Idania Marrero

Slc11a1 Enhances the Autoimmune Diabetogenic T-Cell Response by Altering Processing and Presentation of Pancreatic Islet Antigens

OBJECTIVE—Efforts to map non–major histocompatibility complex (MHC) genes causing type 1 diabetes in NOD mice identified Slc11a1, formerly Nramp1, as the leading candidate gene in the Idd5.2 region. Slc11a1 is a membrane transporter of bivalent cations that is expressed in late endosomes and lysosomes of macrophages and dendritic cells (DCs). Because DCs are antigen-presenting cells (APCs) known to be critically involved in the immunopathogenic events leading to type 1 diabetes, we hypothesized that Slc11a1 alters the processing or presentation of islet-derived antigens to T-cells. RESEARCH DESIGN AND METHODS—NOD mice having wild-type (WT) or mutant Slc11a1 molecules and 129 mice having WT or null Slc11a1 alleles were examined for parameters associated with antigen presentation. RESULTS—We found that Slc11a1 enhanced the presentation of a diabetes-related T-cell determinant of GAD65, and its function contributed to the activation of a pathogenic T-cell clone, BDC2.5. An enhanced generation of interferon (IFN)-γ–producing T-cells was also associated with functional Slc11a1. The alteration of immune responsiveness by Slc11a1 genotype did not correlate with altered MHC class II expression in DCs; however, functional Slc11a1 was associated with accelerated phagocytosis and phagosomal acidification in DCs. CONCLUSIONS—The association of variants encoding Slc11a1 with type 1 diabetes may reflect its function in processing and presentation of islet self-antigens in DCs. Thus, non-MHC genes could affect the MHC-restricted T-cell response through altered antigen processing and presentation.

NKT cell subsets as key participants in liver physiology and pathology

Natural killer T (NKT) cells are innate-like lymphocytes that generally recognize lipid antigens and are enriched in microvascular compartments of the liver. NKT cells can be activated by self- or microbial-lipid antigens and by signaling through toll-like receptors. Following activation, NKT cells rapidly secrete pro-inflammatory or anti-inflammatory cytokines and chemokines, and thereby determine the milieu for subsequent immunity or tolerance. It is becoming clear that two different subsets of NKT cells—type I and type II—have different modes of antigen recognition and have opposing roles in inflammatory liver diseases. Here we focus mainly on the roles of both NKT cell subsets in the maintenance of immune tolerance and inflammatory diseases in liver. Furthermore, how the differential activation of type I and type II NKT cells influences other innate cells and adaptive immune cells to result in important consequences for tissue integrity is discussed. It is crucial that better reagents, including CD1d tetramers, be used in clinical studies to define the roles of NKT cells in liver diseases in patients.

Inhibition of type I natural killer T cells by retinoids or following sulfatide-mediated activation of type II natural killer T cells attenuates alcoholic liver disease in mice.

Innate immune mechanisms leading to liver injury subsequent to chronic alcohol ingestion are poorly understood. Natural killer T (NKT) cells, enriched in the liver and comprised of at least two distinct subsets, type I and II, recognize different lipid antigens presented by CD1d molecules. We have investigated whether differential activation of NKT cell subsets orchestrates inflammatory events leading to alcoholic liver disease (ALD). We found that after chronic plus binge feeding of Lieber‐DeCarli liquid diet in male C57BL/6 mice, type I, but not type II, NKT cells are activated, leading to recruitment of inflammatory Gr‐1highCD11b+ cells into the liver. A central finding is that liver injury after alcohol feeding is dependent upon type I NKT cells. Thus, liver injury is significantly inhibited in Jα18−/− mice deficient in type I NKT cells as well as after their inactivation by sulfatide‐mediated activation of type II NKT cells. Furthermore, we have identified a novel pathway involving all‐trans retinoic acid (ATRA) and its receptor (RARγ) signaling that inhibits type I NKT cells and, consequently, ALD. A semiquantitative polymerase chain reaction analysis of hepatic gene expression of some of the key proinflammatory molecules shared in human disease indicated that their up‐regulation in ALD is dependent upon type I NKT cells. Conclusions: Type I, but not type II, NKT cells become activated after alcohol feeding. Type I NKT cell‐induced inflammation and neutrophil recruitment results in liver tissue damage whereas type II NKT cells protect from injury in ALD. Inhibition of type I NKT cells by retinoids or by sulfatide prevents ALD. Given that the CD1d pathway is highly conserved between mice and humans, NKT cell subsets might be targeted for potential therapeutic intervention in ALD. (Hepatology 2015;61:1357–1369)

Type II NKT Cells in Inflammation, Autoimmunity, Microbial Immunity, and Cancer

Natural killer T cells (NKT) recognize self and microbial lipid antigens presented by non-polymorphic CD1d molecules. Two major NKT cell subsets, type I and II, express different types of antigen receptors (TCR) with distinct mode of CD1d/lipid recognition. Though type II NKT cells are less frequent in mice and difficult to study, they are predominant in human. One of the major subsets of type II NKT cells reactive to the self-glycolipid sulfatide is the best characterized and has been shown to induce a dominant immune regulatory mechanism that controls inflammation in autoimmunity and in anti-cancer immunity. Recently, type II NKT cells reactive to other self-glycolipids and phospholipids have been identified suggesting both promiscuous and specific TCR recognition in microbial immunity as well. Since the CD1d pathway is highly conserved, a detailed understanding of the biology and function of type II NKT cells as well as their interplay with type I NKT cells or other innate and adaptive T cells will have major implications for potential novel interventions in inflammatory and autoimmune diseases, microbial immunity, and cancer.

High-throughput sequencing of islet-infiltrating memory CD4+ T cells reveals a similar pattern of TCR Vβ usage in prediabetic and diabetic NOD mice.

Autoreactive memory CD4+ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4+ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4+CD44high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4+CD44high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4+CD44high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.

N-terminal flanking residues of a diabetes-associated GAD65 determinant are necessary for activation of antigen-specific T cells in diabetes-resistant mice

A diabetes‐associated peptide in the glutamic acid decarboxylase 65 (GAD65) molecule, p524–543, activates two distinct populations of T cells, which apparently play opposite roles in the development of diabetes in NOD mice. By comparing the fine specificity of these two T cell repertoires using a nested set of truncated peptides that cover the p524–543 region, we found, surprisingly, that all clones recognized the same core within this peptide, p530–539. The core itself was non‐immunogenic, but the residues flanking this shared sequence played the crucial role in selecting T cells to activate. A peptide missing N‐terminal flanking residues at position 528 and 529 was stimulatory in NOD but not in MHC‐matched, NOD‐resistant (NOR) mice, suggesting that a protective response in the resistant mice may require T cell recognition of one or more of the N‐terminal flanking residues. T cell repertoire studies demonstrated selective clonal expansions within the BV4 TCR family that dominates the p524–543 response in NOD but not in NOR mice. These data suggest that processing or trimming events affecting T cell recognition of very few flanking residues of diabetes‐associated determinants might be involved in the protective response in NOR mice.

T cell populations in the pancreatic lymph node naturally and consistently expand and contract in NOD mice as disease progresses

Nonobese diabetic (NOD) mice develop spontaneous autoimmune Type 1 diabetes (T1D) that results from the destruction of insulin secreting β cells by diabetogenic T cells. The activation of autoreactive T cells occurs in the pancreatic lymph nodes (PLN) from where effector T cells migrate to the pancreas. This study was designed to explore whether T cell populations in the NOD PLN expand in a predictable and reproducible way during disease progression. Complementary determining region (CDR) 3 length spectratype analysis of 19 TCR Vβ families was used to identify the relative frequency of T populations in PLN of 4 and 10 week old NOD mice and mice at T1D onset. Significant and highly reproducible changes in specific T cell populations were detected in 14 of Vβ families tested at all stages of disease. However, of these, the CDR3 spectratype of only four Vβ families was significantly more perturbed at T1D onset than in 10 week old mice. Intriguingly, when diabetes was induced in 10 week old mice with cyclophosphamide (CYP) the same four Vβ families, Vβ5.1, Vβ9, Vβ10, and Vβ15, were again significantly more perturbed than in the untreated non-diabetic age matched mice. Taken together the data show that while T cell responses in PLN of NOD mice are heterogeneous, they are ordered and consistent throughout disease development. The finding that within this heterogeneous response four Vβ families are significantly more perturbed in diabetic mice, whether spontaneous or induced, strongly suggests their selection as part of the disease process.

High-throughput sequencing reveals restricted TCR Vβ usage and public TCRβ clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes

Islet-reactive memory CD4(+) T cells are an essential feature of type 1 diabetes (T1D) as they are involved in both spontaneous disease and in its recurrence after islet transplantation. Expansion and enrichment of memory T cells have also been shown in the peripheral blood of diabetic patients. Here, using high-throughput sequencing, we investigated the clonal diversity of the TCRβ repertoire of memory CD4(+) T cells in the pancreatic lymph nodes (PaLN) of non-obese diabetic (NOD) mice and examined their clonal overlap with islet-infiltrating memory CD4T cells. Both prediabetic and diabetic NOD mice exhibited a restricted TCRβ repertoire dominated by clones expressing TRBV13-2, TRBV13-1 or TRBV5 gene segments. There is a limited degree of TCRβ overlap between the memory CD4 repertoire of PaLN and pancreas as well as between the prediabetic and diabetic group. However, public TCRβ clonotypes were identified across several individual animals, some of them with sequences similar to the TCRs from the islet-reactive T cells suggesting their antigen-driven expansion. Moreover, the majority of the public clonotypes expressed TRBV13-2 (Vβ8.2) gene segment. Nasal vaccination with an immunodominat peptide derived from the TCR Vβ8.2 chain led to protection from diabetes, suggesting a critical role for Vβ8.2(+) CD4(+) memory T cells in T1D. These results suggest that memory CD4(+) T cells bearing limited dominant TRBV genes contribute to the autoimmune diabetes and can be potentially targeted for intervention in diabetes. Furthermore, our results have important implications for the identification of public T cell clonotypes as potential novel targets for immune manipulation in human T1D.

CD4+ CD44v.low cells are unique peripheral precursors that are distinct from recent thymic emigrants and stem cell-like memory cells

CD4(+) CD44(v.low) cells are peripheral precursor T cells that inhibit lymphopenia by generating a large CD4(+) T cell pool containing balanced numbers of naïve, memory, and regulatory Foxp3(+) cells with a diverse TCR repertoire. Recent thymic emigrants (RTE) and stem cell-like memory T cells (T(SCM)) can also replenish a T cell pool. In this study we formally test whether CD44(v.low) cells are the same population as RTE and T(SCM). Our data show that, in contrast to RTE, CD44(v.low) cells express high levels of CD45RB and low levels of CD24. Moreover, CD44(v.low) cells isolated from mice devoid of RTE retain their capacity to repopulate lymphopenic mice with naïve and memory cells and Foxp3(+) Tregs. In addition, CD44(v.low) cells do not express IL-2Rβ, Sca-1, and CXCR3, the phenotypic hallmarks of T(SCM). Overall, these data demonstrate that CD44(v.low) cells are neither RTE nor T(SCM).

Autoreactivity to self H-2Kb peptides in TAP1−/− mice. Intravenous administration of H-2Kb class I-derived peptides induces long-term survival of grafts from C57BL/6 donors

We and others have previously shown that TAP1–/– mice (H‐2b) reject grafts from donors without major histocompatibility complex (MHC) disparity that express wild‐type levels of H‐2b class I molecules (C57BL/6, TAP1+/+ mice). In this same model, we also showed that subcutaneous priming of TAP1–/– mice with synthetic peptides derived from the H‐2Kb molecule accelerated graft rejection and that in vivo depletion of CD4+ T cells induced a significant prolongation of graft survival, suggesting an important role for CD4 T cells. We hypothesize that, in this model, rejection is triggered by the recognition of class I molecules or derived peptides, in an inflammatory microenvironment, by a functionally altered autoreactive T‐cell repertoire that escapes the control of peripheral regulatory mechanisms. In the present study, we analysed the cellular autoreactivity induced by synthetic peptides derived from the H‐2Kb sequence in naive and TAP1–/– mice transplanted with C57BL/6 grafts, and investigated whether intravenous modulation of autoreactivity to these peptides induced transplantation tolerance. We showed that TAP1–/– mice have peripheral autoreactive T cells that recognize H‐2Kb peptides. A significant amplification of proliferation against these peptides was detected in TAP1–/– mice that rejected grafts, indicating that the inflammatory context of transplantation induced peripheral expansion of these autoreactive T cells. Furthermore, intravenous injection of H‐2Kb‐derived peptides significantly prolonged graft survival in some animals. In these mice (> 100 days graft survival), we observed intragraft inhibition of interferon‐γ and interleukin‐10 expression, suggesting that these cytokines have an active role during the rejection. In conclusion, our present data indicate that inflammatory autoreactive T cells directed against H‐2Kb peptides can be inhibited in the periphery to prolong graft survival in TAP1–/– mice.