单细胞转录组数据揭示PHLDA1是卵巢癌T细胞耗竭的关键分子

Single cell sequencing data reveal PHLDA1 as a critical molecule responsible for T cell exhaustion in ovarian cancer

目的通过绘制高级别浆液性卵巢癌(High-grade serous ovarian cancer,HGSOC)单细胞转录组图谱,筛选并验证与CD8^(+)T细胞耗竭相关的关键基因。方法利用实验室前期的单细胞测序数据(SRA数据库:PRJNA756768),并整合数据库中5例HGSOC测序数据,分析肿瘤微环境中T细胞的具体亚型,拟时序分析探究T细胞亚群分化轨迹,差异基因富集确定免疫抑制的CD8^(+)IL-2^(Low)和CD8^(+)IFN-γ^(Low)细胞亚群及差异基因,再结合患者预后筛选出与CD8^(+)T细胞耗竭密切相关的候选分子。流式分析人PBMC中的T细胞激活到耗竭过程中Pleckstrin同源样结构域家族A成员1(Pleckstrin homology-like domain,family A,member 1,PHLDA1)在CD8^(+)T、CD4^(+)T及Treg细胞上的表达,ELISA检测PHLDA1 High和PHLDA1 Low的CD8^(+)T细胞分泌IFN-γ和IL-2的水平,最后流式数据分析PHLDA1与耗竭标志物PD-1、TIM-3的关联。结果将T细胞按三种方式分群:(1)IL-2 High和IL-2^(Low);(2)IFN-γ^(High)和IFN-γ^(Low);(3)耗竭和杀伤T细胞。随后取其差异基因的交集,最终筛选到关键基因PHLDA1。流式分析提示T细胞激活到耗竭的过程中,PHLDA1在CD8^(+)T、CD4^(+)T及Treg细胞上的表达持续升高;ELISA结果显示CD8^(+)PHLDA1^(High)的T细胞分泌IFN-γ和IL-2的水平显著低于CD8^(+)PHLDA1^(Low)T细胞。同时,CD8^(+)PHLDA1^(High)T细胞亚群可同时覆盖CD8^(+)TIM-3^(+)和CD8^(+)PD-1^(+)的耗竭T细胞类型。结论本研究基于单细胞测序数据筛选到PHLDA1是卵巢癌CD8^(+)T细胞耗竭的关键分子,该研究为卵巢癌的免疫治疗提供了新的思路。

Objective The critical genes associated with exhausted CD8^(+)T cells were screened and validated by mapping the single-cell transcriptome profile of high-grade serous ovarian cancer(HGSOC).Methods The specific subtypes of T cells in the tumor microenvironment were analyzed using the single-cell sequencing data from the early stage of laboratory(SRA database:PRJNA756768)and integrating 5 HGSOC sequencing from the database,and the differentiation trajectory of T cell subsets was explored through pseudotime analysis.Differential gene enrichment was used to determine immunosuppressed CD8^(+)IL-2 Low and CD8^(+)IFN-γ^(Low)T cell subsets and differential genes,and candidate molecules closely related to exhausted CD8^(+)T cells were screened based on patient prognosis.Flow cytometry was used to analyze the expression of PHLDA1 on CD8^(+)T cells,CD4^(+)T cells and Treg cells during the activation to exhaustion process of T cells in human PBMCs.ELISA was used to detect the levels of IFN-γand IL-2 secreted by CD8^(+)T cells in PHLDA1 High and PHLDA1 Low.Finally,flow cytometry was used to analyze the association between PHLDA1 and exhausted markers PD-1 and TIM-3.Results The results showed that T cells were grouped in three ways:(1)IL-2 High and IL-2 Low;(2)IFN-γ^(High)and IFN-γ^(Low);and(3)exhausted and cytotoxic CD8^(+)T cells.Subsequently,the intersection of its differentially expressed genes was taken,and the key gene PHLDA1 was ultimately screened.Flow cytometry analysis suggested that during the process of T cell activation to exhaustion,the expression of PHLDA1 continued to increase on CD8^(+)T cells,CD4^(+)T cells and Treg cells;The ELISA results showed that the levels of IFN-γand IL-2 secreted by CD8^(+)PHLDA1^(High)T cells were significantly lower than those of CD8^(+)PHLDA1 Low T cells.Meanwhile,the CD8^(+)PHLDA1^(High)T cell subset could simultaneously cover the exhausted T cell types of CD8^(+)TIM-3^(+)and CD8^(+)PD-1^(+).Conclusion Based on single-cell sequencing data,this study identified PHLDA1 as a key molecule responsible for CD8^(+)T cell exhaustion in OC,providing new insights for immunotherapy of OC.