Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen-specific CD4 T cells.

The endocrine pancreas is one of the most inaccessible organs of the human body. Its autoimmune attack leads to type 1 diabetes (T1D) in a genetically susceptible population and a lifelong need for exogenous insulin replacement. Monitoring disease progression by sampling peripheral blood would provide key insights into T1D immune-mediated mechanisms and potentially change preclinical diagnosis and the evaluation of therapeutic interventions. This effort has been limited to the measurement of circulating anti-islet antibodies, which despite a recognized diagnostic value, remain poorly predictive at the individual level for a fundamentally CD4 T cell-dependent disease. Here, peptide-major histocompatibility complex tetramers were used to profile blood anti-insulin CD4 T cells in mice and humans. While percentages of these were not directly informative, the state of activation of anti-insulin T cells measured by RNA and protein profiling was able to distinguish the absence of autoimmunity versus disease progression. Activated anti-insulin CD4 T cell were detected not only at time of diagnosis but also in patients with established disease and in some at-risk individuals. These results support the concept that antigen-specific CD4 T cells might be used to monitor autoimmunity in real time. This advance can inform our approach to T1D diagnosis and therapeutic interventions in the preclinical phase of anti-islet autoimmunity.

Identification of an anergic BND cell-derived activated B cell population (BND2) in young-onset type 1 diabetes patients.

Recent evidence suggests a role for B cells in the pathogenesis of young-onset type 1 diabetes (T1D), wherein rapid progression occurs. However, little is known regarding the specificity, phenotype, and function of B cells in young-onset T1D. We performed a cross-sectional analysis comparing insulin-reactive to tetanus-reactive B cells in the blood of T1D and controls using mass cytometry. Unsupervised clustering revealed the existence of a highly activated B cell subset we term BND2 that falls within the previously defined anergic BND subset. We found a specific increase in the frequency of insulin-reactive BND2 cells in the blood of young-onset T1D donors, which was further enriched in the pancreatic lymph nodes of T1D donors. The frequency of insulin-binding BND2 cells correlated with anti-insulin autoantibody levels. We demonstrate BND2 cells are pre-plasma cells and can likely act as APCs to T cells. These findings identify an antigen-specific B cell subset that may play a role in the rapid progression of young-onset T1D.

Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD.

Autoimmune thyroid disease (AITD) is caused by aberrant activation of the immune system allowing autoreactive B and T cells to target the thyroid gland leading to disease. Although AITD is more frequently diagnosed in adults, children are also affected but rarely studied. Here, we performed phenotypic and functional characterization of peripheral blood immune cells from pediatric and adult-onset AITD patients and age-matched controls using mass cytometry. Major findings indicate that unlike adult-onset AITD patients, pediatric AITD patients exhibit a decrease in anergic B cells (BND) and DN2 B cells and an increase in immature B cells compared to age-matched controls. These results indicate alterations in peripheral blood immune cells seen in pediatric-onset AITD could lead to rapid progression of disease. Hence, this study demonstrates diversity of AITD by showing differences in immune cell phenotypes and function based on age of onset, and may inform future therapies.

Hybrid Insulin Peptides Are Autoantigens in Type 1 Diabetes.

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester-based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

Biomarker Discovery in Pre-Type 1 Diabetes; Th40 Cells as a Predictive Risk Factor.

CONTEXT: The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components. OBJECTIVE: More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker. METHODS: Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet. RESULTS: In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases. CONCLUSION: Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

Elevated PTEN expression maintains anergy in human B cells and reveals unexpectedly high repertoire autoreactivity.

It has been reported that 2.5%-30% of human peripheral CD27- B cells are autoreactive and anergic based on unresponsiveness to antigen receptor (BCR) stimulation and autoreactivity of cloned and expressed BCR. The molecular mechanisms that maintain this unresponsiveness are unknown. Here, we showed that in humans anergy is maintained by elevated expression of PTEN, a phosphatidylinositol 3,4,5P-3-phosphatase. Upregulation of PTEN was associated with reduced expression of microRNAs that control its expression. Pharmacologic inhibition of PTEN lead to significant restoration of responsiveness. Consistent with a role in conferring risk of autoimmunity, B cells from type 1 diabetics and autoimmune thyroid disease patients expressed reduced PTEN. Unexpectedly, in healthy individuals PTEN expression was elevated in on average 40% of CD27- B cells, with levels gradually decreasing as IgM levels increase. Our findings suggest that a much higher proportion of the peripheral repertoire is autoreactive than previously thought and that B cells upregulate PTEN in a manner that is proportional to the recognition of autoantigens of increasing avidity, thus tuning BCR signaling to prevent development of autoimmunity while providing a reservoir of cells that can be readily activated to respond when needed.

Loss of B-Cell Anergy in Type 1 Diabetes Is Associated With High-Risk HLA and Non-HLA Disease Susceptibility Alleles.

Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to the development of type 1 diabetes. We previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (BND) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the BND compartment of some first-degree relatives (FDRs) as well as all patients with autoantibody-positive prediabetes and new-onset type 1 diabetes, a time when they are found in pancreatic islets. Loss of BND IBCs is associated with a loss of the entire BND B-cell compartment consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B-cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes-associated risk allele genotypes and loss of BNDs in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles (i.e., PTPN2 [rs1893217], INS [rs689], and IKZF3 [rs2872507]), relevant to B- and T-cell development and function are associated with loss of anergy. Hence, the results suggest a role for risk-conferring alleles in perturbation of B-cell anergy during development of type 1 diabetes.

Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop.

AIMS/HYPOTHESIS: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. METHODS: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. RESULTS: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. CONCLUSIONS/INTERPRETATION: Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

Activation of thyroid antigen-reactive B cells in recent onset autoimmune thyroid disease patients.

Autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is the most common autoimmune disorder in the United States, affecting over 20 million people. At the time of diagnosis, both HD and GD are characterized by the accumulation of B and T lymphocytes in the thyroid gland and production of autoantibodies targeting the thyroid, indicating that a breach in tolerance of autoreactive lymphocytes has occurred. However, few studies have sought to understand the underlying pathogenesis of AITD that ultimately leads to production of autoantibodies and loss of thyroid function. In this study, we analyzed the phenotype of thyroid antigen-reactive B cells in the peripheral blood of recent onset and long standing AITD patients. We found that in recent onset patients thyroid antigen-reactive B cells in blood no longer appear anergic, rather they express CD86, a marker of activation. This likely reflects activation of these cells leading to their production of autoantibodies. Hence, this study reports the early loss of anergy in thyroid antigen-reactive B cells, an event that contributes to development of AITD.

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function.

B cells reactive with a specific antigen usually occur at a frequency of <0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.