Single cell sequencing data reveal PHLDA1 as a critical molecule responsible for T cell exhaustion in ovarian cancer
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 ex-plored through pseudotime analysis.Differential gene enrichment was used to determine immunosuppressed CD8+IL-2Low and CD8+IFN-γLow 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 dur-ing the activation to exhaustion process of T cells in human PBMCs.ELISA was used to detect the levels of IFN-γ and IL-2 secreted by CD8+T cells in PHLDA1High and PHLDA1Low.Finally,flow cytometry was used to analyze the association between PHLDA1 and ex-hausted markers PD-1 and TIM-3.Results The results showed that T cells were grouped in three ways:(1)IL-2High and IL-2Low;(2)IFN-γHigh and IFN-γLow;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-γ and IL-2 secreted by CD8+PHLDA1High T cells were significantly lower than those of CD8+PHLDA1Low T cells.Meanwhile,the CD8+PHLDA1High 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 responsi-ble for CD8+T cell exhaustion in OC,providing new insights for immunotherapy of OC.
Single cell sequencingOvarian cancerPleckstrin homology-like domain,family A,member 1T cell exhaustion
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