The CD226 costimulatory axis in type 1 diabetes

摘要。1型糖尿病（T1 D）是由150多个独立基因座之间的复杂相互作用引起的。 赋予疾病易感性和破坏免疫耐受的环境因素，导致免疫- 介导的破坏产生胰岛素的胰腺β细胞。其中存在少量的编码 变异，这可能是合理的治疗目标，以恢复免疫耐受，但细胞和 风险变体改变免疫功能的分子机制仍然缺乏表征。CD226 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 CD 226作为与负调节因子TIGIT和CD 96竞争的共刺激分子起作用， 用于结合抗原呈递细胞（APC）上表达的CD 155或CD 112。我们已公布的和初步的 由R 01的初始阶段支持的数据表明，CD 226信号转导使调节性T细胞不稳定， （Treg）表型。具体地说，Cd 226基因的缺失减弱了非肥胖者的疾病发展。 T1 D的糖尿病（NOD）小鼠模型，基因组敲除（gKO）和Treg特异性条件性KO（cKO） 细胞系，其中gKO动物的胰腺淋巴结（pLN）中ex-Treg频率降低。此外，CD 226- 人TCL 4显示出增加纯度、稳定性和抑制功能。单细胞转录谱分析和 对从T1 D器官供体胰腺和pLN分离的组织驻留T细胞的流式细胞术分析鉴定了一种新的免疫抑制剂。 CD 8 + T细胞上CD 226和TIGIT表达失衡。我们假设T1 D相关风险 CD 226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T1 D发作之前导致Treg不稳定。为了验证这一点，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，以确定细胞和 CD 226导致NOD小鼠免疫耐受缺陷的分子基础，使用我们的Cd 226 gKO和Treg cKO菌株。2)我们将评估CD 226风险的表达谱和功能影响。 使用来自具有以下特征的个体的库存器官供体组织和人外周血样品， 并有患T1 D的风险。这些努力将涉及基因型选择的胰腺和pLN样本的原位分析 通过空间转录组学和高含量成像，沿着CRISPR/cas9基因编辑原发性肝癌患者的CD 226， 具有T细胞受体（TCR）基因转移的细胞，以产生同基因的自身反应性Treg、CD 4+和CD 8 + T细胞 用于体外功能研究。3)我们将测试靶向CD 226共刺激轴的候选疗法， 体内使用NOD小鼠和体外使用人细胞。因此，拟定的研究将采用新的动物 菌株沿着与基因编辑和TCR基因转移技术在人类淋巴细胞，我们开创了 在先前的资助期限内，告知CD 226/TIGIT/CD 96：CD 155/CD 112免疫的贡献 T1 D发展的检查点，具有阻断CD 226共刺激的可翻译干预的潜力 以阻止免疫介导的对胰腺癌细胞的破坏。

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.