A VISTA on naïve T cell fate T cell quiescence and tolerance restrain the immune system from becoming overactive and attacking healthy tissue. Negative checkpoint regulators normally limit T cell responses to help safeguard against conditions such as autoimmunity. ElTanbouly et al. report that the checkpoint regulator VISTA (V-type immunoglobulin domain-containing suppressor of T cell activation) restricts early stages of T cell activation by shaping the inherent heterogeneity of the naïve CD4+ T cell compartment to one that is more uniformly quiescent and silent (see the Perspective by Brown and Rudensky). Therapeutic targeting of VISTA using an agonistic antibody in mice curbed the development of graft-versus-host disease and promoted the death of naïve T cells abnormally activated by self-antigen. VISTA thus represents a distinctive immunoregulatory molecule that controls naïve T cell function by maintaining quiescence and peripheral tolerance. Science, this issue p. eaay0524; see also p. 247 The immunological checkpoint regulator VISTA silences nonspecific activity of naive CD4+ T cells, limiting immune responses to self-antigens. INTRODUCTION A central tenet of the clonal selection theory (CST), the current cornerstone of adaptive immunity, states that naïve T cells are selected on the basis of specificity to antigen and that the rest of the repertoire remains agnostic to antigen challenge. However, CST and more recent paradigms have not considered the need for negative regulatory signals to maintain quiescence of the unselected T cells as a pivotal aspect of maintaining clonal selection. Silencing of naïve T cells, or tolerance, also becomes critical upon encounter with self-antigen, whereby self-reactive clones cannot be allowed to develop effector function. These events are known to be interrelated because the loss of quiescence precipitates a loss in tolerance. RATIONALE V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is an inhibitory receptor expressed on naïve T lymphocytes, and genetic deletion of VISTA culminates in T cell–mediated autoimmunity. Unlike other negative checkpoint regulators identified to date, VISTA is expressed on naïve T cells, which led us to investigate its function in maintaining naïve T cell quiescence and tolerance. RESULTS Using a combination of single-cell RNA sequencing and single-cell ATAC sequencing technologies coupled with in vivo analyses, we investigated the cell-state phenotype of naïve CD4+ (CD44lo CD62Lhi) T cells in mice and found multiple T cell subpopulations exist within a surprisingly heterogeneous naïve T cell compartment. The vast majority of cells display a quiescent phenotype. However, there were subsets expressing less quiescent, memory-like features, a cluster with a high interferon-I (IFN-I) response signature, and a population with higher early T cell receptor (TCR) signaling genes. We show that genetic deletion of VISTA results in a major redistribution of the naïve T cell subsets with the notable reduction of the quiescent subset and the enhanced presence of a memory-like activated T cell subset. In the absence of intrinsic VISTA expression, naïve T cells were more epigenetically and transcriptionally primed toward stronger responses to TCR and cytokine stimulation, providing a rationale for the enhancement of CD44hi CD4+ memory-like T cells observed in the absence of VISTA. As a consequence of reduced quiescence within the naïve T cell compartment, these naïve T cells were less susceptible to tolerance induction. Genetic deletion or antibody blockade of VISTA resulted in significant expansion and reduced tolerance of antigen-specific T cells. These effects were abolished under inflammatory conditions where VISTA expression on antigen-specific T cells was reduced. By contrast, agonistic engagement of VISTA and antigen exposure increased tolerance induction by enhancing the death and deletion of antigen-specific T cells. Tolerogenic death induced by anti-VISTA was demonstrated in a model of acute graft-versus-host disease (GVHD), whereby deletion of donor host reactive T cells resulted in a pronounced therapeutic benefit and long-term survival. The gene signature of VISTA−/− T cells overlapped significantly with T cells from patients with the autoimmune diseases systemic lupus erythematosus and rheumatoid arthritis, suggesting that VISTA may play a broad regulatory role in suppressing T cell self-reactivity. CONCLUSION We introduce VISTA as the earliest checkpoint regulator of peripheral T cell tolerance identified to date and describe VISTA as the first of a new class of immunoregulatory molecules whose function is to enforce quiescence in naïve T lymphocytes. In addition, comprehensive epigenetic and transcriptional analyses defined fluorescence-activated sorting-purified CD44lo CD62Lhi naïve CD4+ T cells as heterogeneous. Our current in vivo findings also suggest that regulation of quiescence is inextricably linked to the capacity to maintain T cell self-tolerance. Although a critical player in naïve T cell homeostasis, the restraint enforced by VISTA on naïve T cell responses is lost when antigen stimulation is met under inflammatory conditions. Loss of VISTA reduces the quiescence phenotype of the naïve T cells at the transcriptional and epigenetic levels, causing enhancement in TCR and cytokine pathways. (Left) This loss undermines tolerance induction to self-antigen and imparts a more inflammatory phenotype upon activation. KLF2/6, BTG1/2, FOXP1, and SLFN2 are regulators of quiescence. TCF7, BCL2, INFGR1, and SLAMF6 direct memory T cell fate and activity. IFN-γ and CCL5 are effector cytokines. CD69, CD25, and CD40LG are activation and costimulation receptors. (Right) Integration of VISTA with other well-established negative checkpoint regulators of T cell activation. VISTA is constitutively expressed on naïve T cells and plays a critical role in enforcing quiescence. CTLA-4 is expressed on the cell surface briefly after T cell activation and inhibits T cell activation at the priming stage by limiting costimulation. PD-1 is expressed later during priming and inhibits T cells at the effector stage. Negative checkpoint regulators (NCRs) temper the T cell immune response to self-antigens and limit the development of autoimmunity. Unlike all other NCRs that are expressed on activated T lymphocytes, V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is expressed on naïve T cells. We report an unexpected heterogeneity within the naïve T cell compartment in mice, where loss of VISTA disrupted the major quiescent naïve T cell subset and enhanced self-reactivity. Agonistic VISTA engagement increased T cell tolerance by promoting antigen-induced peripheral T cell deletion. Although a critical player in naïve T cell homeostasis, the ability of VISTA to restrain naïve T cell responses was lost under inflammatory conditions. VISTA is therefore a distinctive NCR of naïve T cells that is critical for steady-state maintenance of quiescence and peripheral tolerance.

中文翻译：

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VISTA 是幼稚 T 细胞静止和外周耐受的检查点调节剂

幼稚 T 细胞命运的 VISTA T 细胞静止和耐受性可抑制免疫系统变得过度活跃并攻击健康组织。阴性检查点调节因子通常会限制 T 细胞反应，以帮助防范自身免疫等疾病。埃尔坦布利等人。报告称，检查点调节因子 VISTA（含有 V 型免疫球蛋白结构域的 T 细胞激活抑制因子）通过将初始 CD4+ T 细胞区室的固有异质性塑造为更均匀的静止和沉默状态，从而限制 T 细胞激活的早期阶段（参见布朗和鲁登斯基的观点）。在小鼠体内使用激动性抗体进行 VISTA 治疗，可抑制移植物抗宿主病的发展，并促进被自身抗原异常激活的幼稚 T 细胞死亡。因此，VISTA 代表了一种独特的免疫调节分子，它通过维持静止和外周耐受来控制幼稚 T 细胞的功能。科学，本期第 14 页。eaay0524；另见 p. 247 免疫检查点调节剂 VISTA 会抑制初始 CD4+ T 细胞的非特异性活性，限制对自身抗原的免疫反应。简介 克隆选择理论 (CST) 是当前适应性免疫的基石，其核心原则指出，幼稚 T 细胞是根据对抗原的特异性进行选择的，而其余的细胞库仍然与抗原攻击无关。然而，CST 和最近的范式并未考虑到需要负调控信号来维持未选择的 T 细胞的静止，作为维持克隆选择的关键方面。幼稚 T 细胞的沉默或耐受在遇到自身抗原时也变得至关重要，因此自身反应性克隆不能发展出效应功能。已知这些事件是相互关联的，因为静止的丧失会导致耐受性的丧失。基本原理 含有 V 型免疫球蛋白结构域的 T 细胞激活抑制因子 (VISTA) 是一种在幼稚 T 淋巴细胞上表达的抑制性受体，VISTA 的基因缺失最终导致 T 细胞介导的自身免疫。与迄今为止发现的其他阴性检查点调节因子不同，VISTA 在幼稚 T 细胞上表达，这促使我们研究其在维持幼稚 T 细胞静止和耐受性方面的功能。结果 通过结合单细胞 RNA 测序和单细胞 ATAC 测序技术以及体内分析，我们研究了小鼠中幼稚 CD4+ (CD44lo CD62Lhi) T 细胞的细胞状态表型，并发现多个 T 细胞亚群存在于小鼠体内。令人惊奇的是，幼稚 T 细胞区室具有异质性。绝大多数细胞表现出静止表型。然而，有些亚群表达较少的静态、类似记忆的特征，一个簇具有高干扰素-I (IFN-I) 反应特征，以及一个群体具有较高的早期 T 细胞受体 (TCR) 信号基因。我们发现 VISTA 的基因删除导致幼稚 T 细胞亚群的重大重新分布，其中静止亚群显着减少，记忆样激活 T 细胞亚群的存在增强。在缺乏内在 VISTA 表达的情况下，幼稚 T 细胞在表观遗传和转录上更加容易对 TCR 和细胞因子刺激产生更强的反应，这为在缺乏 VISTA 的情况下观察到的 CD44hi CD4+ 记忆样 T 细胞的增强提供了理论依据。由于幼稚 T 细胞室内的静止减少，这些幼稚 T 细胞不太容易受到耐受诱导的影响。VISTA 的基因缺失或抗体阻断导致抗原特异性 T 细胞显着扩增并降低耐受性。在炎症条件下，抗原特异性 T 细胞上的 VISTA 表达减少，这些作用被消除。相比之下，VISTA 和抗原暴露的激动作用通过增强抗原特异性 T 细胞的死亡和缺失来增加耐受诱导。抗VISTA诱导的耐受性死亡在急性移植物抗宿主病（GVHD）模型中得到证实，其中供体宿主反应性T细胞的缺失导致显着的治疗益处和长期存活。VISTA−/− T 细胞的基因特征与患有自身免疫性疾病系统性红斑狼疮和类风湿性关节炎患者的 T 细胞显着重叠，表明 VISTA 可能在抑制 T 细胞自身反应性方面发挥广泛的调节作用。结论 我们将 VISTA 介绍为迄今为止发现的最早的外周 T 细胞耐受检查点调节剂，并将 VISTA 描述为第一个新型免疫调节分子，其功能是强制初始 T 淋巴细胞的静止。此外，综合表观遗传学和转录分析将荧光激活分选纯化的 CD44lo CD62Lhi 幼稚 CD4+ T 细胞定义为异质性。我们目前的体内研究结果还表明，静止的调节与维持 T 细胞自我耐受的能力有着千丝万缕的联系。尽管 VISTA 在幼稚 T 细胞稳态中发挥着关键作用，但当在炎症条件下遇到抗原刺激时，VISTA 对幼稚 T 细胞反应的限制就会消失。VISTA 的缺失会降低初始 T 细胞在转录和表观遗传水平上的静止表型，从而导致 TCR 和细胞因子途径增强。（左）这种损失破坏了对自身抗原的耐受性诱导，并在激活后产生更具炎症的表型。KLF2/6、BTG1/2、FOXP1 和 SLFN2 是静止调节因子。TCF7、BCL2、INFGR1 和 SLAMF6 直接记忆 T 细胞的命运和活性。IFN-γ 和 CCL5 是效应细胞因子。CD69、CD25 和 CD40LG 是激活和共刺激受体。（右）VISTA 与其他成熟的 T 细胞激活负检查点调节因子的整合。VISTA 在初始 T 细胞上组成型表达，在强制静止中发挥着关键作用。CTLA-4 在 T 细胞激活后短暂地在细胞表面表达，并通过限制共刺激在启动阶段抑制 T 细胞激活。PD-1 在启动过程中后期表达，并在效应阶段抑制 T 细胞。阴性检查点调节因子 (NCR) 会调节 T 细胞对自身抗原的免疫反应，并限制自身免疫的发展。与在活化 T 淋巴细胞上表达的所有其他 NCR 不同，含有 V 型免疫球蛋白结构域的 T 细胞活化抑制因子 (VISTA) 在初始 T 细胞上表达。我们报告了小鼠幼稚 T 细胞区室中意想不到的异质性，其中 VISTA 的缺失破坏了主要的静态幼稚 T 细胞子集并增强了自身反应性。激动性 VISTA 参与通过促进抗原诱导的外周 T 细胞缺失来增加 T 细胞耐受性。尽管 VISTA 在幼稚 T 细胞稳态中发挥着关键作用，但在炎症条件下会失去抑制幼稚 T 细胞反应的能力。因此，VISTA 是幼稚 T 细胞的独特 NCR，对于静态维持和外周耐受性至关重要。