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

摘要 在皮质中经历阳性选择后，胸腺细胞迁移到髓质中， 耐受性通过展示多种自身抗原的抗原呈递细胞（APC）来增强。树突状 细胞（DC）和胸腺髓质上皮细胞（mTEC）是诱导免疫应答的两种主要的髓质APC。 中央宽容总的来说，mTEC表达大部分蛋白质组，但任何个体的自身抗原都是 仅由这些细胞的1- 3%表达。胸腺DC呈递自身抗原， 蛋白质组，但也从血液中获得的，从mTEC，和从外周组织。因此，mTEC DC显示自身抗原的镶嵌，胸腺细胞必须导航以扫描自身反应性。这 这一过程对于自身耐受性至关重要，因为胸腺中即使一种自身抗原的表达缺失也会导致 外周自身免疫在这个提议中，我们将研究趋化因子受体 CCR 4和CCR 7改变胸腺细胞定位和与不同APC的相互作用以增强中枢耐受性。 有充分证据表明，CCR 7促进胸腺细胞髓质定位，因此，CCR 7对胸腺细胞髓质定位呈阴性。 选择.在上一个资助期间，我们也确定了CCR 4在这些过程中的关键作用。我们 数据提示了一种新的模型，其中CCR 4促进阳性选择后胸腺细胞的髓质进入 和与DC的相互作用，而CCR 7维持成熟胸腺细胞的髓质定位，并促进 通过与mTEC相互作用，以诱导不同TCR库的负选择和Treg分化。在目标1中， 我们将结合使用双光子显微镜、TCR库测序和TCR逆转录骨 骨髓嵌合体，以测试CCR 4和CCR 7对胸腺细胞定位、与APC的相互作用以及 中央宽容值得注意的是，我们已经开发了一种新的双光子显微镜方法来量化 不同的APC对阴性选择的贡献，并将扩大这种方法，以确定所需的APC 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 髓质，分别由CCR 4和CCR 7驱动。在目标2中，我们将测试这个新模型，它与 普遍的观点认为，选择的早期和晚期阶段分别发生在皮质和髓质。超过 在上一个资助期，我们确定了胸腺DCs表达CCR 7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，功能测定，TCR库， 测序和逆转录骨髓嵌合体，以检验胸腺DCs的CCR 7表达 促进它们的存活，并且是获得和展示mTEC衍生的自身抗原所必需的，因此 影响库选择。总之，拟议的实验将阐明机制， CCR 4和CCR 7促进中枢耐受性，并将测试我们的数据所建议的创新模型， 中心耐受分为两个阶段，第一阶段由DC介导的较不成熟的缺失主导， 然后通过mTEC介导的成熟胸腺细胞的缺失来确保自身耐受性。

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.