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

摘要项目3 围产期胸腺在促进中枢耐受方面起着独特的作用。中心公差产生于 自身反应性常规T细胞(Tconv)和调节性T细胞(Treg)的阴性选择。 值得注意的是，在围产期选择的树突状细胞更具自身反应性，是生命所独有的。 对自身免疫的长期保护。围产期的Tconv细胞也有更多的自身反应，并具有独特的 将其分子和功能特性与其幼体/成体进行比较。两者都是负选择 当胸腺细胞遇到髓质胸腺上皮细胞递呈的自身抗原时，Treg诱导发生 细胞(MTECs)或造血抗原提呈细胞(HAPC)，包括树突状细胞(DC)。TECS和 胸腺HAPC在围产期到幼年期的转变过程中发生了深刻的变化。RP3的基本原理是 胸腺HAPC在围产期的细胞组成和分子图谱上是不同的，当 胸腺的选择对于第一波Tconv细胞的分化和自我耐受的建立至关重要。 我们将检验这一假设，即围产期mTECs和HAPC的独特属性创建了一个 中枢耐受诱导的环境改变，并负责选择Tconv和Treg细胞 具有更强的自我反应性和特定年龄的功能特性。在目标1中，我们将使用单细胞 转录图谱(scRNA-seq)和多重成像以识别细胞、转录和 小鼠和人类HAPC在围产期到幼年期的转变过程中发生的组织变化 可能会影响中枢耐受性。在目标2中，我们将使用表型分析、转录图谱、遗传 模型和功能分析，以确定负责选择组织保护性Treg的APC 围产期。我们还将确定CCR7与TEC之间的串扰是否会改变DC 围产期小鼠体内的隔室，对Treg的选择有下游影响。在目标3中，我们将确定 如果围产儿对一系列亲缘关系中的抗原的负面选择受到损害。活细胞2- 光子显微镜将被用来确定不同的APC是否会在围产期诱导负选择。 RP3与所有项目和核心都有多个交叉点。我们将使用scRNA-seq来鉴定 小鼠和人类(核心B和C)在围产期HAPC分子特征的改变 可能影响中枢耐受性的青少年过渡。来自RP1和RP2的并行数据将提供 TECs和胸腺基质细胞在围产期到幼年移植过程中变化的综合图谱 可以改变中枢耐受性。我们还将与曼利实验室(RP2)合作进行Micasa分析，以 揭示围产期向青少年过渡期间胸腺环境的组织变化。结果 在RP3中生成的是实现阐明机制的总体计划目标所不可或缺的，通过这些机制 胸腺基质特性改变了围产期Treg和Tconv细胞的分化和选择 对设计合理的治疗策略以安全地提高T细胞产量的启示。

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.