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.

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.

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.

PD-1 regulates extrathymic regulatory T-cell differentiation.

Treg cells and the programed death-1/programed death ligand-1 (PD-1/PD-L1) pathway are both critical for maintaining peripheral tolerance to self-Ags. A significant subset of Treg cells constitutively expresses PD-1, which prompted an investigation into the role of PD-1/PD-L1 interactions in Treg-cell development, function, and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD-1-deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD-1 deficiency. The suppressive function of PD-1(-/-) Treg cells was similar to their PD-1(+/+) counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD-1(-/-) conventional CD4(+) T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD-1 is dispensable for thymic Treg-cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that Ab blockade of the PD-1/PD-L1 pathway may augment T-cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells.

Effector T cell responses unleashed by regulatory T cell ablation exacerbate oral squamous cell carcinoma.

Immune suppression by CD4+FOXP3+ regulatory T (Treg) cells and tumor infiltration by CD8+ effector T cells represent two major factors impacting response to cancer immunotherapy. Using deconvolution-based transcriptional profiling of human papilloma virus (HPV)-negative oral squamous cell carcinomas (OSCCs) and other solid cancers, we demonstrate that the density of Treg cells does not correlate with that of CD8+ T cells in many tumors, revealing polarized clusters enriched for either CD8+ T cells or CD4+ Treg and conventional T cells. In a mouse model of carcinogen-induced OSCC characterized by CD4+ T cell enrichment, late-stage Treg cell ablation triggers increased densities of both CD4+ and CD8+ effector T cells within oral lesions. Notably, this intervention does not induce tumor regression but instead induces rapid emergence of invasive OSCCs via an effector T cell-dependent process. Thus, induction of a T cell-inflamed phenotype via therapeutic manipulation of Treg cells may trigger unexpected tumor-promoting effects in OSCC.

Altered selection on a single self-ligand promotes susceptibility to organ-specific T cell infiltration.

For the large array of self-peptide/MHC class II (pMHC-II) complexes displayed in the body, it is unclear whether CD4+ T cell tolerance must be imparted for each individual complex or whether pMHC-II-nonspecific bystander mechanisms are sufficient to confer tolerance by acting broadly on T cells reactive to multiple self-pMHC-II ligands. Here, via reconstitution of T cell-deficient mice, we demonstrate that altered T cell selection on a single prostate-specific self-pMHC-II ligand renders recipient mice susceptible to prostate-specific T cell infiltration. Mechanistically, this self-pMHC-II complex is required for directing antigen-specific cells into the Foxp3+ regulatory T cell lineage but does not induce clonal deletion to a measurable extent. Thus, our data demonstrate that polyclonal T reg cells are unable to functionally compensate for a breach in tolerance to a single self-pMHC-II complex in this setting, revealing vulnerabilities in antigen-nonspecific bystander mechanisms of immune tolerance.

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.