Integrated Analysis of the Pancreas and Islets Reveals Unexpected Findings in Human Male With Type 1 Diabetes.

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor's diabetes. Examination of the donor's pancreatic tissue found substantial but reduced ß-cell mass with some islets devoid of ß cells (29.3% of 311 islets) while other islets had many ß cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor's clinical insulin deficiency was most likely the result autoimmune-mediated ß-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet ß-Cells From Donors With Type 1 Diabetes.

Type 1 diabetes studies consistently generate data showing islet ß-cell dysfunction and T cell-mediated anti-ß-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the ß-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single ß-cells. Consistent with immunohistological studies, ß-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These ß-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- ß-cells, specifically found in islets with lymphocytic infiltrates. ß-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ ß-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic ß-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. ß-Cell expression of Class II molecules suggests that ß-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.

Cystic fibrosis-related diabetes is caused by islet loss and inflammation.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of ß cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine ß cells did not affect ß cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of ß cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by ß cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

Deciphering the Pathogenesis of Human Type 1 Diabetes (T1D) by Interrogating T Cells from the "Scene of the Crime".

PURPOSE OF REVIEW: Autoimmune-mediated destruction of insulin-producing ß-cells within the pancreas results in type 1 diabetes (T1D), which is not yet preventable or curable. Previously, our understanding of the ß-cell specific T cell repertoire was based on studies of autoreactive T cell responses in the peripheral blood of patients at risk for, or with, T1D; more recently, investigations have included immunohistochemical analysis of some T cell specificities in the pancreas from organ donors with T1D. Now, we are able to examine live, islet-infiltrating T cells from donors with T1D. RECENT FINDINGS: Analysis of the T cell repertoire isolated directly from the pancreatic islets of donors with T1D revealed pro-inflammatory T cells with targets of known autoantigens, including proinsulin and glutamic acid decarboxylase, as well as modified autoantigens. We have assayed the islet-infiltrating T cell repertoire for autoreactivity and function directly from the inflamed islets of T1D organ donors. Design of durable treatments for prevention of or therapy for T1D requires understanding this repertoire.

Narrowing in on the anti-ß cell-specific T cells: looking 'where the action is'.

PURPOSE OF REVIEW: By necessity, the vast majority of information we have on autoreactive T cells in human type 1 diabetes (T1D) has come from the study of peripheral blood of donors with T1D. It is not clear how representative the peripheral autoreactive T-cell repertoire is of the autoreactive T cells infiltrating the islets in T1D. We will summarize and discuss what is known of the immunohistopathology of insulitis, the T-cell receptor repertoire expressed by islet-infiltrating T cells, and the autoreactivity and function of islet-infiltrating T cells in T1D. RECENT FINDINGS: Recovery and analysis of live, islet-infiltrating T cells from the islets of cadaveric donors with T1D revealed a broad repertoire and proinflammatory phenotype of CD4 T-cell autoreactivity to peptide targets from islet proteins, including proinsulin, as well as CD4 T-cell reactivity to a number of post-translationally modified peptides, including peptides with citrullinations and hybrid insulin peptide fusions. Islet-infiltrating CD8 T cells were also derived and required further isolation and characterization. SUMMARY: The recovery of live, islet-infiltrating T cells from donors with T1D, reactive with a broad range of known targets and post-translationally modified peptides, allows for the specific functional analysis of islet-infiltrating T cells for the development of antigen-specific immunotherapies.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes.

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 cells, 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 insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. 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.

Tolerogenic nanoparticles inhibit T cell-mediated autoimmunity through SOCS2.

Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing ß 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 ß 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.

Cross-reactive human B cell and T cell epitopes between influenza A and B viruses.

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.

A human CD4+ T cell epitope in the influenza hemagglutinin is cross-reactive to influenza A virus subtypes and to influenza B virus.

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4(+) T-cell responses to internal proteins, hemagglutinins, and neuraminidases.

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 identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed 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. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.

In vitro evidence that commercial influenza vaccines are not similar in their ability to activate human T cell responses.

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 IFNgamma producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.

Influenza A virus matrix protein 1-specific human CD8+ T-cell response induced in trivalent inactivated vaccine recipients.

Among 17 HLA-A2-positive healthy adults, CD8+ T-cell responses against an HLA-A2-restricted matrix protein 1 (M1) epitope increased after immunization with trivalent inactivated influenza vaccine (TIV) in two individuals. The presence of M1 in TIV was confirmed by Western blotting. T-cell cytotoxicity assays showed that TIV is processed and the epitope is presented by antigen-presenting cells to an M1 epitope-specific CD8+ T-cell line for specific lysis. These data show that TIV, which is formulated to contain surface glycoproteins to induce serotype-specific antibody responses, also contains M1, capable of inducing subtype cross-reactive CD8+ T-cell responses in some vaccinees.

Transcription factor modularity in a gene-centered C. elegans core neuronal protein-DNA interaction network.

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.