A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells1–3, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis4–8, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors9 and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D10. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

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Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing β cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3(+) regulatory T (Treg) cells or tolerogenic dendritic cells (DCs) that promote Treg cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the β cell antigen proinsulin (NPITE+Ins) to induce a tolerogenic phenotype in DCs and promote Treg cell generation in vivo. NPITE+Ins administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NPITE+Ins induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3(+) Treg cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders.

Transcription regulatory networks play a pivotal role in the development, function, and pathology of metazoan organisms. Such networks are comprised of protein-DNA interactions between transcription factors (TFs) and their target genes. An important question pertains to how the architecture of such networks relates to network functionality. Here, we show that a Caenorhabditis elegans core neuronal protein-DNA interaction network is organized into two TF modules. These modules contain TFs that bind to a relatively small number of target genes and are more systems specific than the TF hubs that connect the modules. Each module relates to different functional aspects of the network. One module contains TFs involved in reproduction and target genes that are expressed in neurons as well as in other tissues. The second module is enriched for paired homeodomain TFs and connects to target genes that are often exclusively neuronal. We find that paired homeodomain TFs are specifically expressed in C. elegans and mouse neurons, indicating that the neuronal function of paired homeodomains is evolutionarily conserved. Taken together, we show that a core neuronal C. elegans protein-DNA interaction network possesses TF modules that relate to different functional aspects of the complete network.

Type 1 diabetes studies consistently generate data showing islet b-cell dysfunction and T cell-mediated anti-b-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the b-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single b-cells. Consistent with immunohistological studies, b-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These b-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin + CD68 2 b-cells, specifically found in islets with lymphocytic infiltrates. b-Cell surface expression of HLA Class II was detected on a portion of CD45 2 insulin + b-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic b-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. b-Cell expression of Class II molecules suggests that b-cells may interact directly with islet-infiltrating CD4 + T cells and may play an immunopathogenic role. The immune system plays a critical role in human type 1 diabetes pathogenesis. Varying proportions of T-cell subsets (CD8 + and CD4 +) and B cells infiltrate the pancreatic islets (1) and target b-cells by recognizing type 1 diabetes-associated autoantigens (2,3). The immunological mechanisms recruiting these cells to the islets had remained incompletely understood because, until recently, islets from donors with type 1 diabetes were not available for study. Antigen presentation to T cells is mediated by antigen-presenting cells (APCs) via two classes of HLA molecules: HLA Class I, recognized by CD8 +-expressing T cells (Class I is present on nearly all nucleated cells), and

We performed a genome-wide screening for T cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA) (H1, H3 and H5) and neuraminidase (NA) (human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We found a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in IFN-γ ELISPOT assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, had many T cell epitopes. HA and matrix protein 1 had more T cell epitopes than other viral proteins, most of which were recognized by CD4 + T cells. We established several cytotoxic CD4 + T cell lines from these donors. We also analyzed H1 and H3 HA-specific T cell responses using the peripheral blood mononuclear cells of 30 hospital workers. 53% of donors gave a positive response to H3 HA peptides, while 17% gave a positive response to H1 HA peptides. Our genomewide screening is useful in identifying T cell epitopes and complementary to the approach based on the predicted binding peptides to well-studied HLA-A, B and DR alleles.

We evaluated three commercial trivalent inactivated vaccines (TIVs) from the 2007-2008 season in terms of their ability to elicit in vitro T cell responses. T cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of pandemic or epidemic influenza. Human cytotoxic T cell lines demonstrated differences in matrix protein 1 and nucleocapsid protein recognition of autologous target cells. Peripheral blood mononuclear cells stimulated with each of the TIVs showed statistically significant differences between the vaccines in the numbers of IFNγ producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.

Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4+ T cell epitopes conserved between the two genera were identified in humans. A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently.