Neonatal T Cells: A Reinterpretation.

Neonatal CD4+ and CD8+ T cells have historically been characterized as immature or defective. However, recent studies prompt a reinterpretation of the functions of neonatal T cells. Rather than a population of cells always falling short of expectations set by their adult counterparts, neonatal T cells are gaining recognition as a distinct population of lymphocytes well suited for the rapidly changing environment in early life. In this review, I will highlight new evidence indicating that neonatal T cells are not inert or less potent versions of adult T cells but instead are a broadly reactive layer of T cells poised to quickly develop into regulatory or effector cells, depending on the needs of the host. In this way, neonatal T cells are well adapted to provide fast-acting immune protection against foreign pathogens, while also sustaining tolerance to self-antigens.

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.

Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function.

Dynamic tuning of cellular responsiveness as a result of repeated stimuli improves the ability of cells to distinguish physiologically meaningful signals from each other and from noise. In particular, lymphocyte activation thresholds are subject to tuning, which contributes to maintaining tolerance to self-antigens and persisting foreign antigens, averting autoimmunity and immune pathogenesis, but allowing responses to strong, structured perturbations that are typically associated with acute infection. Such tuning is also implicated in conferring flexibility to positive selection in the thymus, in controlling the magnitude of the immune response, and in generating memory cells. Additional functional properties are dynamically and differentially tuned in parallel via subthreshold contact interactions between developing or mature lymphocytes and self-antigen-presenting cells. These interactions facilitate and regulate lymphocyte viability, maintain their functional integrity, and influence their responses to foreign antigens and accessory signals, qualitatively and quantitatively. Bidirectional tuning of T cells and antigen-presenting cells leads to the definition of homeostatic set points, thus maximizing clonal diversity.

Adoptive immunotherapy for cancer or viruses.

Adoptive immunotherapy, or the infusion of lymphocytes, is a promising approach for the treatment of cancer and certain chronic viral infections. The application of the principles of synthetic biology to enhance T cell function has resulted in substantial increases in clinical efficacy. The primary challenge to the field is to identify tumor-specific targets to avoid off-tumor, on-target toxicity. Given recent advances in efficacy in numerous pilot trials, the next steps in clinical development will require multicenter trials to establish adoptive immunotherapy as a mainstream technology.

Metabolic regulation of T lymphocytes.

T cell activation leads to dramatic shifts in cell metabolism to protect against pathogens and to orchestrate the action of other immune cells. Quiescent T cells require predominantly ATP-generating processes, whereas proliferating effector T cells require high metabolic flux through growth-promoting pathways. Further, functionally distinct T cell subsets require distinct energetic and biosynthetic pathways to support their specific functional needs. Pathways that control immune cell function and metabolism are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell functions. As a result of these findings, cell metabolism is now appreciated as a key regulator of T cell function specification and fate. This review discusses the role of cellular metabolism in T cell development, activation, differentiation, and function to highlight the clinical relevance and opportunities for therapeutic interventions that may be used to disrupt immune pathogenesis.

The Immunology of Multisystem Inflammatory Syndrome in Children with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is typically very mild and often asymptomatic in children. A complication is the rare multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19, presenting 4-6 weeks after infection as high fever, organ dysfunction, and strongly elevated markers of inflammation. The pathogenesis is unclear but has overlapping features with Kawasaki disease suggestive of vasculitis and a likely autoimmune etiology. We apply systems-level analyses of blood immune cells, cytokines, and autoantibodies in healthy children, children with Kawasaki disease enrolled prior to COVID-19, children infected with SARS-CoV-2, and children presenting with MIS-C. We find that the inflammatory response in MIS-C differs from the cytokine storm of severe acute COVID-19, shares several features with Kawasaki disease, but also differs from this condition with respect to T cell subsets, interleukin (IL)-17A, and biomarkers associated with arterial damage. Finally, autoantibody profiling suggests multiple autoantibodies that could be involved in the pathogenesis of MIS-C.

BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection.

During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108-CX3CR1- terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.

Developmental Origin Governs CD8+ T Cell Fate Decisions during Infection.

Heterogeneity is a hallmark feature of the adaptive immune system in vertebrates. Following infection, naive T cells differentiate into various subsets of effector and memory T cells, which help to eliminate pathogens and maintain long-term immunity. The current model suggests there is a single lineage of naive T cells that give rise to different populations of effector and memory T cells depending on the type and amounts of stimulation they encounter during infection. Here, we have discovered that multiple sub-populations of cells exist in the naive CD8+ T cell pool that are distinguished by their developmental origin, unique transcriptional profiles, distinct chromatin landscapes, and different kinetics and phenotypes after microbial challenge. These data demonstrate that the naive CD8+ T cell pool is not as homogeneous as previously thought and offers a new framework for explaining the remarkable heterogeneity in the effector and memory T cell subsets that arise after infection.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing.

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8+ T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

A two-amino-acid substitution in the transcription factor RORγt disrupts its function in TH17 differentiation but not in thymocyte development.

The transcription factor RORγt regulates differentiation of the TH17 subset of helper T cells, thymic T cell development and lymph-node genesis. Although elimination of RORγt prevents TH17 cell-mediated experimental autoimmune encephalomyelitis (EAE), it also disrupts thymocyte development, which could lead to lethal thymic lymphoma. Here we identified a two-amino-acid substitution in RORγt (RORγtM) that 'preferentially' disrupted TH17 differentiation but not thymocyte development. Mice expressing RORγtM were resistant to EAE associated with defective TH17 differentiation but maintained normal thymocyte development and normal lymph-node genesis, except for Peyer's patches. RORγtM showed less ubiquitination at Lys69 that was selectively required for TH17 differentiation but not T cell development. This study will inform the development of treatments that selectively target TH17 cell-mediated autoimmunity but do not affect thymocyte development or induce lymphoma.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

Spatial map of human T cell compartmentalization and maintenance over decades of life.

Mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis.

Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-ß.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-ß1 (TGF-ß) for epidermal residence. Here we found that integrins αvß6 and αvß8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-ß. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvß6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-ß, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-ß by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire. CTLA-4 imposes major boundaries on CD4+ T cell phenotypes, whereas PD-1 subtly limits CD8+ T cell phenotypes. By computationally reconstructing T cell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in T cell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of T cell differentiation and suggest that checkpoint blockade might work in part by altering the limits of T cell phenotypes.

Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.

A naive CD4(+) T cell population specific for a microbial peptide:major histocompatibility complex II ligand (p:MHCII) typically consists of about 100 cells, each with a different T cell receptor (TCR). Following infection, this population produces a consistent ratio of effector cells that activate microbicidal functions of macrophages or help B cells make antibodies. We studied the mechanism that underlies this division of labor by tracking the progeny of single naive T cells. Different naive cells produced distinct ratios of macrophage and B cell helpers but yielded the characteristic ratio when averaged together. The effector cell pattern produced by a given naive cell correlated with the TCR-p:MHCII dwell time or the amount of p:MHCII. Thus, the consistent production of effector cell subsets by a polyclonal population of naive cells results from averaging the diverse behaviors of individual clones, which are instructed in part by the strength of TCR signaling.

Understanding Subset Diversity in T Cell Memory.

Considerable advances have been made in recent years in understanding the generation and function of memory T cells. Memory T cells are typically parsed into discreet subsets based on phenotypic definitions that connote distinct roles in immunity. Here we consider new developments in the field and focus on how emerging differences between memory cells with respect to their trafficking, metabolism, epigenetic regulation, and longevity may fail to fit into small groups of "memory subsets." Rather, the properties of individual memory T cells fall on a continuum within each of these and other parameters. We discuss how this continuum influences the way that the efficacy of vaccination is assessed, as well as the suitability of a memory population for protective immunity.

T-Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation.

Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.

Molecular regulation of effector and memory T cell differentiation.

Immunological memory is a cardinal feature of adaptive immunity and an important goal of vaccination strategies. Here we highlight advances in the understanding of the diverse T lymphocyte subsets that provide acute and long-term protection from infection. These include new insights into the transcription factors, and the upstream 'pioneering' factors that regulate their accessibility to key sites of gene regulation, as well as metabolic regulators that contribute to the differentiation of effector and memory subsets; ontogeny and defining characteristics of tissue-resident memory lymphocytes; and origins of the remarkable heterogeneity exhibited by activated T cells. Collectively, these findings underscore progress in delineating the underlying pathways that control diversification in T cell responses but also reveal gaps in the knowledge, as well as the challenges that arise in the application of this knowledge to rationally elicit desired T cell responses through vaccination and immunotherapy.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.