The CD226 costimulatory axis in type 1 diabetes

抽象的。1型糖尿病(T1D)是由150多个独立基因座之间复杂的相互作用引起的 传递疾病易感性和破坏免疫耐受性的环境因素，导致免疫- 介导对产生胰岛素的胰腺细胞的破坏。其中存在少量的编码 变异体，可能代表恢复免疫耐受的合理治疗靶点，但细胞和 风险变异改变免疫功能的分子机制仍未得到很好的描述。CD226 候选基因包含一个蛋白质编码变异体(Rs763361)，与多种自身免疫性疾病有关，包括 T1D。CD226作为共刺激分子与负调控因子TIGIT和CD96竞争， 用于与抗原提呈细胞(APC)上表达的CD155或CD112结合。我们已出版的和初步的 R01的初始阶段支持的数据表明，CD226信号会破坏调节性T细胞的稳定 (Treg)表型。具体地说，在非肥胖者中，cd226基因缺失减缓了疾病的发展。 糖尿病(NOD)小鼠T1D模型，包括基因组敲除(GKO)和Treg特异性条件性KO(CKO) 在GKO动物的胰腺淋巴结(PLN)中，ex-Treg频率降低。此外，CD226- 人类Tregs显示出更高的纯度、稳定性和抑制功能。单细胞转录图谱和 从T1D器官供者胰腺和PLN分离的组织驻留T细胞的流式细胞术分析 CD8 T细胞表面CD226和TIGIT表达失衡我们假设与T1D相关的风险 CD226基因变异导致免疫检查点失调，导致外周免疫缺陷 耐受性，特别是在T1D发作之前导致Treg不稳定。为了检验这一点，我们提出了三个具体目标。 1)我们将进行单细胞多基因组图谱和过继转移研究，以确定细胞和 CD226在NOD小鼠缺陷免疫耐受中的分子基础 GKO和Treg CKO菌株。2)我们将评估CD226风险的表达谱和功能影响 变种使用储存的器官捐赠者组织和来自患有 并有患T1D的风险。这些努力将包括对选定基因的胰腺和PLN样本进行原位分析 通过空间转录和高含量成像，以及CD226的CRISPR/Cas9基因编辑 T细胞受体(TCR)基因转移细胞产生等基因、自身反应性Treg、CD4和CD8T细胞 用于体外功能研究。3)我们将测试针对CD226共刺激轴的候选疗法 体内使用NOD小鼠，体外使用人类细胞。因此，拟议的研究将使用新的动物 菌株以及我们首创的人类淋巴细胞的基因编辑和TCR基因转移技术 在之前的赠款期限内，告知CD226/TIGIT/CD96的贡献：CD155/CD112免疫 T1D发育的检查点，可能会有可翻译的干预措施阻止CD226共刺激 阻止免疫介导的对胰腺细胞的破坏。

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.