In vivo development and reactivity of human autoreactive T cells

摘要 在胸腺生成过程中，自身抗原识别不足可导致异常高频率的 自身反应性T细胞，当与免疫缺陷机制结合时，可导致自身免疫性疾病。 易感个体的外周耐受性。人类自身反应性T细胞的胸腺选择过程 由于缺乏体内模型，在很大程度上还不了解，迄今为止只能从啮齿动物研究中推断。 对人胸腺中自身抗原表达的分析提供了一个不完整的图像， 这表明这并不一定与T细胞缺失相关。由于自身反应性T细胞也被发现 在"健康"个体中，不完全胸腺缺失（或调节性T细胞）的程度尚不清楚， （Treg）选择）有助于自身免疫。 我们提出的研究是基于人源化小鼠重建与胎儿造血干细胞（HSC） 和胸腺组织，由此可以转导HSC以表达自身反应性T细胞受体（TCR）和/或 这个TCR所识别的实际表位。然后，HSC在正常人胸腺中发育成T细胞。 体内，其中一部分显示自身反应性TCR。在建立了具有特异性T细胞的模型后， 黑素细胞自身抗原不能经历胸腺缺失，除非该表位也被引入HSC，我们 将使该系统适用于在1型糖尿病胰岛浸润中鉴定的TCR集合 患者选择这些TCR是因为还不知道它们的特异性表位是否被呈递 并在正常人胸腺中被识别因此，该模型将有助于确定T细胞是否携带那些 TCR通过将被测试的许多机制（缺乏表达、交叉表达、免疫抑制、免疫抑制）逃避胸腺缺失。 反应性或新表位形成）。在HSC中引入同源表位将使我们能够评估它们的表位。 对发展自身反应性T细胞的影响（TcR的缺失与阳性选择），并进一步探讨 抗原呈递细胞（APC）的特定亚群在这一过程中的独特贡献，包括它们的能力， 从血液中转移到胸腺来影响胸腺的选择。使用能够 为了将抗原呈递给人类T细胞和两个物种之间交叉反应的抗原，我们将 评估激活自身反应性T细胞攻击靶组织所需的条件。 这项工作将采用免疫荧光成像，多参数流式细胞术和单细胞RNA 测序分析，以分析人APC亚群，所述人APC亚群分布在胸腺和外周淋巴组织中 并参与人类自身反应性T细胞，并评估这些自身反应性T细胞的表型和分子特征， T细胞基于不同的抗原接触条件（胸腺与外周、APC类型等）。总体而言，这些 研究将为人类自身反应性T细胞的产生提供有价值的见解， 疾病，它们对体内自身抗原的反应性，并将促进新的 靶向自身反应性T细胞的缺失或耐受的治疗方法。

Abstract Inadequate recognition of autoantigens during thymopoiesis can result in abnormally high frequency of autoreactive T cells, which can lead to autoimmune diseases when combined with defective mechanisms of peripheral tolerance in susceptible individuals. The process of thymic selection of autoreactive T cells in humans is largely not understood due to lack of in vivo models and could so far only be extrapolated from rodent studies. Analysis of autoantigen expression in the human thymus has provided an incomplete picture based on increasing evidence that this does not necessarily correlate with T cell deletion. As autoreactive T cells are also found circulating in `healthy' individuals, it is unclear to what extent incomplete thymic deletion (or regulatory T cell (Treg) selection) contributes to autoimmunity. Our proposed research is based on humanized mice reconstituted with fetal hematopoietic stem cells (HSCs) and thymic tissue, whereby HSCs can be transduced to express an autoreactive T cell receptor (TCR) and/or the actual epitope recognized by this TCR. The HSCs then develop into T cells in a normal human thymus in vivo, a fraction of which display the autoreactive TCR. Having established the model with T cells specific for a melanocyte autoantigen that fail to undergo thymic deletion unless the epitope is also introduced in HSCs, we will adapt this system to a collection of TCRs that have been identified in islet infiltrates of Type 1 diabetes patients. These TCRs were selected because it is yet unknown whether their specific epitopes are presented and recognized in a normal human thymus. Thus the model will help determine whether T cells bearing those TCRs escape thymic deletion through a number of mechanisms that will be tested (lack of expression, of cross- reactivity or of neoepitope formation). Introduction of the cognate epitopes in HSCs will allow us to evaluate their effect on developing autoreactive T cells (deletion versus positive selection of Tregs), and further explore the unique contribution of specific subsets of antigen-presenting cells (APCs) in this process, including their ability to relocate to the thymus from the bloodstream to influence thymic selection. Using recipient mice that are able to present antigens to human T cells and antigens that are cross-reactive between the two species, we will assess the conditions that are needed to activate autoreactive T cells to attack their target tissue. This work will feature immunofluorescence imaging, multi-parametric flow cytometry and single-cell RNA sequencing analysis to analyze human APC subsets that populate the thymus and peripheral lymphoid tissues and engage human autoreactive T cells, and to assess the phenotype and molecular profile of these autoreactive T cells based on different conditions of antigen encounter (thymus vs periphery, type of APC, etc). Overall, these studies will provide valuable insights into the generation of human autoreactive T cells causing autoimmune diseases, their reactivity to autoantigens in vivo, and will facilitate the development and evaluation of new therapeutic approaches targeting autoreactive T cells for deletion or tolerance.