Kruppel-like factor-2 CD4+ T cells and intestinal inflammation

像肠道这样的粘液组织含有数以万亿计的抗原性外来微生物。不可回避 在这种情况下，与肠道微生物的近似强调了扩大外周免疫的需要。 宽容然而，关于这种生理上的必要性是如何产生的， 办妥了一批填补这些知识空白有巨大的潜力，揭示新的见解免疫- 炎症性肠病和其他人类自身炎症性疾病的发病机制。我们目前 研究耐受性如何扩大以适应肠道微生物的框架是 主要集中在称为调节性T细胞（Tcells）的CD 4 + T细胞的FOXP 3+抑制性亚群。 然而，一些不一致的地方也突出表明，将植物耐药性完全归因于 FOXP 3+细胞。考虑到这些因素，我们的初步研究转向研究特定的 CD 4 + T细胞，无FOXP 3偏倚，使用指导性模型， 特异性可以被精确识别。利用重组白色念珠菌建立肠道定植 用MHC II类四聚体追踪内源性CD 4 + T细胞，我们对基因表达的初步分析 结果显示，单细胞RNA-seq图谱显示，在CD 4+细胞中，Treg分化最小（<5%）， 的特异性相反，RNA分析显示，近一半的外周细胞在对细胞因子的反应中扩增， 刺激不根据其他谱系定义标记的表达进行分类，而是通过表达进行统一 锌指转录因子Kruppel样因子2（KLF 2）。抗原经历的KLF 2 + CD 4 + T细胞是 进一步显示在体外共培养期间有效抑制应答T细胞增殖。T的必要性 细胞表达的KLF 2进一步突出了自发性肠道炎症， T细胞中的条件性KLF 2缺陷，或诱导的小鼠中疾病的快速（10天内）发作 CD 4+细胞中的KLF 2缺陷。在缺乏KLF 2 + CD 4 + T细胞的情况下发生的肠道炎症， 由肠道微生物引发，因为使用抗菌剂鸡尾酒消除它们， 炎症，有效地绕过T细胞表达KLF 2的必要性。因此，我们的总体假设是， KLF 2鉴定了维持CD 4 + T细胞的FOXP 3阴性免疫抑制亚群， 对肠道微生物的耐受性旨在进一步发展这一潜在基础的三个相互关联的目标： 提出了一个突破性的假设，其中包括建立KLF 2 + CD 4 + T细胞 介导抑制，以及KLF 2是否是促进功能性抑制所必需的和/或足够的（目的 1），KLF 2 + CD 4 + T细胞如何在体内保护肠道炎症的分子基础（Aim 2），沿着 KLF 2 + CD 4 + T细胞之间的基因表达、染色质可及性和KLF 2 DNA结合差异 与FOXP 3 + T细胞亚群和肠和淋巴组织中的其他CD 4 + T细胞分化亚群相比， 作为其独特的生物功能特性的基础（目标3）。

Abstract Mucosal tissues like the intestine harbor trillions of antigenically foreign microbes. Unavoidable approximation with commensal microbes in this context highlights the need for expanded peripheral immune tolerance. However, fundamental gaps in knowledge remain as to how this physiological imperative is achieved. Filling these knowledge gaps have enormous potential to reveal novel insights on the immune- pathogenesis of inflammatory bowel disease and other human autoinflammatory disorders. Our current framework for investigating how tolerance expands to accommodate commensal intestinal microbes is primarily focused on the FOXP3+ suppressive subset of CD4+ T cells called regulatory T cells (Tregs). However, several inconsistencies also highlight the limitations attributing commensal tolerance exclusively to FOXP3+ cells. With these considerations, our preliminary studies pivoted to investigate commensal specific CD4+ T cells, without a FOXP3 bias, using an instructive model whereby CD4+ T cells with commensal specificity can be precisely identified. Using recombinant Candida albicans to establish intestinal colonization and tracking endogenous CD4+ T cells with MHC class II tetramers, our initial analysis of gene expression profiles (single-cell RNA-seq) shows minimal (<5%) Treg differentiation amongst CD4+ cells with commensal specificity. Instead, RNA profiling showed nearly half of peripheral cells that expand in response to commensal stimulation are not classified based on expression of other lineage-defining markers, and unified by expression of the zinc finger transcription factor Kruppel-like factor-2 (KLF2). Antigen-experienced KLF2+ CD4+ T cells are further shown to potently suppress responder T cell proliferation during in vitro co-culture. The necessity for T cell expressed KLF2 is further highlighted by spontaneous intestinal inflammation that develops in mice with conditional KLF2 deficiency in T cells, or the rapid (within 10 days) onset of disease in mice with induced KLF2-deficiency in CD4+ cells. Intestinal inflammation that occurs in the absence of KLF2+ CD4+ T cells is triggered by commensal microbes since their elimination using a cocktail of antimicrobials averts intestinal inflammation, efficiently bypassing the necessity for T cell expressed KLF2. Thus, our overall hypothesis is that KLF2 identifies a FOXP3-negative immune-suppressive subset of CD4+ T cells essential for sustaining tolerance to intestinal microbes. Three inter-related aims designed to further develop this potentially ground- breaking hypothesis are proposed which include establishing the molecular basis for how KLF2+ CD4+ T cells mediate suppression, and whether KLF2 is necessary and/or sufficient to promote functional suppression (Aim 1), the molecular basis for how KLF2+ CD4+ T cells protect against intestinal inflammation in vivo (Aim 2), along with gene expression, chromatin accessibility, and KLF2 DNA binding distinctions between KLF2+ CD4+ T cells compared with FOXP3+ Tregs, and other CD4+ T cell differentiation subsets in intestinal and lymphoid tissue as a basis for their unique biological functional properties (Aim 3).