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OBJECTIVE: CD8(+) T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8(+) T-cells will require the identification of disease-relevant epitopes. RESEARCH DESIGN AND METHODS: We used islet-infiltrating CD8(+) T-cells from HLA-A*0201 transgenic NOD mice in an interferon-gamma enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike. RESULTS: We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3-11, Ins1 B5-14, and Ins1/2 A2-10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5-14 and Ins1/2 A2-10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process. CONCLUSIONS: The human versions of B5-14 and A2-10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8(+) T-cell targets in HLA-A*0201-positive type 1 diabetic patients. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton is necessary for cell viability and plays crucial roles in cell motility, endocytosis, growth, and cytokinesis. Hence visualization of dynamic changes in F-actin distribution in vivo is of central importance in cell biology. This has been accomplished by the development of fluorescent protein fusions to actin itself or to various actin-binding proteins, actin cross-linking proteins, and their respective actin-binding domains (ABDs). Although these protein fusions have been shown to bind to F-actin in vivo, we show that the fluorescent protein used for visualization changes the subset of F-actin labeled by an F-actin ABD probe. Further, different amino acid linkers between the fluorescent protein and ABD induced a similar change in localization. Although different linkers and fluorescent proteins can alter the subset of actin bound by a particular ABD, in most cases, the fusion protein did not label all of a cell's F-actin all of the time. Even LimEΔcoil and GFP-actin, which have been used extensively for cytoskeletal visualization, were highly variable in the subsets of actin that they labeled. Lifeact, conversely, clearly labeled cortical F-actin as well as F-actin in the anterior pseudopods of motile cells and in macropinocytotic cups. We conclude that Lifeact most accurately labels F-actin and is the best currently available probe for visualization of dynamic changes in F-actin networks., (Copyright © 2013 Wiley Periodicals, Inc.)

Clostridium difficile can infect the large intestine and cause colitis when the normal intestinal microbiota is altered by antibiotic administration. Little is known about the innate immune signaling pathways that marshal inflammatory responses to C. difficile infection and whether protective and pathogenic inflammatory responses can be dissociated. Toll-like receptors predominantly signal via the MyD88 adaptor protein and are important mediators of innate immune signaling in the intestinal mucosa. Here, we demonstrate that MyD88-mediated signals trigger neutrophil and CCR2-dependent Ly6C(hi) monocyte recruitment to the colonic lamina propria (cLP) during infection, which prevent dissemination of bystander bacteria to deeper tissues. Mortality is markedly increased in MyD88-deficient mice following C. difficile infection, as are parameters of mucosal tissue damage and inflammation. Antibody-mediated depletion of neutrophils markedly increases mortality, while attenuated recruitment of Ly6C(hi) monocytes in CCR2-deficient mice does not alter the course of C. difficile infection. Expression of CXCL1, a neutrophil-recruiting chemokine, is impaired in the cLP of MyD88(-/-) mice. Our studies suggest that MyD88-mediated signals promote neutrophil recruitment by inducing expression of CXCL1, thereby providing critical early defense against C. difficile-mediated colitis.

Type 1 diabetes is an autoimmune disease characterized by T cell responses to β cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for β cell-specific CD8(+) T cells in the pathogenic process. As CD8(+) T cells specific for β cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β(2)-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify β cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8(+) T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.

Antibiotic-induced changes in the intestinal microbiota predispose mammalian hosts to infection with antibiotic-resistant pathogens. Clostridium difficile is a Gram-positive intestinal pathogen that causes colitis and diarrhea in patients following antibiotic treatment. Clindamycin predisposes patients to C. difficile colitis. Here, we have used Roche-454 16S rRNA gene pyrosequencing to longitudinally characterize the intestinal microbiota of mice following clindamycin treatment in the presence or absence of C. difficile infection. We show that a single dose of clindamycin markedly reduces the diversity of the intestinal microbiota for at least 28 days, with an enduring loss of ca. 90% of normal microbial taxa from the cecum. Loss of microbial complexity results in dramatic sequential expansion and contraction of a subset of bacterial taxa that are minor contributors to the microbial consortium prior to antibiotic treatment. Inoculation of clindamycin-treated mice with C. difficile (VPI 10463) spores results in rapid development of diarrhea and colitis, with a 4- to 5-day period of profound weight loss and an associated 40 to 50% mortality rate. Recovering mice resolve diarrhea and regain weight but remain highly infected with toxin-producing vegetative C. difficile bacteria and, in comparison to the acute stage of infection, have persistent, albeit ameliorated cecal and colonic inflammation. The microbiota of "recovered" mice remains highly restricted, and mice remain susceptible to C. difficile infection at least 10 days following clindamycin, suggesting that resolution of diarrhea and weight gain may result from the activation of mucosal immune defenses.

The microbial communities that inhabit the intestinal tract are essential for mammalian health. Communication between the microbiota and the host establishes and maintains immune homeostasis, enabling protective immune responses against pathogens while preventing adverse inflammatory responses to harmless commensal microbes. Specific bacteria, such as segmented filamentous bacteria, Clostridium species, and Bacteroides fragilis, are key contributors to immune homeostasis in the gut. The cellular and molecular interactions between intestinal microbes and the immune system are rapidly being elucidated. Here, we review advances in our understanding of the microbial populations that shape the mucosal immune system and create a protective defense that prevents infection while tolerating friendly commensals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Clostridium difficile is a spore-forming bacterium that infects the lower intestinal tract of humans and is the most common known cause of diarrhea among hospitalized patients. Clostridium difficile colitis is mediated by toxins and develops during or following antibiotic administration. We have used a murine model of C. difficile infection, which reproduces the major features of the human disease, to study the effect of innate immune activation on resistance to C. difficile infection. We found that administration of purified Salmonella-derived flagellin, a Toll-like receptor 5 (TLR5) agonist, protects mice from C. difficile colitis by delaying C. difficile growth and toxin production in the colon and cecum. TLR5 stimulation significantly improves pathological changes in the cecum and colon of C. difficile-infected mice and reduces epithelial cell loss. Flagellin treatment reduces epithelial apoptosis in the large intestine, thereby protecting the integrity of the intestinal epithelial barrier during C. difficile infection. We demonstrate that restoring intestinal innate immune tone by TLR stimulation in antibiotic-treated mice ameliorates intestinal inflammation and prevents death from C. difficile colitis, potentially providing an approach to prevent C. difficile-induced pathology.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

Type 1 diabetes results from the autoimmune destruction of insulin-producing beta cells by T cells specific for beta cell Ags, including insulin. In humans, the non-MHC locus conferring the strongest disease susceptibility is the insulin gene, and alleles yielding lower thymic insulin expression are predisposing. We sought to incorporate this characteristic into an HLA-transgenic model of the disease and to determine the influence of reduced thymic insulin expression on CD8+ T cell responses to preproinsulin. We examined NOD.Ins2(-/-) mice, which do not express insulin in the thymus and show accelerated disease, to determine whether they exhibit quantitative or qualitative differences in CD8+ T cell responses to preproinsulin. We also generated NOD.Ins2(-/-) mice expressing type 1 diabetes-associated HLA-A*0201 (designated NOD.beta2m(-/-).HHD.Ins2(-/-)) in an effort to obtain an improved humanized disease model. We found that CD8+ T cell reactivity to certain insulin peptides was more readily detected in NOD.Ins2(-/-) mice than in NOD mice. Furthermore, the proportion of insulin-reactive CD8+ T cells infiltrating the islets of NOD.Ins2(-/-) mice was increased. NOD.beta2m(-/-).HHD.Ins2(-/-) mice exhibited rapid onset of disease and had an increased proportion of HLA-A*0201-restricted insulin-reactive T cells, including those targeting the clinically relevant epitope Ins B10-18. Our results suggest that insulin alleles that predispose to type 1 diabetes in humans do so, at least in part, by facilitating CD8+ T cell responses to the protein. We propose the NOD.beta2m(-/-).HHD.Ins2(-/-) strain as an improved humanized disease model, in particular for studies seeking to develop therapeutic strategies targeting insulin-specific T cells.

Members of the TNF-ligand and receptor superfamilies are important in the pathogenesis of lupus nephritis, a major cause of mortality and morbidity in SLE. TWEAK, a member of the TNF-ligand superfamily, is markedly increased in urine from patients with active lupus nephritis, and urinary TWEAK levels significantly correlate with renal disease activity. To support a possible role of TWEAK in the pathogenesis of lupus nephritis and other inflammatory nephritides, we examined the effects of TWEAK in human kidney mesangial cells, podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. We found that TWEAK induces human kidney cells to express multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1alpha, ICAM-1, and VCAM-1. Cytokine production is mediated through NF-kappaB activation, and is inhibited by anti-TWEAK monoclonal antibodies. TWEAK stimulated chemokines induced migration of human PBMC, particularly monocytes/macrophages. Furthermore, we found that TWEAK promotes kidney infiltration of inflammatory cells, and stimulates proliferation of kidney cells in vitro and in vivo. Thus, TWEAK may play an important pathogenic role in the development of glomerulonephritis by promoting a local inflammatory environment and inducing kidney cell proliferation. Blocking TWEAK/Fn14 interactions may be a promising therapeutic target in immune-mediated renal diseases.

Type 1 diabetes is an autoimmune disorder characterized by progressive destruction of insulin-secreting beta cells of the pancreas, in which CD8(+) T cells play a critical role. The diversity in the HLA alleles expressed among various racial and ethnic groups leads to great variability in antigen presentation and recognition by CD8(+) T cells in the context of MHC class I molecules. To date, studies aimed at identifying disease-relevant antigenic epitopes have focused on using mice transgenic for HLA-A*0201, a common allele, particularly among Caucasians. We present HLA class I typing data from 88 children with type 1 diabetes at the Children's Hospital at Montefiore, where the patient population is ethnically diverse, but largely minority. When categorized into the HLA supertypes A2, A3, B7, and C1, 77% of those studied have alleles belonging to at least one supertype, and of these patients, 65% do not belong to the A2 supertype, which is the supertype represented by the HLA-A*0201 allele. These results support the need for studies using HLA transgenic mice expressing MHC molecules representative of a variety of HLA supertypes, particularly when searching for antigenic epitopes applicable for study among largely urban, minority pediatric populations.

During progression to type 1 diabetes (T1D), the pancreatic islets of humans and of the widely studied mouse model of T1D, the nonobese diabetic (NOD) mouse, are infiltrated by cells of the immune system. Here we report that infiltrated mouse islets ("translucent islets") can be identified visually. We demonstrate the use of an efficient method for the isolation and culture of the islet-infiltrating cells of NOD mice, which results in a high percentage of CD8(+) T cells after seven days, with minimal manipulation. We show that islet-infiltrating cells exit the islets soon after they are placed in culture and can be used in flow cytometric experiments several hours later. Importantly, we demonstrate that the cultured cells are antigen-responsive and that specificities present at the beginning of the culture are generally still present on day seven. In addition, some reactivities are undetected without culture, supporting the validity of the seven-day expansion period. This technique greatly facilitates investigations of the CD8(+) T cell reactivities that play a pivotal role in the demise of pancreatic beta cells leading to the development of T1D.

CD8(+) T cells play an important role in the development of type 1 diabetes (T1D) in NOD mice and humans. IGRP (islet-specific glucose-6-phosphatase catalytic subunit-related protein) has emerged in recent years as a major antigen in NOD mice. Therefore, we aimed to determine if IGRP is an antigen in T1D patients and to identify the HLA-A2-restricted IGRP epitopes targeted. Using IFN-gamma ELISPOT assay, we tested PBMC from recent-onset pediatric T1D patients and healthy controls for reactivity to four IGRP peptides directly ex vivo. Importantly, 65% of patients and 0% of controls were positive for at least one IGRP peptide. Two of these have not been reported previously. These data provide evidence that IGRP is a CD8(+) T cell antigen in humans, contributing to the understanding of the underlying disease process as well as to future directions for diagnosis and monitoring disease progression in T1D patients.

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet beta cells. Cytotoxic CD8(+) T cells, reactive to beta cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8(+) T cells specific for beta cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8(+) T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with beta cell antigens leads to deletion of autoreactive CD8(+) T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.