Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

IL-6-targeted therapies to block the cytokine or its receptor drive distinct alterations in T cell function.

Therapeutics that inhibit IL-6 at different points in its signaling pathway are in clinical use, yet whether the immunological effects of these interventions differ based on their molecular target is unknown. We performed short-term interventions in individuals with type 1 diabetes using anti-IL-6 (siltuximab) or anti-IL-6 receptor (IL-6R; tocilizumab) therapies and investigated the impact of this in vivo blockade on T cell fate and function. Immune outcomes were influenced by the target of the therapeutic intervention (IL-6 versus IL-6R) and by peak drug concentration. Tocilizumab reduced ICOS expression on T follicular helper cell populations and T cell receptor-driven (TCR-driven) STAT3 phosphorylation. Siltuximab reversed resistance to Treg-mediated suppression and increased TCR-driven phosphorylated STAT3 and production of IL-10, IL-21, and IL-27 by T effectors. Together, these findings indicate that the context of IL-6 blockade in vivo drives distinct T cell-intrinsic changes that may influence therapeutic outcomes.

Dynamic Immune Phenotypes of B and T Helper Cells Mark Distinct Stages of T1D Progression.

Multiple studies of B- and T-cell compartments and their response to stimuli demonstrate alterations in established type 1 diabetes (T1D). Yet it is not known whether these alterations reflect immune mechanisms that initiate islet autoimmunity, promote disease progression, or are secondary to disease. To address these questions, we used samples from the TrialNet Pathway to Prevention study to investigate T-cell responses to interleukin (IL)-2 and regulatory T cell-mediated suppression, the composition of the B-cell compartment, and B-cell responses to B-cell receptor and IL-21 receptor engagement. These studies revealed stage-dependent T- and B-cell functional and immune phenotypes; namely, early features that differentiate autoantibody-positive at-risk first-degree relatives (FDRs) from autoantibody-negative FDRs and persisted through clinical diagnosis; late features that arose at or near T1D diagnosis; and dynamic features that were enhanced early and blunted at later disease stages, indicating evolving responses along the continuum of T1D. We further explored how these specific phenotypes are influenced by therapeutic interventions. Our integrated studies provide unique insights into stable and dynamic stage-specific immune states and define novel immune phenotypes of potential clinical relevance.

A novel and rapid method to quantify Treg mediated suppression of CD4 T cells.

Measuring regulatory T cell suppression provides important insight into T cell dysfunction in autoimmune disease. However, to date, suppression assays are limited by the requirement for freshly isolated cells, and significant cell numbers. Here, we present a novel and rapid in vitro assay using effector T cell surface expression of both CD25 and CD134 as a surrogate marker of regulatory T cell-mediated suppression. This surface marker-based suppression assay works for frozen samples and for samples with limited cell numbers. It is also shorter taking two days to complete compared to the four days required for proliferation-based assays. Furthermore, this assay works with both in vitro expanded and natural Tregs, as well as anti-CD3/anti-CD28 bead-based and APC stimulation conditions. In conclusion, we have developed and validated a new suppression assay for cryopreserved samples with limited cell numbers that may be helpful to investigate T cell regulation in the context of infection or autoimmune diseases.

Citrulline-specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy.

OBJECTIVE: Rheumatoid arthritis (RA) is thought to be a T cell-mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell-directed therapies, and the presence of CD4+ T cells in rheumatoid joints. The presence of anti-citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA-DR4 alleles implicate citrulline-specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA. METHODS: We developed a panel of HLA-DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01-transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of citrulline-specific (Cit-specific) T cells in patients with RA and healthy subjects with DRB1*04:01 haplotypes, using a magnetic enrichment procedure. RESULTS: Cit-specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of Cit-specific T cells, and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA patients, the frequency of Cit-specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic agents, irrespective of disease duration. CONCLUSION: These findings link the presence of ACPAs in RA with Th1 cells specific for citrullinated epitopes and provide tools for disease-specific immunomonitoring of autoreactive T cells.

Multiple autoimmune-associated variants confer decreased IL-2R signaling in CD4+ CD25(hi) T cells of type 1 diabetic and multiple sclerosis patients.

IL-2 receptor (IL-2R) signaling is essential for optimal stability and function of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Treg); a cell type that plays an integral role in maintaining tolerance. Thus, we hypothesized that decreased response to IL-2 may be a common phenotype of subjects who have autoimmune diseases associated with variants in the IL2RA locus, including T1D and MS, particularly in cells expressing the high affinity IL-2R alpha chain (IL-2RA or CD25). To examine this question we used phosphorylation of STAT5 (pSTAT5) as a downstream measure of IL-2R signaling, and found a decreased response to IL-2 in CD4(+)CD25(hi) T cells of T1D and MS, but not SLE patients. Since the IL2RArs2104286 haplotype is associated with T1D and MS, we measured pSTAT5 in controls carrying the rs2104286 risk haplotype to test whether this variant contributed to reduced IL-2 responsiveness. Consistent with this, we found decreased pSTAT5 in subjects carrying the rs2104286 risk haplotype. Reduced IL-2R signaling did not result from lower CD25 expression on CD25(hi) cells; instead we detected increased CD25 expression on naive Treg from controls carrying the rs2104286 risk haplotype, and subjects with T1D and MS. However the rs2104286 risk haplotype correlated with increased soluble IL-2RA levels, suggesting that shedding of the IL-2R may account in part for the reduced IL-2R signaling associated with the rs2104286 risk haplotype. In addition to risk variants in IL2RA, we found that the T1D-associated risk variant of PTPN2rs1893217 independently contributed to diminished IL-2R signaling. However, even when holding genotype constant at IL2RA and PTPN2, we still observed a significant signaling defect in T1D and MS patients. Together, these data suggest that multiple mechanisms converge in disease leading to decreased response to IL-2, a phenotype that may eventually lead to loss of tolerance and autoimmunity.

Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells.

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.

Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in CD4(+)CD25(+) regulatory T-cells of type 1 diabetic subjects.

OBJECTIVE: In humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2-dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects. RESEARCH DESIGN AND METHODS: Persistence of Tregs was assessed by culturing sorted CD4(+)CD25(hi) natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4(+)CD25(-) T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis. RESULTS: Maintenance of FOXP3 expression in CD4(+)CD25(+) Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4(+) T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling. CONCLUSIONS: Aberrant IL-2R signaling in CD4(+) T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes.