Project 3 - Differential contribution of thymic APCs to central tolerance during the perinatal to adult transition

项目3 围产期胸腺在促进中枢耐受中起着独特的作用。中心公差的结果来自 自身反应性常规T细胞（Tconv）的阴性选择和调节性T细胞（Treg）的分化。 值得注意的是，在围产期选择的睾酮更具自身反应性，并且是生命唯一需要的- 长期保护自身免疫。围产期Tconv细胞也更具自身反应性，具有独特的 分子和功能特性进行比较，其少年/成人同行。既负选择 当胸腺细胞遇到胸腺髓质上皮细胞呈递的自身抗原时， 细胞（mTEC）或造血抗原呈递细胞（HAPC），包括树突状细胞（DC）。TEC和 胸腺HAPCs在围产期到幼年期的转变过程中经历深刻的变化。RP 3的基本原理是 胸腺HAPCs在围产期的细胞组成和分子特征是不同的， 胸腺选择对于Tconv细胞的第一波分化和自身耐受性的建立是关键的。 我们将检验这一假设，即围产期mTECs和HAPCs的独特性质产生了 改变了中枢耐受诱导的环境，并负责选择Tconv和Treg细胞 具有增加的自反应性和年龄特异性功能特性。在目标1中，我们将使用单细胞 转录谱分析（scRNA-seq）和多重成像，以确定细胞，转录， 在小鼠和人类HAPC中发生的从围产期到幼年期的组织变化， 会影响中枢耐受性在目标2中，我们将使用表型分析，转录谱分析，遗传分析， 模型和功能测定，以鉴定负责选择组织保护性Treg的APC。 围产期我们还将确定CCR 7介导的与TEC的串扰是否改变了DC 在围产期小鼠中的隔室，具有Treg选择的下游后果。在目标3中，我们将确定 如果围产期动物在对抗原的反应中负选择在一系列亲和力上受损。活细胞2- 光子显微镜将用于确定不同的APC是否在围产期诱导负选择。 RP 3与所有项目和核心有多个交叉点。我们将使用scRNA-seq来识别 在围产期至分娩期间，小鼠和人（具有核心B和C）中HAPC的分子特征改变， 可能影响中枢耐受性的幼年期转变。来自RP 1和RP 2的并行数据将提供 从围产期到幼年期TEC和胸腺基质细胞变化的综合图谱， 可以改变中枢耐受性。我们还将与Manley实验室（RP 2）合作进行MiCasa分析， 揭示胸腺环境的组织变化，在围产期到青少年的过渡。结果 在RP 3中生成的信息对于实现阐明机制的总体计划目标是不可或缺的， 胸腺基质特性改变围产期Treg和Tconv细胞的分化和选择， 这对设计合理的治疗策略以安全地增强T细胞输出具有重要意义。

ABSTRACT Project 3 The perinatal thymus plays a unique role in promoting central tolerance. Central tolerance results from negative selection of autoreactive conventional T cells (Tconv) and differentiation of regulatory T cells (Treg). Notably, Tregs selected during the perinatal period are more autoreactive and are uniquely required for life- long protection against autoimmunity. Perinatal Tconv cells are also more autoreactive and have unique molecular and functional properties compared with their juvenile/adult counterparts. Both negative selection and Treg induction occur when thymocytes encounter auto-antigens presented by medullary thymic epithelial cells (mTECs) or hematopoietic antigen presenting cells (HAPCs), including dendritic cells (DCs). TECs and thymic HAPCs undergo profound changes during the perinatal to juvenile transition. The rationale for RP3 is that thymic HAPCs are distinct in cellular composition and molecular profiles in the perinatal period, when thymic selection is critical for differentiation of the first wave of Tconv cells and establishment of self-tolerance. We will test the hypothesis that unique properties of mTECs and HAPCs in the perinatal period create an altered environment for central tolerance induction and are responsible for selection of Tconv and Treg cells with increased self-reactivity and age-specific functional properties. In Aim 1, we will use single cell transcriptional profiling (scRNA-seq) and multiplex imaging to identify the cellular, transcriptional, and organizational changes that occur in mouse and human HAPCs over the perinatal to juvenile transition which could impact central tolerance. In Aim 2, we will use phenotypic analyses, transcriptional profiling, genetic models, and functional assays to identify the APCs responsible for selecting tissue-protective Treg in the perinatal period. We will also determine whether CCR7-mediated cross-talk with TECs alters the DC compartment in perinatal mice, with downstream consequences for Treg selection. In Aim 3, we will determine if negative selection is impaired in perinates in response to antigens across a range of affinities. Live-cell 2- photon microscopy will be used to determine if distinct APCs induce negative selection in the perinatal period. RP3 has multiple points of intersection with all Projects and Cores. We will use scRNA-seq to identify altered molecular signatures of HAPCs in mice and humans (with Cores B and C) during the perinatal to juvenile transition that could impact central tolerance. Parallel data from RP1 and RP2 will provide a comprehensive map of changes in TECs and thymic stromal cells over the perinatal to juvenile transtition that could to alter central tolerance. We will also collaborate with the Manley lab (RP2) for MiCasa analysis to reveal organizational changes in the thymic environment during the perinatal to juvenile transition. Results generated in RP3 are integral to achieving the overall Program goals of elucidating mechanisms by which thymic stromal properties alter Treg and Tconv cell differentiation and selection in the perinatal period, with implications for devising rational therapeutic strategies to safely enhance T cell output.