Generation of self-reactive, shared T-cell receptor α chains in the human thymus.

The T-cell receptor (TCR) repertoire is generated in a semistochastic process of gene recombination and pairing of TCRα to TCRß chains with the estimated total TCR diversity of >108. Despite this high diversity, similar or identical TCR chains are found to recur in immune responses. Here, we analyzed the thymic generation of TCR sequences previously associated with recognition of self- and nonself-antigens, represented by sequences associated with autoimmune diabetes and HIV, respectively. Unexpectedly, in the CD4+ compartment TCRα chains associated with the recognition of self-antigens were generated in significantly higher numbers than TCRα chains associated with the recognition of nonself-antigens. The analysis of the circulating repertoire further showed that these chains are not lost in negative selection nor predominantly converted to the regulatory T-cell lineage. The high abundance of self-reactive TCRα chains in multiple individuals suggests that the human thymus has a predilection to generate self-reactive TCRα chains independently of the HLA-type and that the individual risk of autoimmunity may be modulated by the TCRß repertoire associated with these chains.

Effects of galloflavin and ellagic acid on sirtuin 6 and its anti-tumorigenic activities.

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates distinct cellular functions; genome stability, DNA repair, and inflammation related diseases. Recently, we demonstrated that anthocyanidins in berries induce the catalytic activity of SIRT6. In this study, we explored the effects of Galloflavin and Ellagic acid, the most common polyphenols in berries, on SIRT6. SIRT6 deacetylation was investigated using HPLC and immunoblotting assays. The expression levels of SIRT6, glycolytic proteins and cellular metabolism were studied on human colon adenocarcinoma cells (Caco2). Molecular docking studies were carried out to study possible interactions of the compounds with sirtuins. Ellagic acid increased the deacetylase activity of SIRT6 by up to 50-fold; it showed moderate inhibition of SIRT1-3. Galloflavin and Ellagic acid showed anti-proliferative effects on Caco2. The compounds also upregulated SIRT6 expression whereas key proteins in glycolysis were downregulated. Galloflavin decreased glucose transporter 1 (GLUT1) expression, and Ellagic acid affected the expression of protein dehydrogenase kinase 1 (PDK1). Interestingly, both compounds caused reduction in glucose uptake and lactate production. Both Galloflavin and Ellagic acid were able to form hydrogen bonds with Asp188 and Gly6 in SIRT6. In this study, we showed that Galloflavin and Ellagic acid increased SIRT6 activity and decreased the expression of SIRT6 associated proteins involved in cancer development. Taken together, Galloflavin and Ellagic acid targeting SIRT6 activity may provide a new insight in the development of anti-cancer therapy.

Characterization of Proinsulin T Cell Epitopes Restricted by Type 1 Diabetes-Associated HLA Class II Molecules.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which the insulin-producing ß cells within the pancreas are destroyed. Identification of target Ags and epitopes of the ß cell-reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific immunotherapies for the disease. Several studies suggest that proinsulin is an early and integral target autoantigen in T1D. However, proinsulin epitopes recognized by human CD4+ T cells have not been comprehensively characterized. Using a dye dilution-based T cell cloning method, we generated and characterized 24 unique proinsulin-specific CD4+ T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted epitopes within the C-peptide (C25-35) or A-chain (A1-15) of proinsulin. However, we also characterized DR3-restricted epitopes within both the B-chain (B16-27 and B22-C3) and C-peptide (C25-35). Moreover, we identified DQ2-restricted epitopes within the B-chain and several DQ2- or DQ8-restricted epitopes within the C-terminal region of C-peptide that partially overlap with previously reported DQ-restricted epitopes. Two of the DQ2-restricted epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the proinsulin-specific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported epitopes but also identified novel (to our knowledge) epitopes within proinsulin, which are presented by HLA class II molecules associated with T1D risk.

Type 1 diabetes linked PTPN22 gene polymorphism is associated with the frequency of circulating regulatory T cells.

Dysfunction of FOXP3-positive regulatory T cells (Tregs) likely plays a major role in the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). Whether genetic polymorphisms associated with the risk of autoimmune diseases affect Treg frequency or function is currently unclear. Here, we analysed the effect of T1D-associated major HLA class II haplotypes and seven single nucleotide polymorphisms in six non-HLA genes [INS (rs689), PTPN22 (rs2476601), IL2RA (rs12722495 and rs2104286), PTPN2 (rs45450798), CTLA4 (rs3087243), and ERBB3 (rs2292239)] on peripheral blood Treg frequencies. These were determined by flow cytometry in 65 subjects who had progressed to T1D, 86 islet autoantibody-positive at-risk subjects, and 215 islet autoantibody-negative healthy controls. The PTPN22 rs2476601 risk allele A was associated with an increase in total (p = 6 × 10-6 ) and naïve (p = 4 × 10-5 ) CD4+CD25+CD127lowFOXP3+ Treg frequencies. These findings were validated in a separate cohort comprising ten trios of healthy islet autoantibody-negative children carrying each of the three PTPN22 rs2476601 genotypes AA, AG, and GG (p = 0.005 for total and p = 0.03 for naïve Tregs, respectively). In conclusion, our analysis implicates the autoimmune PTPN22 rs2476601 risk allele A in controlling the frequency of Tregs in human peripheral blood.

Circulating CXCR5-PD-1hi peripheral T helper cells are associated with progression to type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is preceded by a period of asymptomatic autoimmunity characterised by positivity for islet autoantibodies. Therefore, T helper cell responses that induce B cell activation are likely to play a critical role in the disease process. Here, we aimed to evaluate the role of a recently described subset, C-X-C motif chemokine receptor type 5-negative, programmed cell death protein 1-positive (CXCR5-PD-1hi) peripheral T helper (Tph) cells, in human type 1 diabetes. METHODS: The phenotype of blood CXCR5-PD-1hi CD4+ T cells was analysed by multicolour flow cytometry. The frequencies of circulating CXCR5-PD-1hi T cells were analysed in a cohort of 44 children with newly diagnosed type 1 diabetes, 40 autoantibody-positive (AAb+) at-risk children and 84 autoantibody-negative healthy control children, and the findings were replicated in a separate cohort of 15 children with newly diagnosed type 1 diabetes and 15 healthy control children. RESULTS: Circulating CXCR5-PD-1hi Tph cells share several features associated with B cell helper function with circulating CXCR5+PD-1hi follicular T helper (Tfh) cells. Moreover, the frequency of circulating Tph cells was increased in children with newly diagnosed type 1 diabetes, especially in those who are positive for multiple autoantibodies. Importantly, circulating Tph cells were also increased in autoantibody-positive at-risk children who later progressed to type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our results demonstrate that circulating CXCR5-PD-1hi Tph cells are associated with progression to clinical type 1 diabetes. Consequently, Tph cells could have potential both as a biomarker of disease progression and as a target for immunotherapy in type 1 diabetes.

FOXP3+ Regulatory T Cell Compartment Is Altered in Children With Newly Diagnosed Type 1 Diabetes but Not in Autoantibody-Positive at-Risk Children.

The dysfunction of FOXP3-positive regulatory T cells (Tregs) plays a key role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D). However, previous studies analyzing the peripheral blood Treg compartment in patients with T1D have yielded partially conflicting results. Moreover, the phenotypic complexity of peripheral blood Tregs during the development of human T1D has not been comprehensively analyzed. Here, we used multi-color flow cytometry to analyze the frequency of distinct Treg subsets in blood samples from a large cohort comprising of 74 children with newly diagnosed T1D, 76 autoantibody-positive children at-risk for T1D and 180 age- and HLA-matched control children. The frequency of CD4+CD25+CD127lowFOXP3+ Tregs was higher in children with T1D compared to control children, and this change was attributable to a higher proportion of naïve Tregs in these subjects. Further longitudinal analyses demonstrated that the increase in Treg frequency correlated with disease onset. The frequencies of the minor subsets of CD25+FOXP3low memory Tregs as well as CD25lowCD127lowFOXP3+ Tregs were also increased in children with T1D. Moreover, the ratio of CCR6-CXCR3+ and CCR6+CXCR3- memory Tregs was altered and the frequency of proliferating Ki67-positive and IFN-γ producing memory Tregs was decreased in children with T1D. The frequency of CXCR5+FOXP3+ circulating follicular T regulatory cells was not altered in children with T1D. Importantly, none of the alterations observed in children with T1D were observed in autoantibody-positive at-risk children. In conclusion, our study reveals multiple alterations in the peripheral blood Treg compartment at the diagnosis of T1D that appear not to be features of early islet autoimmunity.

Effector T Cell Resistance to Suppression and STAT3 Signaling during the Development of Human Type 1 Diabetes.

Dysregulation of regulatory T cell (Treg)-mediated suppression and, in particular, resistance of CD4+ effector T cells (Teffs) to suppression have been implicated in the pathogenesis of human type 1 diabetes (T1D). However, the mechanistic basis behind this resistance and the time frame during which it develops in relation to the onset of clinical T1D remain unclear. In this study, we analyzed the capacity of peripheral blood Teffs isolated both from patients with T1D and from prediabetic at-risk subjects positive for multiple diabetes-associated autoantibodies (AAb+) to be suppressed by Tregs. Because STAT3 activation through IL-6 has previously been implicated in mediating Teff resistance, we also investigated the surface expression of IL-6R as well as IL-6- and TCR-mediated phosphorylation of STAT3 in T cells from our study subjects. Teff resistance to suppression was observed both in patients with newly diagnosed and long-standing T1D but not in AAb+ subjects and was shown to be STAT3 dependent. No alterations in IL-6R expression or IL-6-mediated STAT3 activation were observed in T cells from patients with T1D or AAb+ subjects. However, faster STAT3 activation after TCR stimulation without concomitant increase in IL-6 expression was observed in T cells from patients with T1D. These experiments suggest that Teff resistance in T1D patients is STAT3 dependent but not directly linked with the capacity of Teffs to produce or respond to IL-6. In conclusion, Teff resistance to Treg-mediated suppression is likely a feature of disease progression in human T1D and can potentially be targeted by immune therapies that block STAT3 activation.

Circulating CXCR5+PD-1+ICOS+ Follicular T Helper Cells Are Increased Close to the Diagnosis of Type 1 Diabetes in Children With Multiple Autoantibodies.

Although type 1 diabetes (T1D) is primarily perceived as a T cell-driven autoimmune disease, islet autoantibodies are the best currently available biomarker for autoimmunity and disease risk. These antibodies are produced by autoreactive B cells, the activation of which is largely dependent on the function of CD4+CXCR5+ follicular T helper cells (Tfh). In this study, we have comprehensively characterized the Tfh- as well as B-cell compartments in a large cohort of children with newly diagnosed T1D or at different stages of preclinical T1D. We demonstrate that the frequency of CXCR5+PD-1+ICOS+-activated circulating Tfh cells is increased both in children with newly diagnosed T1D and in autoantibody-positive at-risk children with impaired glucose tolerance. Interestingly, this increase was only evident in children positive for two or more biochemical autoantibodies. No alterations in the circulating B-cell compartment were observed in children with either prediabetes or diabetes. Our results demonstrate that Tfh activation is detectable in the peripheral blood close to the presentation of clinical T1D but only in a subgroup of children identifiable by positivity for multiple autoantibodies. These findings suggest a role for Tfh cells in the pathogenesis of human T1D and carry important implications for targeting Tfh cells and/or B cells therapeutically.

Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals.

Bacteria rely mainly on enzymes, glutathione and other low-molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and trimers of 3-hydroxybutyrate (ME-3HB), produced by bacteria capable of polyhydroxybutyrate biosynthesis, have 3-fold greater hydroxyl radical-scavenging activity than glutathione and 11-fold higher activity than vitamin C or the monomer 3-hydroxybutyric acid. We found that ME-3HB oligomers protect hypersensitive yeast deletion mutants lacking oxidative stress-response genes from hydroxyl radical stress. Our results show that phaC and phaZ, encoding polymerase and depolymerase, respectively, are activated and polyhydroxybutyrate reserves are degraded for production of ME-3HB oligomers in bacteria infecting plant cells and exposed to hydroxyl radical stress. We found that ME-3HB oligomer production is widespread, especially in bacteria adapted to stressful environments. We discuss how ME-3HB oligomers could provide opportunities for numerous applications in human health.

The intracellular Scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus and encodes eukaryote-like proteins.

UNLABELLED: Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. IMPORTANCE: All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside all tissues. M. extorquens DSM13060 occupies an unusual niche inside cells of the dividing shoot tissues of a pine and stimulates seedling growth without producing cytokinin, auxin, or other plant hormones commonly synthesized by plant-associated bacteria. Here, we tracked the bacteria using a fluorescent tag and confocal laser scanning microscopy and found that they localize near the nucleus of the plant cell. This prompted us to sequence the genome and identify proteins that may affect host growth by targeting processes in the host cytoplasm and nucleus. We found many novel genes whose products may modulate plant processes from within the plant cell. Our results open up new avenues to better understand how bacteria assist in plant growth, with broad implications for plant science, forestry, and agriculture.