Follicular helper T cells as drivers of epitope spreading

项目总结： 在一系列自身免疫性疾病中，自身抗体介导组织损伤、器官衰竭和临床衰退。 这些自身抗体的反应性可能会随着时间的推移而改变，导致不断演变的自身免疫后遗症， 称为表位扩散。表位扩散与疾病严重程度相关，可用于临床 诊断和预后，但自身抗体反应和表位扩散的驱动力仍然存在 不清楚。这些机制的特征可能为预防和治疗提供洞察力。 自身抗体介导的疾病的治疗。我们开发了一种表位在小鼠体内传播的模型，在 混合骨髓嵌合小鼠产生针对不同自身抗原的自身抗体。至 为了描述这种克隆进化的细胞机制，我进行了单细胞RNA 滤泡T细胞测序(scRNA-seq)。初步分析发现，卵泡辅助T细胞(TFH) 来自自身免疫嵌合体的细胞和卵泡调节性T(TFR)细胞表达增加的几个 长的非编码RNA(LncRNAs)。同时，我对滤泡T细胞进行了TCR测序，以提供 配对克隆型信息，揭示了丰富的自身免疫嵌合体以及差异的T细胞克隆 基因在个体克隆类型中的表达。这些初步发现表明，滤泡T细胞是 在B细胞驱动的自身免疫性疾病中，转录和克隆是不同的。我假设InncRNA 自身反应性TFH细胞的表达促进外周耐受性的丧失，作为一种必要和 自身反应性生发中心的表位扩散有足够的驱动力。这项提案旨在确定 LncRNA表达是否改变TFH细胞功能(目标1)，TFH细胞是否在B细胞驱动下发生自身反应 自身免疫性疾病(目标2)，以及这些TFH细胞是否负责表位扩散(目标3)。目标 1将测试lncRNAs是否可以通过逆转录病毒转导初级T细胞并鉴定其功能来改变Tfh功能 它们在体外和体内的功能。目标2将确定自身免疫相关的TFH克隆型的反应性 通过在体外表达这些TCR并利用MHC-TCR嵌合受体筛选其反应性。目标3将 确定TFH细胞通过生成混合骨促进表位扩散的必要性和充分性 缺乏TFH细胞的骨髓嵌合小鼠，并过继将TFH细胞转移到这些小鼠中并测量 自身抗体的产生。拟议的项目旨在提供对细胞和分子的洞察 表位扩散机制，同时可能揭示自身抗体的新治疗策略- 介导性疾病和其他生发中心功能障碍的疾病。

Project Summary: Autoantibodies mediate tissue damage, organ failure, and clinical decline in a range of autoimmune diseases. The reactivities of these autoantibodies may change over time leading to evolving autoimmune sequalae, termed epitope spreading. Epitope spreading correlates with disease severity and can be used for both clinical diagnosis and prognosis, but the driving forces of autoantibody responses and epitope spreading remain unclear. Characterization of these mechanisms might provide therapeutic insight to the prevention and treatment of autoantibody-mediated diseases. We have developed a model of epitope spreading in mice, in which mixed bone marrow chimeric mice develop autoantibodies to a diverse set of self-antigens. To characterize the cellular mechanisms contributing to this clonal evolution, I performed single cell RNA sequencing (scRNA-seq) of follicular T cells. Preliminary analyses have found that follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells from autoimmune chimeras expressed increased levels of several long non-coding RNAs (lncRNAs). In parallel, I have performed TCR sequencing of follicular T cells to provide paired clonotypic information, revealing T cell clones enriched in autoimmune chimeras as well as differential gene expression within individual clonotypes. These initial findings suggest that follicular T cells are transcriptionally and clonally distinct in B cell-driven autoimmune disease. I hypothesize that lncRNA expression in autoreactive Tfh cells promotes loss of peripheral tolerance, serving as a necessary and sufficient driver of epitope spreading in autoreactive germinal centers. This proposal seeks to determine whether lncRNA expression alters Tfh cell function (Aim 1), whether Tfh cells are autoreactive in B cell-driven autoimmune disease (Aim 2), and whether these Tfh cells are responsible for epitope spreading (Aim 3). Aim 1 will test if lncRNAs can alter Tfh functionality by retrovirally transducing primary T cells and characterizing their function in vitro and in vivo. Aim 2 will determine the reactivity of autoimmune-associated Tfh clonotypes by expressing these TCRs in vitro and screening their reactivity using MHC-TCR chimeric receptors. Aim 3 will determine the necessity and sufficiency of Tfh cells to promote epitope spreading by generating mixed bone marrow chimeric mice that lack Tfh cells, and adoptively transferring Tfh cells into these mice and measuring autoantibody production. The proposed project aims to provide insight into the cellular and molecular mechanisms of epitope spreading, while potentially revealing new therapeutic strategies for both autoantibody- mediated disease and other diseases of germinal center dysfunction.