In vivo development and reactivity of human autoreactive T cells

摘要 在胸腺生成过程中对自身抗原的识别不足会导致异常高频率的 自身反应性T细胞，可导致自身免疫性疾病，当与缺陷的机制 易感人群的外周耐受性。人类胸腺选择自身反应性T细胞的过程 由于缺乏活体模型，很大程度上还不清楚，到目前为止，只能从啮齿动物的研究中推断出来。 对人类胸腺中自身抗原表达的分析提供了一个不完整的图景，其基础是 证据表明，这并不一定与T细胞缺失相关。因为还发现了自身反应性T细胞 目前尚不清楚胸腺缺失(或调节性T细胞)在多大程度上不完全缺失 (Treg)选择)有助于自身免疫。 我们建议的研究是基于用胎儿造血干细胞(HSCs)重组的人源化小鼠 和胸腺组织，借此HSCs可被转导以表达自身反应性T细胞受体(TCR)和/或 该TCR识别的实际表位。然后，HSCs在正常人的胸腺中发育成T细胞 Vivo，其中一小部分显示了自身反应的TCR。在建立了针对A细胞的T细胞模型后 黑素细胞自身抗原无法进行胸腺删除，除非表位也被引入HSCs，我们说 将使这个系统适用于在1型糖尿病胰岛浸润液中发现的一组TCR 病人。之所以选择这些TCR，是因为尚不清楚是否呈现了它们的特定表位 并在正常人的胸腺中被识别。因此，该模型将有助于确定T细胞是否携带这些 TCR通过多种机制逃脱胸腺缺失，这些机制将被测试(缺乏表达，交叉- 新表位形成的反应性)。在造血干细胞中引入同源表位将使我们能够评估它们的 对自身反应性T细胞发育的影响(Tregs缺失与阳性选择)，并进一步探讨 特定的抗原提呈细胞亚群(APC)在这一过程中的独特贡献，包括它们的能力 从血液重新定位到胸腺，以影响胸腺的选择。使用的受体小鼠能够 为了将抗原呈现给人类T细胞，以及在两个物种之间发生交叉反应的抗原，我们将 评估激活自身反应性T细胞攻击其靶组织所需的条件。 这项工作将以免疫荧光成像、多参数流式细胞术和单细胞RNA为特色 用于分析胸腺和外周淋巴组织中的人APC亚群的测序分析 并激发人类自身反应性T细胞，并评估这些自身反应性T细胞的表型和分子图谱 T细胞基于不同的抗原相遇条件(胸腺与外周、APC类型等)。总的来说，这些 研究将为人类自身反应性T细胞的产生引起自身免疫提供有价值的见解 疾病，它们对体内自身抗原的反应性，并将促进新的开发和评估 针对自身反应性T细胞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.