here, we describe that nearby construction significantly impedes the progression to overt diabetes in female NOD mice offspring. We demonstrate that this was not due to a genetic drift or to particularities associated with our specific mouse colony. Interestingly, although the glycemia levels remained low in mice born from mothers subject to construction stress during gestation, we detected an active autoimmune reaction towards pancreatic islet cells, as measured by both the degree of insulitis and the presence of insulin autoantibody levels in the serum. These results suggest that the external stress imposed during embryonic development does not prevent but significantly delays the autoimmune process. Together, our findings emphasize the impact of surrounding factors during in vivo studies and are in agreement with the hypothesis that both environmental and genetic cues contribute to autoimmune diabetes development.

Idd13 is involved in determining immunoregulatory DN T-cell number in NOD mice

Immunoregulatory T cells have been identified as key modulators of peripheral tolerance and participate in preventing autoimmune diseases. CD4−CD8− (double negative, DN) T cells compose one of these immunoregulatory T-cell subsets, where the injection of DN T cells confers protection from autoimmune diabetes progression. Interestingly, genetic loci defining the function and number of CD4+CD25+Foxp3+ regulatory T cells (Tregs) coincide with at least some autoimmune disease susceptibility loci. Herein, we investigate the impact of major insulin-dependent diabetes (Idd) loci in defining the number of DN T cells. We demonstrate that although Idd3, Idd5 and Idd9 loci do not regulate DN T-cell number, NOD mice congenic for diabetes resistance alleles at the Idd13 locus show a partial restoration in DN T-cell number. Moreover, competitive and non-competitive bone marrow chimera experiments reveal that DN T-cell number is defined by a bone marrow-intrinsic, but DN T-cell-extrinsic, factor. This suggests that non-autonomous candidate genes define DN T-cell number in secondary lymphoid organs. Together, our results show that the regulation of DN T-cell number in NOD mice is at least partially conferred by alleles at the Idd13 locus.

NK Cell Proportion and Number Are Influenced by Genetic Loci on Chromosomes 8, 9, and 17

NK cells play a crucial role in innate immunity due to their direct cytotoxicity toward tumors, virally infected cells, and stressed cells, and they also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. In secondary lymphoid organs, NK cells compose the third most abundant lymphocyte subset after T cells and B cells. In this study, we perform an unbiased linkage analysis to determine the genetic loci that may limit the size of the NK cell compartment. Specifically, we exploit differences in NK cell proportion and absolute number between the C57BL/6 and the NOD mice. In addition to the previously identified linkage to chromosome 8, we find that a locus on chromosome 17, which encompasses the MHC locus, impacts NK cell number. Moreover, we identify a locus on mouse chromosome 9 that is strongly linked to the proportion and absolute number of NK cells. Using NOD congenic mice, we validate that both the MHC and the chromosome 9 loci influence the proportion and absolute number of NK cells. We have thus identified additional loci specifically linked to the proportion of NK cells and present some of the potential candidate genes comprised within these loci.

Unusual selection and peripheral homeostasis for immunoregulatory CD4(-) CD8(-) T cells.

Immunoregulatory CD4− CD8− (double‐negative; DN) T cells exhibit a unique antigen‐specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T‐cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2k MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2b MHC haplotype. Herein, we take advantage of this well‐defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC‐selecting haplotype. By defining DN T‐cell differentiation in 3A9 TCR transgenic CD47‐deficient mice as well as in mice bearing the NOD.H2k genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.

An Unbiased Linkage Approach Reveals That the p53 Pathway Is Coupled to NK Cell Maturation

Natural killer cells constitute potent innate lymphoid cells that play a major role in both tumor immunosurveillance and viral clearance via their effector functions. A four-stage model of NK cell functional maturation has been established according to the expression of CD11b and CD27, separating mature NK (mNK) cells into distinct populations that exhibit specific phenotypic and functional properties. To identify genetic factors involved in the regulation of NK cell functional maturation, we performed a linkage analysis on F2 (B6.Rag1−/− × NOD.Rag1−/− intercross) mice. We identified six loci on chromosomes 2, 4, 7, 10, 11, and 18 that were linked to one or more mNK cell subsets. Subsequently, we performed an in silico analysis exploiting mNK cell subset microarray data, highlighting various genes and microRNAs as potential regulators of the functional maturation of NK cells. Together, the combination of our unbiased genetic linkage study and the in silico analysis positions genes known to affect NK cell biology along the specific stages of NK cell functional maturation. Moreover, this approach allowed us to uncover a novel candidate gene in the regulation of NK cell maturation, namely Trp53. Using mice deficient for Trp53, we confirm that this tumor suppressor regulates NK cell functional maturation. Additional candidate genes revealed in this study may eventually serve as targets for the modulation of NK cell functional maturation to potentiate both tumor immunosurveillance and viral clearance.

TCR transgenic mice reveal the impact of type 1 diabetes loci on early and late disease checkpoints

Linkage analysis studies for autoimmune diabetes have revealed multiple non‐major histocompatibility complex (MHC) chromosomal regions linked to disease susceptibility. To date, more than 20 insulin‐dependent diabetes (Idd) loci linked to diabetes susceptibility have been identified in NOD mice and validated via congenic breeding. Importantly, evidence suggests that Idd loci may regulate at least two pathological steps during autoimmune diabetes development, namely the onset of insulitis and the transition from insulitis to overt diabetes. Here we assess the role of various non‐MHC Idd diabetes‐resistance loci, which have been validated in the non‐transgenic setting, on autoimmune diabetes progression in the transgenic setting. Specifically, we generated multiple Idd congenic strains in the 3A9‐TCR:insHEL NOD.H2k transgenic model and monitored their diabetes incidence. We show that 3A9‐TCR:insHEL NOD.H2k mice congenic for Idd3 or Idd5 display a reduction in diabetes development, whereas mice congenic for Idd9 or Idd13 exhibit an increase, in comparison with 3A9‐TCR:insHEL NOD.H2k mice. These results suggest that the presence of the 3A9‐TCR and hen egg lysosyme transgenes can offset the regulatory function of certain diabetes‐resistance genetic variants contained within the Idd loci, including Idd9 and Idd13. We propose the antigen‐specific 3A9‐TCR:insHEL transgenic model as a useful tool for the study of the genetics of autoimmune diabetes development.