A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4⁺ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

免疫的一个核心过程是通过 T 细胞受体 (TCR) 与抗原呈递细胞 (APC) 表面的激动性肽-主要组织相容性复合物 (pMHC) 的相互作用来激活 T 细胞。TCR-pMHC 结合触发了称为免疫突触的两个细胞之间广泛接触的形成，该突触作为整合确定细胞结果的多种信号的平台，包括来自多种共刺激/抑制受体的信号。对此的贡献者包括许多趋化因子受体，尤其是 CXC-趋化因子受体 4 (CXCR4)，以及 G 蛋白偶联受体 (GPCR) 家族的其他成员。尽管最能表征为配体依赖性趋化性的介质，但一些趋化因子受体也被募集到突触并在没有连接的情况下有助于信号传导。这些和其他 GPCRs 如何整合到突触的动态结构中是未知的，它们通常迁移的 Gαi 耦合信号如何在招募时终止。在这里，我们报告了几个 GPCR 的时空组织，重点是 CXCR4，以及初级人类 CD4 突触内的 G 蛋白 Gαi2⁺支持的脂质双层上的 T 细胞，使用标准和超分辨率荧光显微镜。我们发现 CXCR4 经历了精心策划的重组阶段，最终招募到 TCR 丰富的中心。从纳米尺度来看，这似乎依赖于 CXCR4 泛素化，并且不涉及与 TCR 微团簇的稳定相互作用。突变对该过程的破坏会损害 CXCR4 对细胞活化的贡献。Gαi2 从突触中主动排除，从中央积累的 CXCR4 中分离出来。使用 CRISPR-Cas9 敲除筛选，我们确定了几种对 T 细胞活化有贡献的不同 GPCR，最显着的是 1-磷酸鞘氨醇受体 S1PR1 和氧甾醇受体 GPR183。这些和其他 GPCR 的组织结构类似于 CXCR4；包括初始排除，向心运输，缺乏受体-TCR相互作用。这些构成了对突触内 GPCR 动力学的首次观察，并深入了解了这些受体如何有助于 T 细胞活化。观察到广泛的 GPCR 对 T 细胞活化的贡献也开启了响应细胞状态或环境调节 GPCR 表达可能直接调节对 pMHC 的反应性的可能性。