Regulatory T Cell Development.

Foxp3-expressing CD4+ regulatory T (Treg) cells play key roles in the prevention of autoimmunity and the maintenance of immune homeostasis and represent a major barrier to the induction of robust antitumor immune responses. Thus, a clear understanding of the mechanisms coordinating Treg cell differentiation is crucial for understanding numerous facets of health and disease and for developing approaches to modulate Treg cells for clinical benefit. Here, we discuss current knowledge of the signals that coordinate Treg cell development, the antigen-presenting cell types that direct Treg cell selection, and the nature of endogenous Treg cell ligands, focusing on evidence from studies in mice. We also highlight recent advances in this area and identify key unanswered questions.

A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells.

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Eomes identifies thymic precursors of self-specific memory-phenotype CD8+ T cells.

Unprimed mice harbor a substantial population of 'memory-phenotype' CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.

Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.

Aire Enforces Immune Tolerance by Directing Autoreactive T Cells into the Regulatory T Cell Lineage.

The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3(+) regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire(-/-) mice revealed an unexpected third possibility. We found that the predominant conventional T cell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3(+) Treg cells in Aire(+/+) mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive T cell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional T cells.

Dendritic Cells Coordinate the Development and Homeostasis of Organ-Specific Regulatory T Cells.

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance.

Close Encounters of the Tertiary Kind.

Elucidating the function of tumor-infiltrating regulatory T (Treg) cells has been difficult. In this issue of Immunity, Joshi et al. (2015) demonstrate that Treg cells associated with murine lung cancers are found within tertiary lymphoid structures and actively restrain effector T cells at the tumor site.

A robust and interpretable gene signature for predicting the lymph node status of primary T1/T2 oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) affects more than 30 000 individuals in the United States annually, with smoking and alcohol consumption being the main risk factors. Management of early-stage tumors usually includes surgical resection followed by postoperative radiotherapy in certain cases. The cervical lymph nodes (LNs) are the most common site for local metastasis, and elective neck dissection is usually performed if the primary tumor thickness is greater than 3.5 mm. However, postoperative histological examination often reveals that many patients with early-stage disease are negative for neck nodal metastasis, posing a pressing need for improved risk stratification to either avoid overtreatment or prevent the disease progression. To this end, we aimed to identify a primary tumor gene signature that can accurately predict cervical LN metastasis in patients with early-stage OSCC. Using gene expression profiles from 189 samples, we trained K-top scoring pairs models and identified six gene pairs that can distinguish primary tumors with nodal metastasis from those without metastasis. The signature was further validated on an independent cohort of 35 patients using real-time polymerase chain reaction (PCR) in which it achieved an area under the receiver operating characteristic (ROC) curve and accuracy of 90% and 91%, respectively. These results indicate that such signature holds promise as a quick and cost effective method for detecting patients at high risk of developing cervical LN metastasis, and may be potentially used to guide the neck treatment regimen in early-stage OSCC.

Application of liquid biopsy as multi-functional biomarkers in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a molecularly heterogeneous disease, with a 5-year survival rate that still hovers at ~60% despite recent advancements. The advanced stage upon diagnosis, limited success with effective targeted therapy and lack of reliable biomarkers are among the key factors underlying the marginally improved survival rates over the decades. Prevention, early detection and biomarker-driven treatment adaptation are crucial for timely interventions and improved clinical outcomes. Liquid biopsy, analysis of tumour-specific biomarkers circulating in bodily fluids, is a rapidly evolving field that may play a striking role in optimising patient care. In recent years, significant progress has been made towards advancing liquid biopsies for non-invasive early cancer detection, prognosis, treatment adaptation, monitoring of residual disease and surveillance of recurrence. While these emerging technologies have immense potential to improve patient survival, numerous methodological and biological limitations must be overcome before their implementation into clinical practice. This review outlines the current state of knowledge on various types of liquid biopsies in HNSCC, and their potential applications for diagnosis, prognosis, grading treatment response and post-treatment surveillance. It also discusses challenges associated with the clinical applicability of liquid biopsies and prospects of the optimised approaches in the management of HNSCC.

Negligible Role for Deletion Mediated by cDC1 in CD8+ T Cell Tolerance.

Deletion of CD8+ T cells by dendritic cells (DCs) is recognized as a critical mechanism of immune tolerance to self-antigens. Although DC-mediated peripheral deletion of autoreactive CD8+ T cells has been demonstrated using T cells reactive to model Ags, its role in shaping the naturally occurring polyclonal CD8+ T cell repertoire has not been defined. Using Batf3-/- mice lacking cross-presenting CD8α+ and CD103+ DCs (also known as type 1 conventional [cDC1]), we demonstrate that peripheral deletion of CD8+ T cells reactive to a model tissue Ag is dependent on cDC1. However, endogenous CD8+ T cells from the periphery of Batf3-/- mice do not exhibit heightened self-reactivity, and deep TCR sequencing of CD8+ T cells from Batf3-/- and Batf3+/+ mice reveals that cDC1 have a minimal impact on shaping the peripheral CD8+ T cell repertoire. Thus, although evident in reductionist systems, deletion of polyclonal self-specific CD8+ T cells by cDC1 plays a negligible role in enforcing tolerance to natural self-ligands.

T cell surveillance of oncogene-induced prostate cancer is impeded by T cell-derived TGF-ß1 cytokine.

Tolerance induction in T cells takes place in most tumors and is thought to account for tumor evasion from immune eradication. Production of the cytokine TGF-ß is implicated in immunosuppression, but the cellular mechanism by which TGF-ß induces T cell dysfunction remains unclear. With a transgenic model of prostate cancer, we showed that tumor development was not suppressed by the adaptive immune system, which was associated with heightened TGF-ß signaling in T cells from the tumor-draining lymph nodes. Blockade of TGF-ß signaling in T cells enhanced tumor antigen-specific T cell responses and inhibited tumor development. Surprisingly, T cell- but not Treg cell-specific ablation of TGF-ß1 was sufficient to augment T cell cytotoxic activity and blocked tumor growth and metastases. These findings reveal that T cell production of TGF-ß1 is an essential requirement for tumors to evade immunosurveillance independent of TGF-ß produced by tumors.

Unlocking the Complexities of Tumor-Associated Regulatory T Cells.

Regulatory T (Treg) cells are found at elevated densities in many human cancers and are thought to be a major barrier to the generation of robust antitumor T cell responses. In this review, we discuss recent advances in the understanding of tumor-associated Treg cell diversity and function. Emerging evidence indicates that the transcriptional program of Treg cells infiltrating human cancers may represent a composite program blending a tissue-associated expression signature with an additional tumor-specific signature common to Treg cells from multiple cancer types. Studies in mouse models have defined unique molecular pathways required for Treg cell function in the tumor context that can be manipulated to selectively dampen intratumoral Treg cell activity. Finally, an expanding body of work has revealed diverse functions for Treg cells in nonlymphoid tissues that are unrelated to immune suppression, suggesting a need to explore functions of intratumoral Treg cells beyond the regulation of antitumor immunity.

Review of methods for measuring ß-cell function: Design considerations from the Restoring Insulin Secretion (RISE) Consortium.

The Restoring Insulin Secretion (RISE) study was initiated to evaluate interventions to slow or reverse the progression of ß-cell failure in type 2 diabetes (T2D). To design the RISE study, we undertook an evaluation of methods for measurement of ß-cell function and changes in ß-cell function in response to interventions. In the present paper, we review approaches for measurement of ß-cell function, focusing on methodologic and feasibility considerations. Methodologic considerations included: (1) the utility of each technique for evaluating key aspects of ß-cell function (first- and second-phase insulin secretion, maximum insulin secretion, glucose sensitivity, incretin effects) and (2) tactics for incorporating a measurement of insulin sensitivity in order to adjust insulin secretion measures for insulin sensitivity appropriately. Of particular concern were the capacity to measure ß-cell function accurately in those with poor function, as is seen in established T2D, and the capacity of each method for demonstrating treatment-induced changes in ß-cell function. Feasibility considerations included: staff burden, including time and required methodological expertise; participant burden, including time and number of study visits; and ease of standardizing methods across a multicentre consortium. After this evaluation, we selected a 2-day measurement procedure, combining a 3-hour 75-g oral glucose tolerance test and a 2-stage hyperglycaemic clamp procedure, augmented with arginine.

Shaping the repertoire of tumor-infiltrating effector and regulatory T cells.

Many tumors express antigens that can be specifically or selectively recognized by T lymphocytes, suggesting that T-cell-mediated immunity may be harnessed for the immunotherapy of cancer. However, since tumors originate from normal cells and evolve within the context of self-tissues, the immune mechanisms that prevent the autoimmune attack of normal tissues function in parallel to restrict anti-tumor immunity. In particular, the purging of autoreactive T cells and the development of immune-suppressive regulatory T cells (Tregs) are thought to be major barriers impeding anti-tumor immune responses. Here, we discuss current understanding regarding the antigens recognized by tumor-infiltrating T-cell populations, the mechanisms that shape the repertoire of these cells, and the role of the transcription factor autoimmune regulator (Aire) in these processes. Further elucidation of these principles is likely to be critical for optimizing emerging cancer immunotherapies, and for the rational design of novel therapies exhibiting robust anti-tumor activity with limited toxicity.

Tumor antigenicity revealed.

The extent to which mutated peptide antigens encoded by somatic mutations contribute to tumor antigenicity in human cancer patients has been unclear. Two recent studies using tumor exome sequencing to predict candidate mutated antigens provide insight into this question.

Detecting prediabetes among Hispanics/Latinos from diverse heritage groups: Does the test matter? Findings from the Hispanic Community Health Study/Study of Latinos.

The objectives of this analysis were to compare the ability of fasting plasma glucose (FPG), post oral load plasma glucose (2hPG), and hemoglobin A1c (HbA1c) to identify U.S. Hispanic/Latino individuals with prediabetes, and to assess its cardiovascular risk factor correlates. This is a cross-sectional analysis of baseline data from 15,507 adults without self-reported diabetes mellitus from six Hispanic/Latino heritage groups, enrolled in the Hispanic Community Health Study/Study of Latinos, which takes place in four U.S. communities. The prevalence of prediabetes was determined according to individual or combinations of ADA-defined cut points: FPG=5.6-7.0mmol/L, 2hPG=7.8-11.1mmol/L, and HbA1c=5.7%-6.4% (39-46mmol/mol). The sensitivity of these criteria to detect prediabetes was estimated. The prevalence ratios (PRs) for selected cardiovascular risk factors were compared among alternative categories of prediabetes versus normoglycemia [FPG<5.6mmol/L and 2hPG<7.8mmol/L and HbA1c<5.7% (39mmol/mol)]. Approximately 36% of individuals met any of the ADA prediabetes criteria. Using 2hPG as the gold standard, the sensitivity of FPG was 40.1%, HbA1c was 45.6%, and that of HbA1c+FPG was 62.2%. The number of significant PRs for cardiovascular risk factors was higher among individuals with isolated 2hPG=7.8-11.1mmol/L, FPG=5.6-7.0mmol/L+HbA1c=5.7%-6.4%, or those who met the three prediabetes criteria. Assessing FPG, HbA1c, and cardiovascular risk factors in Hispanics/Latinos at risk might enhance the early prevention of diabetes mellitus and cardiovascular complications in this young and growing population, independent of their heritage group.

HEMOGLOBIN A1C, BLOOD PRESSURE, AND LDL-CHOLESTEROL CONTROL AMONG HISPANIC/LATINO ADULTS WITH DIABETES: RESULTS FROM THE HISPANIC COMMUNITY HEALTH STUDY/STUDY OF LATINOS (HCHS/SOL).

OBJECTIVE: To determine the prevalence of Hispanic/Latino adults with diabetes who meet target hemoglobin A1c, blood pressure (BP), and low-density-lipoprotein cholesterol (LDL-C) recommendations, and angiotensin-converting enzyme (ACE) inhibitors/angiotensin receptor blocker (ARB) and statin medication use by heritage and sociodemographic and diabetes-related characteristics. METHODS: Data were cross-sectional, collected between 2008 and 2011, and included adults age 18 to 74 years who reported a physician diagnosis of diabetes in the Hispanic Community Health Study/Study of Latinos (N = 2,148). Chi-square tests compared the prevalence of hemoglobin A1c, BP, and LDL-C targets and ACE/ARB and statin use across participant characteristics. Predictive margins regression was used to determine the prevalence adjusted for sociodemographic characteristics. RESULTS: The overall prevalence of A1c <7.0% (53 mmol/mol), BP <130/80 mm Hg, and LDL-C <100 mg/dL was 43.0, 48.7, and 36.6%, respectively, with 8.4% meeting all three targets. Younger adults aged 18 to 39 years with diabetes were less likely to have A1c <7.0% (53 mmol/mol) or LDL-C <100 mg/dL compared to those aged 65 to 74 years; younger adults were more likely to have BP <130/80 mm Hg (P<.05 for all). Individuals of Mexican heritage were significantly less likely to have A1c <7.0% (53 mmol/mol) compared to those with Cuban heritage, but they were more likely to have BP <130/80 mm Hg compared to those with Dominican, Cuban, or Puerto Rican heritage (P<.05 for all); there was no difference in LDL-C by heritage. Overall, 38.2% of adults with diabetes were taking a statin, and 50.5% were taking ACE/ARB medications. CONCLUSION: Hemoglobin A1c, BP, and LDL-C control are suboptimal among Hispanic/Latinos with diabetes living in the U.S. With 8.4% meeting all three recommendations, substantial opportunity exists to improve diabetes control in this population. ABBREVIATIONS: A1c = hemoglobin A1c; ABC = hemoglobin A1c, blood pressure, low-density-lipoprotein cholesterol; ACE = angiotensin-converting enzyme; ADA = American Diabetes Association; ARB = angiotensin receptor blocker; BMI = body mass index; BP = blood pressure; CHD = coronary heart disease; CVD = cardiovascular disease; HCHS/SOL = Hispanic Community Health Study/Study of Latinos; LDL-C = low-density-lipoprotein cholesterol; NHANES = National Health and Nutrition Examination Survey; PAD = peripheral artery disease.

Basic principles of tumor-associated regulatory T cell biology.

Due to the critical role of forkhead box (Fox)p3(+) regulatory T cells (Tregs) in the regulation of immunity and the enrichment of Tregs within many human tumors, several emerging therapeutic strategies for cancer involve the depletion or modulation of Tregs, with the aim of eliciting enhanced antitumor immune responses. Here, we review recent advances in understanding of the fundamental biology of Tregs, and discuss the implications of these findings for current models of tumor-associated Treg biology. In particular, we discuss the context-dependent functional diversity of Tregs, the developmental origins of these cells, and the nature of the antigens that they recognize within the tumor environment. In addition, we highlight critical areas of focus for future research.

IDENTIFYING PROBABLE DIABETES MELLITUS AMONG HISPANICS/LATINOS FROM FOUR U.S. CITIES: FINDINGS FROM THE HISPANIC COMMUNITY HEALTH STUDY/STUDY OF LATINOS.

OBJECTIVE: The aim of this study was to compare the ability of American Diabetes Association (ADA) diagnostic criteria to identify U.S. Hispanics/Latinos from diverse heritage groups with probable diabetes mellitus and assess cardiovascular risk factor correlates of those criteria. METHODS: Cross-sectional analysis of data from 15,507 adults from 6 Hispanic/Latino heritage groups, enrolled in the Hispanic Community Health Study/Study of Latinos. The prevalence of probable diabetes mellitus was estimated using individual or combinations of ADA-defined cut points. The sensitivity and specificity of these criteria at identifying diabetes mellitus from ADA-defined prediabetes and normoglycemia were evaluated. Prevalence ratios of hypertension, abnormal lipids, and elevated urinary albumin-creatinine ratio for unrecognized diabetes mellitus-versus prediabetes and normoglycemia-were calculated. RESULTS: Among Hispanics/Latinos (mean age, 43 years) with diabetes mellitus, 39.4% met laboratory test criteria for probable diabetes, and the prevalence varied by heritage group. Using the oral glucose tolerance test as the gold standard, the sensitivity of fasting plasma glucose (FPG) and hemoglobin A1c-alone or in combination-was low (18, 23, and 33%, respectively) at identifying probable diabetes mellitus. Individuals who met any criterion for probable diabetes mellitus had significantly higher (P<.05) prevalence of most cardiovascular risk factors than those with normoglycemia or prediabetes, and this association was not modified by Hispanic/Latino heritage group. CONCLUSION: FPG and hemoglobin A1c are not sensitive (but are highly specific) at detecting probable diabetes mellitus among Hispanics/Latinos, independent of heritage group. Assessing cardiovascular risk factors at diagnosis might prompt multitarget interventions and reduce health complications in this young population. ABBREVIATIONS: 2hPG = 2-hour post-glucose load plasma glucose ADA = American Diabetes Association BMI = body mass index CV = cardiovascular FPG = fasting plasma glucose HbA1c = hemoglobin A1c HCHS/SOL = Hispanic Community Health Study/Study of Latinos HDL-C = high-density-lipoprotein cholesterol NGT = normal glucose tolerance NHANES = National Health and Nutrition Examination Survey OGTT = oral glucose tolerance test TG = triglyceride UACR = urine albumin-creatinine ratio.