HLA-DO / H2-O: modulation of MHC-II peptide diversity and Treg population control

Hla-DO/H2-O(DO)是一种非经典的MHCII蛋白，其功能是作为一种特异性的竞争性抑制因子。 肽交换因子HLA-DM/H2-M(DM)，调节抗原-MHCII分子上的多肽负载- 呈现细胞。在CD4T细胞发育过程中，胸腺细胞被选择转化为传统的幼稚细胞 CD4T细胞或自身耐受性调节性T细胞，或被阴性选择删除，取决于强度 与胸腺中抗原提呈细胞上显示的MHCII结合多肽的相互作用。我们洗脱了 从野生型C57BL/6和DO基因敲除的胸腺中提取多肽，并发现多肽的光谱发生了变化 由MHCII分子提出。我们检测了DO缺乏小鼠的CD4T细胞发育，发现 Treg数量和功能的改变，以及对病毒感染的敏感性增加。最重要的假设 DO通过抑制糖尿病小鼠体内的多肽编辑活性来调节抗原提呈。 以允许呈现在细胞表面的多肽的广泛表示，并且这种活性对于 适当选择胸腺中具有自我耐受性的常规和调节性T细胞。有两个具体目标。目标1 目的是评价DO依赖的抗原提呈在选择CD4Tconv和Treg群体中的作用。 我们将识别在有或没有DO的情况下胸腺中出现的差异多肽，如下所示 在WT和DO-KO TCR限制性小鼠中进行胸腺选择的单个TCR克隆型，结合 这些结果确定了导致Treg偏斜的DO多肽，并最终表征了DO的功能效应 DO-KO小鼠的Treg失调。这些实验将检验这样一种假设，即缩小肽 DO缺失诱导的MHCII分子提供的谱系导致胸腺选择和 调节性T细胞的外周调节。目的2确定人类白细胞抗原DO/H2-O在调节T细胞转化中的作用 以及感染模型中的Treg种群。我们将评估DO诱导的功能后果 IL/6小鼠TCR谱系和CD4T细胞群激活状态的变化 原发流感感染。此前已有研究表明，Tregs可以调节CD4和CD8T细胞的反应，并 先天效应细胞的招募，我们在DO-KO小鼠中观察到这些效应的变化，提示 在没有DO的情况下，Tregs的调节失调。我们将使用FoxP3-DTR系统交换 老鼠之间的特雷格隔间。这一目标的第二部分是评估DO的功能后果- 使用流感感染模型诱导抗原提呈和TCR谱系的改变。长的- 本项目的学期目标是定义DO对DM介导肽编辑的控制如何调节选择 MHCII上显示的自身和病原体衍生的多肽，并破译DO调节的影响 介绍CD4T细胞库的发展和功能。 1

HLA-DO / H2-O (DO) is a non-classical MHCII protein that functions as a specific competitive inhibitor of the peptide exchange factor HLA-DM / H2-M (DM), regulating peptide loading onto MHCII molecules in antigen- presenting cells. During CD4 T cell development, thymocytes are selected for conversion to conventional naïve CD4 T cells or self-tolerant regulatory T cells, or are deleted by negative selection, depending on the strength of interaction with MHCII-bound peptides displayed on antigen presenting cells in the thymus. We eluted peptides from wild-type C57BL/6 and DO-knockout thymus, and found alterations in the spectrum of peptides presented by MHCII molecules. We examined CD4 T cell development in DO-deficient mice, and found alterations in Treg number and function, and increased sensitivity to viral infection. The overarching hypothesis of this proposal is that DO regulates antigen presentation by restraining the peptide editing activity of DM in order to allow a broad representation of peptides presented at the cell surface, and this activity is essential for proper thymic selection of self-tolerant conventional and regulatory T cells. There are two specific aims. Aim 1 is to evaluate the role of DO-dependent antigen presentation in selection of CD4 Tconv and Treg populations. We will identify peptides differentially presented in the thymus in the presence or absence of DO, follow individual TCR clonotypes as they undergo thymic selection in WT and DO-KO TCR-restricted mice, combine these results to identity DO-peptides responsible for Treg skewing, and finally characterize functional effects of Treg dysregulation in DO-KO mice. These experiments will test the hypothesis that narrowing of the peptide repertoire presented by MHCII molecules induced by deletion of DO results in altered thymic selection and peripheral regulation of regulatory T cells. Aim 2 is to determine the role of HLA-DO / H2-O in regulating Tconv and Treg populations in an infection model. We will evaluate the functional consequences of DO-induced changes in the TCR repertoire and activation state of CD4 T cell populations in the response of BL/6 mice to primary influenza infection. Tregs have been shown previously to regulate CD4 and CD8 T cell responses and recruitment of innate effector cells, and we observe alterations in these effects in DO-KO mice, suggesting a dysregulation of Tregs in the absence of DO. We will test this hypothesis, using a FoxP3-DTR system to swap Treg compartments between mice. A second part of this aim is to evaluate the functional consequences of DO- induced alterations in antigen presentation and TCR repertoires using the influenza infection model. The long- term goals of this project are to define how control of DM-mediated peptide editing by DO regulates selection of self- and pathogen-derived peptides displayed on MHCII, and to decipher the impact of DO-regulated peptide presentation on the development and function of the CD4 T cell repertoire. 1