The CD226 costimulatory axis in type 1 diabetes

1 型糖尿病中的 CD226 共刺激轴

基本信息

批准号：
10594278
负责人：
Todd Michael Brusko
金额：
$ 62.69万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10594278
关键词：
Adoptive Cell Transfers Adoptive Transfer Animals Antigen-Presenting Cells Antigens Antithymoglobulin Attenuated Autoimmune Autoimmune Diseases Autoimmunity Beta Cell Binding Biological Biological Assay Blocking Antibodies Blood specimen CD3 Antigens CD8-Positive T-Lymphocytes CRISPR/Cas technology Candidate Disease Gene Cell Lineage Cell Separation Cell physiology Cells Chimeric Proteins Clinical Code Complex Cytokine Signaling Data Defect Development Diabetes Mellitus Diabetes prevention Disease Disease Progression Disease susceptibility Dose Environmental Risk Factor Female Flow Cytometry Frequencies Gene Expression Profiling Gene Transfer Gene Transfer Techniques Genes Genetic Genetic Diseases Genetic Risk Genome Genomics Genotype Goals Grant HLA Antigens Histocompatibility Antigens Class II Histology Human Image Immune Immune Tolerance Immune mediated destruction In Situ In Vitro Inbred NOD Mice Incidence Individual Insulin Insulin-Dependent Diabetes Mellitus Intervention Investigation Knock-out Link Lymphocyte Lymphocyte Function Methods Modeling Modernization Molecular Molecular Biology Multiple Sclerosis Natural History Non obese Organ Donor Pancreas Pathogenesis Pathway interactions Peripheral Phase Phenotype Proteins Proto-Oncogene Proteins c-akt Publishing Regulatory T-Lymphocyte Rheumatoid Arthritis Risk Role Safety Sampling Signal Transduction Specificity Spleen T-Cell Receptor T-Cell Receptor Genes T-Lymphocyte T-Lymphocyte Subsets Testing Th1 Cells Therapeutic Intervention Tissue Donors Tissue imaging Tissues Variant autoreactive T cell autoreactivity cell killing conditional knockout cytotoxic CD8 T cells cytotoxicity diabetes pathogenesis diabetic diabetogenic draining lymph node drug mechanism effector T cell experimental study genomic locus human tissue immune checkpoint immune function immunoregulation in vivo in vivo Model insight insulin dependent diabetes mellitus onset insulitis islet islet cell antibody knockout animal knockout gene lymph nodes mouse model multiple omics novel peripheral blood peripheral tolerance pre-clinical prevent programs protective allele risk variant targeted agent targeted treatment therapeutic candidate therapeutic target trafficking transcriptomics translational potential

项目摘要

ABSTRACT. Type 1 diabetes (T1D) results from complex interactions between over 150 independent loci imparting disease susceptibility and environmental factors that break immune tolerance, leading to the immune- mediated destruction of insulin-producing pancreatic -cells. Among these exists a small number of coding variants, which may represent rational therapeutic targets to restore immune tolerance, yet the cellular and molecular mechanisms by which risk variants alter immune function remain poorly characterized. The CD226 candidate gene contains a protein coding variant (rs763361) linked to multiple autoimmune disorders, including T1D. CD226 functions as a costimulatory molecule that competes with the negative regulators, TIGIT and CD96, for binding to CD155 or CD112 expressed on antigen presenting cells (APCs). Our published and preliminary data, supported by the initial phase of this R01, suggest that CD226 signaling destabilizes the regulatory T cell (Treg) phenotype. Specifically, genetic deletion of Cd226 attenuated disease development in the non-obese diabetic (NOD) mouse model of T1D, both in genomic knockout (gKO) and Treg-specific conditional KO (cKO) lines, with reduced ex-Treg frequency in the pancreatic lymph nodes (pLN) of gKO animals. Moreover, CD226– human Tregs display increased purity, stability, and suppressive function. Single-cell transcriptional profiling and flow cytometric analysis of tissue-resident T cells isolated from T1D organ donor pancreas and pLN identified an imbalance of CD226 and TIGIT expression on CD8+ T cells. We hypothesize that the T1D-associated risk variant in CD226 results in immune checkpoint dysregulation that leads to defects in peripheral immune tolerance, specifically resulting in Treg instability prior to T1D onset. To test this, we propose three Specific Aims. 1) We will perform single cell multi-omic profiling and adoptive transfer studies to identify the cellular and molecular basis by which CD226 contributes to defective immune tolerance in the NOD mouse, using our Cd226 gKO and Treg cKO strains. 2) We will assess the expression profile and functional impact of the CD226 risk variant using banked organ donor tissues and human peripheral blood samples derived from individuals with and at risk for T1D. These efforts will involve in situ profiling of genotype-selected pancreas and pLN samples via spatial transcriptomics and high-content imaging, along with CRISPR/cas9 gene-editing of CD226 in primary cells with T cell receptor (TCR) gene transfer to generate isogenic, autoreactive Treg, CD4+, and CD8+ T cells for in vitro functional studies. 3) We will test candidate therapeutics targeting the CD226 costimulatory axis in vivo using NOD mice and in vitro using human cells. Hence, the proposed studies will employ novel animal strains along with gene editing and TCR gene transfer techniques in human lymphocytes, which we pioneered over the prior grant term, to inform on the contributions of the CD226/TIGIT/CD96:CD155/CD112 immune checkpoint to T1D development, with the potential for translatable interventions blocking CD226 co-stimulation to halt the immune-mediated destruction of pancreatic -cells.

摘要：1 型糖尿病 (T1D) 由 150 多个独立基因座之间的复杂相互作用所致。赋予疾病易感性和破坏免疫耐受的环境因素，导致免疫介导的产生胰岛素的胰腺  细胞的破坏其中存在少量编码。变体，这可能代表恢复免疫耐受的合理治疗靶标，但细胞和风险变异改变免疫功能的分子机制仍不清楚。候选基因包含与多种自身免疫性疾病相关的蛋白质编码变体（rs763361），包括 T1D。CD226 作为一种共刺激分子，与负调节因子 TIGIT 和 CD96 竞争，用于与抗原呈递细胞 (APC) 上表达的 CD155 或 CD112 结合。该 R01 初始阶段的数据表明 CD226 信号传导破坏了调节性 T 细胞的稳定性 (Treg) 表型具体来说，Cd226 的基因缺失可减弱非肥胖者的疾病发展。 T1D 糖尿病 (NOD) 小鼠模型，包括基因组敲除 (gKO) 和 Treg 特异性条件性敲除 (cKO) 此外，CD226– 的胰腺淋巴结 (pLN) 中的前 Treg 频率降低。人类 Tregs 显示出更高的纯度、稳定性和抑制功能。对从 T1D 器官供体胰腺和 pLN 中分离的组织驻留 T 细胞进行流式细胞术分析，确定了 CD8+ T 细胞上的 CD226 和 TIGIT 表达失衡，我们发现了 T1D 相关风险。 CD226 的变异导致免疫检查点失调，从而导致外周免疫缺陷为了测试这一点，我们提出了三个具体目标。 1) 我们将进行单细胞多组学分析和过继转移研究，以鉴定细胞和 CD226 导致 NOD 小鼠免疫耐受缺陷的分子基础，使用我们的 Cd226 gKO 和 Treg cKO 菌株 2) 我们将评估 CD226 风险的表达谱和功能影响。使用储存的器官捐献组织和来自患有以下疾病的个体的人外周血样本进行变体这些工作将涉及对基因型选择的胰腺和 pLN 样本进行原位分析。通过空间转录组学和高内涵成像，以及原代细胞中 CD226 的 CRISPR/cas9 基因编辑具有 T 细胞受体 (TCR) 基因转移的细胞可产生同基因、自身反应性 Treg、CD4+ 和 CD8+ T 细胞 3) 我们将测试针对 CD226 共刺激轴的候选治疗方法。体内使用 NOD 小鼠，体外使用人类细胞，因此，拟议的研究将采用新的动物。菌株以及我们首创的人类淋巴细胞基因编辑和 TCR 基因转移技术在先前的拨款期限内，告知 CD226/TIGIT/CD96:CD155/CD112 免疫的贡献 T1D 发展的检查点，具有阻断 CD226 共刺激的可转化干预措施的潜力阻止免疫介导的胰腺  细胞破坏。