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 naive 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-gamma 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.Peer reviewe

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-1(hi)) peripheral T helper (Tph) cells, in human type 1 diabetes. Methods The phenotype of blood CXCR5(-)PD-1(hi) CD4(+) T cells was analysed by multicolour flow cytometry. The frequencies of circulating CXCR5(-)PD-1(hi) 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-1(hi) Tph cells share several features associated with B cell helper function with circulating CXCR5(+)PD-1(hi) 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-1(hi) 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.</p

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 naive 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-gamma 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

Circulating CXCR5(-)PD-1(hi) 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-1(hi)) peripheral T helper (Tph) cells, in human type 1 diabetes. Methods The phenotype of blood CXCR5(-)PD-1(hi) CD4(+) T cells was analysed by multicolour flow cytometry. The frequencies of circulating CXCR5(-)PD-1(hi) 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-1(hi) Tph cells share several features associated with B cell helper function with circulating CXCR5(+)PD-1(hi) 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-1(hi) 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.Peer reviewe

Circulating CXCR5(-)PD-1(hi) 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-1(hi)) peripheral T helper (Tph) cells, in human type 1 diabetes. Methods The phenotype of blood CXCR5(-)PD-1(hi) CD4(+) T cells was analysed by multicolour flow cytometry. The frequencies of circulating CXCR5(-)PD-1(hi) 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-1(hi) Tph cells share several features associated with B cell helper function with circulating CXCR5(+)PD-1(hi) 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-1(hi) 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.Peer reviewe

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.</p

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

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

The T-cell receptor (TCR) repertoire is generated in a semistochastic process of gene recombination and pairing of TCR? to TCR? 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 TCR? chains associated with the recognition of self-antigens were generated in significantly higher numbers than TCR? 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 TCR? chains in multiple individuals suggests that the human thymus has a predilection to generate self-reactive TCR? chains independently of the HLA-type and that the individual risk of autoimmunity may be modulated by the TCR? repertoire associated with these chains.Peer reviewe

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

Abstract 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