背景:T细胞受体(TCR)信号传导和T细胞激活受到看门人的严格调节,以维持免疫耐受并避免自身免疫。TRAIL受体(TRAIL-R)是转导凋亡信号以诱导细胞死亡的TNF家族死亡受体。最近的研究表明,TRAIL-R通过直接抑制T细胞活化而不诱导细胞凋亡来调节T细胞介导的免疫应答;调节T细胞活化的独特信号通路仍不清楚.在这项研究中,我们筛选了T细胞内的细胞内TRAIL-R结合蛋白,以探索直接抑制T细胞活化的TRAIL-R转导的新信号通路.
方法:使用全转录组RNA测序来鉴定T细胞活化过程中与TRAIL-R信号相关的基因表达特征。使用质谱高通量筛选来鉴定T细胞内的新型TRAIL-R结合蛋白。免疫共沉淀,脂筏分离,进行共聚焦显微镜分析以验证TRAIL-R与T细胞内鉴定的结合蛋白之间的关联。
结果:TRAIL参与下调TCR信号通路中的基因特征,并在不诱导细胞死亡的情况下深刻抑制TCR近端酪氨酸激酶的磷酸化。酪氨酸磷酸酶SHP-1被鉴定为T细胞内主要的TRAIL-R结合蛋白,使用基于高通量质谱的蛋白质组学分析。此外,Lck在活化的T细胞中与TRAIL-R/SHP-1复合物共免疫沉淀。TRAIL参与极大地抑制了Lck(Y394)的磷酸化,并抑制了Lck在活化T细胞中脂质筏的募集,导致近端TCR信号的中断和随后的T细胞激活。
结论:TRAIL-R与磷酸酶SHP-1相关,并转导独特且独特的免疫看门人信号,通过失活Lck抑制TCR信号和T细胞活化。因此,我们的结果将TRAIL-R定义为一类新的抑制T细胞活化的免疫检查点受体,和TRAIL-R/SHP-1轴可以作为免疫介导的疾病的潜在治疗靶标。
BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation.
METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells.
RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation.
CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.