Felce James H, Parolini Lucia, Sezgin Erdinc, Céspedes Pablo F, Korobchevskaya Kseniya, Jones Mathew, Peng Yanchun, Dong Tao, Fritzsche Marco, Aarts Dirk, Frater John, Dustin Michael L
Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom.
Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Front Cell Dev Biol. 2021 Jan 18;8:608484. doi: 10.3389/fcell.2020.608484. eCollection 2020.
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体（pMHC）相互作用来激活T细胞。TCR-pMHC结合触发了两个细胞之间广泛接触的形成，即免疫突触，它作为一个整合多种信号的平台，这些信号决定了细胞的结果，包括来自多个共刺激/抑制受体的信号。促成这一过程的因素包括一些趋化因子受体，特别是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。尽管趋化因子受体最典型的作用是作为配体依赖性趋化作用的介质，但一些趋化因子受体也会被招募到突触中，并在没有配体结合的情况下参与信号传导。这些以及其他GPCR如何整合到突触的动态结构中尚不清楚，它们通常迁移的Gαi偶联信号在被招募后如何终止也不清楚。在这里，我们使用标准和超分辨率荧光显微镜，报道了几种GPCR的时空组织，重点是CXCR4，以及原代人CD4 T细胞在支持脂质双分子层上的突触内的G蛋白Gαi2。我们发现CXCR4经历了精心编排的重组阶段，最终被招募到富含TCR的中心。这似乎依赖于CXCR4的泛素化，并且在纳米尺度上观察到，不涉及与TCR微簇的稳定相互作用。通过突变破坏这一过程会损害CXCR4对细胞激活的作用。Gαi2被主动排除在突触之外，与在中心积累的CXCR4分离。使用CRISPR-Cas9基因敲除筛选，我们鉴定出几种对T细胞激活有作用的不同GPCR，最显著的是鞘氨醇-1-磷酸受体S1PR1和氧化甾醇受体GPR183。这些以及其他GPCR经历了与CXCR4类似的组织过程；包括最初的排除、向心运输以及缺乏受体-TCR相互作用。这些构成了对突触内GPCR动态变化的首次观察，并深入了解了这些受体如何可能对T细胞激活起作用。对广泛的GPCR对T细胞激活的作用的观察也开启了一种可能性，即根据细胞状态或环境调节GPCR表达可能直接调节对pMHC的反应性。
