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

Kruppel 样因子 2 CD4 T 细胞和肠道炎症

• 批准号: 10730990
• 负责人: Sing Sing Way
• 金额: $ 70.59万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730990
• 关键词: ATAC-seq; Antibodies; Antigens; Autoimmunity; Binding; Biological; Biopsy; Bypass; CD4 Positive T Lymphocytes; CTLA4 gene; Candida albicans; Cell Differentiation process; Cell Lineage; Cells; ChIP-seq; Chromatin; Coculture Techniques; Colitis; Crohn' s disease; Cytoprotection; DNA Binding; Data; Engineering; Enterobacteria phage P1 Cre recombinase; Epigenetic Process; FOXP3 gene; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Silencing; Germ-Free; Homeostasis; House mice; Human; Immune; Immune Tolerance; Immune system; Immunologics; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-10; Intestinal Diseases; Intestines; Irritable Bowel Syndrome; Knowledge; Kruppel-like transcription factors; Link; LoxP-flanked allele; Lymphoid Tissue; MHC Class II Genes; Mediating; Memory; Microbe; Modeling; Molecular; Mucous Membrane; Mus; OX40; Onset of illness; Pathogenesis; Peripheral; Phenotype; Physiological; Production; Property; RNA; Recombinants; Regulatory T-Lymphocyte; Specificity; T cell differentiation; T-Cell Proliferation; T-Lymphocyte; T-Lymphocyte Subsets; Tissues; Ulcerative Colitis; Zinc Fingers; antimicrobial; autoinflammatory diseases; commensal microbes; design; experience; gut colonization; gut inflammation; gut microbiota; in vivo; inducible gene expression; insight; interleukin-10 receptor; mesenteric lymph node; novel; overexpression; reconstitution; response; selective expression; single-cell RNA sequencing; stem

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).

抽象的粘膜组织，例如肠道携带数万亿抗抗原的外国微生物。不可避免的 在这种情况下，与共生微生物的近似值强调了需要扩展的外周免疫 宽容。但是，知识的基本差距仍然是关于这种生理命令的方式 成就了。填补这些知识差距具有巨大的潜力，可以揭示有关免疫的新见解。 炎症性肠病和其他人类自身炎症性疾病的发病机理。我们的目前 调查耐受性如何扩展以适应共生肠道微生物的框架是 主要集中在CD4+ T细胞的Foxp3+抑制子集上，称为调节T细胞（Tregs）。 但是，几个不一致也强调了归因于共同公差的局限性 FOXP3+细胞。通过这些考虑，我们的初步研究枢纽研究了共生特异性 CD4+ T细胞，没有FOXP3偏见，使用具有指导性的CD4+ T细胞的指导性模型 可以精确识别特异性。使用重组白色白色念珠菌建立肠道定植 并使用MHC II类四聚体跟踪内源性CD4+ T细胞，我们对基因表达的初步分析 轮廓（单细胞RNA-Seq）在CD4+细胞之间显示最小（<5％）的Treg分化 特异性。相反，RNA分析显示出近一半的外围细胞，这些细胞响应于共生 刺激不是根据其他定义标记的表达来分类的，而是通过表达统一 锌指转录因子Kruppel样因子2（KLF2）经过抗原经验的KLF2+ CD4+ T细胞是 进一步显示可以在体外共培养过程中有效抑制响应者T细胞增殖。 T的必要性 自发性肠道炎症进一步强调了表达KLF2的细胞 T细胞中有条件的KLF2缺乏症，或诱导的小鼠疾病的快速发作（在10天内） CD4+细胞中的KLF2缺乏。在没有KLF2+ CD4+ T细胞的情况下发生的肠炎是 由共生微生物触发，因为它们使用抗菌鸡尾酒消除了肠道 炎症，有效地绕开了表达KLF2的T细胞的必要性。因此，我们的总体假设是 KLF2识别CD4+ T细胞的FOXP3阴性免疫抑制子集 对肠道微生物的耐受性。三个相互关联的目的旨在进一步发展这一潜在的基础 - 提出了打破假设，其中包括建立KLF2+ CD4+ T细胞的分子基础 介导抑制作用，以及KLF2是否需要和/或足以促进功能抑制（目标 1），KLF2+ CD4+ T细胞如何预防体内肠炎（AIM 2）的分子基础 具有基因表达，染色质的可及性和KLF2 DNA的结合区分KLF2+ CD4+ T细胞之间的区别 与肠和淋巴组织中的其他CD4+ T细胞分化子集相比 作为其独特的生物学功能特性的基础（AIM 3）。