Distinct contributions of CCR4 versus CCR7 to thymocyte localization and central tolerance

摘要 在大脑皮层经历阳性选择后，胸腺细胞迁移到中央位置的髓质 耐受性是通过抗原提呈细胞(APC)显示不同的自身抗原阵列来加强的。树枝状 树突状细胞(DC)和胸腺髓质上皮细胞(MTECs)是诱导 中心容忍。总的来说，mTEC表达大部分蛋白质组，但任何个体的自身抗原都是 只有1-3%的这些细胞表达。胸腺树突状细胞不仅存在自身来源的自身抗原 蛋白质组，也包括从血液、mTECs和外周组织获得的蛋白质组。因此，mTECs 树突状细胞显示出自身抗原的马赛克，胸腺细胞必须导航这些抗原才能扫描自身反应。这 过程是自我耐受的关键，因为胸腺中即使失去一个自我抗原的表达也会导致 外周自身免疫。在这项计划中，我们将研究趋化因子受体 CCR4和CCR7改变胸腺细胞的定位和与不同的APC的相互作用，以加强中枢耐受。 众所周知，CCR7促进胸腺细胞的髓质定位，因此，阴性 选择。在过去的筹资期间，我们还确定了CCR4在这些进程中的关键作用。我们的 数据提示CCR4促进阳性选择后胸腺细胞髓质进入的新模型 并与DC相互作用，而CCR7维持成熟胸腺细胞的髓质定位并促进 MTECs的相互作用，诱导不同TCR谱系的负选择和Treg分化。在目标1中， 我们将使用双光子显微镜、TCR图谱测序和TCR逆生骨的组合 骨髓嵌合体以测试CCR4和CCR7对胸腺细胞定位、与APC相互作用的影响，以及 中心容忍。值得注意的是，我们开发了一种新的双光子显微镜方法来量化 不同的APC对否定选择的贡献，并将扩展这一方法以确定 Treg选择。我们最近的数据还表明，负选择的早期和晚期都发生在 延髓，分别由CCR4和CCR7驱动。在目标2中，我们将测试这个新模型，它与 一种流行的观点，认为选择的早期和晚期分别发生在皮质和髓质。完毕 在上一个资助期，我们确定了胸腺树突状细胞CCR7表达在调节Treg中的新作用 选择。在目标3中，我们将使用现有的和新的遗传小鼠模型、功能分析、TCR谱系 测序和反基因骨髓嵌合体测试胸腺树突状细胞表达CCR7的假设 促进它们的存活，并需要获得和展示mTEC衍生的自身抗原，因此 影响曲目选择。总之，拟议的实验将阐明 CCR4和CCR7促进中央容忍，并将测试我们的数据建议的创新模式，即 中枢耐受分为两个阶段，首先是DC介导的不成熟基因的缺失 胸腺细胞，然后通过mTEC介导的成熟胸腺细胞的删除来确保自我耐受。

ABSTRACT After undergoing positive selection in the cortex, thymocytes migrate into the medulla where central tolerance is enforced by antigen presenting cells (APCs) displaying a diverse array of auto-antigens. Dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) are the two predominant medullary APCs that induce central tolerance. Collectively, mTECs express the majority of the proteome, but any individual self- antigen is expressed by only 1-3 % of these cells. Thymic DCs present self-antigens derived not only from their own proteome, but also those acquired from the blood, from mTECs, and from peripheral tissues. Thus, mTECs and DCs display a mosaic of self-antigens, which thymocytes must navigate to scan for auto-reactivity. This process is critical for self-tolerance, as lost expression of even one self-antigen in the thymus can result in peripheral autoimmunity. In this proposal, we will investigate mechanisms by which the chemokine receptors CCR4 and CCR7 alter thymocyte localization and interactions with distinct APCs to enforce central tolerance. It is well-documented that CCR7 promotes thymocyte medullary localization and thus, negative selection. Over the last funding period, we identified a critical role for CCR4 in these processes as well. Our data suggest a novel model in which CCR4 promotes medullary entry of post-positive selection thymocytes and interactions with DCs, while CCR7 sustains medullary localization of mature thymocytes and promotes interactions mTECs, to induce negative selection and Treg differentiation of distinct TCR repertoires. In Aim 1, we will use a combination of 2-photon microscopy, TCR repertoire sequencing, and TCR retrogenic bone marrow chimeras to test the impact of CCR4 and CCR7 on thymocyte localization, interactions with APCs, and central tolerance. Notably, we have developed a novel 2-photon microscopy approach to quantify the contribution of distinct APCs to negative selection, and will expand this approach to identify APCs required for Treg selection. Our recent data also suggest that both early and late stages of negative selection occur in the medulla, driven by CCR4 and CCR7, respectively. In Aim 2, we will test this novel model, which contrasts with the prevailing view that early and late stages of selection occur in the cortex and medulla, respectively. Over the last funding period, we identified a novel role for CCR7 expression by thymic DCs in regulating Treg selection. In Aim 3, we will use existing and novel genetic mouse models, functional assays, TCR repertoire sequencing, and retrogenic bone marrow chimeras to test the hypothesis that CCR7 expression by thymic DCs promotes their survival and is required for acquisition and display of mTEC-derived self-antigens, thus impacting repertoire selection. Altogether, the proposed experiments will elucidate mechanisms by which CCR4 and CCR7 promote central tolerance and will test the innovative model, suggested by our data, that central tolerance is separated into two stages, first dominated by DC-mediated deletion of less mature thymocytes, and then by mTEC-mediated deletion of mature thymocytes to ensure self- tolerance.