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

Kruppel 样因子 2 CD4 T 细胞和肠道炎症

基本信息

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
项目类别：
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&apos 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

项目摘要

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).

摘要像肠道这样的粘膜组织蕴藏着数万亿的抗原性外来微生物。不可避免的在这种情况下与共生微生物的近似强调了扩大外周免疫的需要宽容。然而，对于这种生理必要性如何发挥作用，仍然存在根本性的知识空白。实现了。填补这些知识空白具有揭示免疫相关新见解的巨大潜力。炎症性肠病和其他人类自身炎症性疾病的发病机制。我们目前的研究耐受性如何扩展以适应共生肠道微生物的框架主要关注 CD4+ T 细胞的 FOXP3+ 抑制子集，称为调节性 T 细胞 (Treg)。然而，一些不一致之处也凸显了将共生耐受完全归因于的局限性。 FOXP3+ 细胞。考虑到这些因素，我们的初步研究转向调查共生特异性 CD4+ T 细胞，没有 FOXP3 偏差，使用指导性模型，其中 CD4+ T 细胞与共生体可以精确识别特异性。使用重组白色念珠菌建立肠道定植并用 MHC II 类四聚体追踪内源性 CD4+ T 细胞，这是我们对基因表达的初步分析谱（单细胞 RNA-seq）显示共生 CD4+ 细胞中 Treg 分化程度极低（<5%）特异性。相反，RNA 分析显示近一半的外周细胞响应共生体而扩增。刺激不根据其他谱系定义标记的表达进行分类，并根据表达进行统一锌指转录因子 Kruppel 样因子 2 (KLF2)。经历过抗原的 KLF2+ CD4+ T 细胞是进一步显示在体外共培养期间可有效抑制应答 T 细胞增殖。 T的必要性小鼠中发生的自发性肠道炎症进一步凸显了细胞表达的 KLF2 T 细胞中条件性 KLF2 缺陷，或诱导小鼠发病迅速（10 天内） CD4+ 细胞中 KLF2 缺陷。在缺乏 KLF2+ CD4+ T 细胞的情况下发生的肠道炎症是由共生微生物引发，因为使用抗生素混合物消除共生微生物可避免肠道炎症，有效绕过 T 细胞表达 KLF2 的必要性。因此，我们的总体假设是 KLF2 识别出 FOXP3 阴性的 CD4+ T 细胞亚群，对于维持免疫系统至关重要对肠道微生物的耐受性。旨在进一步开发这一潜在基础的三个相互关联的目标提出了突破性假设，其中包括建立 KLF2+ CD4+ T 细胞如何发挥作用的分子基础介导抑制，以及 KLF2 是否必要和/或足以促进功能性抑制（目标 1)，KLF2+ CD4+ T 细胞如何预防体内肠道炎症的分子基础（目标 2），以及 KLF2+ CD4+ T 细胞之间的基因表达、染色质可及性和 KLF2 DNA 结合差异与肠道和淋巴组织中的 FOXP3+ Tregs 和其他 CD4+ T 细胞分化亚群相比作为其独特生物功能特性的基础（目标 3）。