Abstract: 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^LowT 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-γ and 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-γ and IL-2 secreted by CD8⁺PHLDA1^HighT cells were significantly lower than those of CD8⁺ PHLDA1^LowT cells.Meanwhile,the CD8⁺PHLDA1^HighT 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.

Key words: Single cell sequencing, Ovarian cancer, Pleckstrin homology-like domain,family A,member 1, T cell exhaustion