Effect of targeting DDR pathway on the late-phase autophagy and the killing effect of CD8+ T cells.

e14646 Background: Deficiency in DDR signaling pathways has been reported to be associated with better response to immunotherapy. This study aims to investigate whether inhibition of the key mediator of DDR pathways ATR could enhance the function of CD8+ T cells and its underlying mechanism. Methods: The CD8+ T cell-killing experiment was conducted in vitro to demonstrate the efficiency in the presence of ATR inhibitors. To investigate the regulatory mechanism, RNA Sequencing analysis was performed to identify gene changes after ATR inhibition. Subsequently, Western blotting and immunofluorescence assays were performed to access the autophagy flux based on the results of the RNA sequencing analysis. Flow cytometry was used to detect the surface MHC-I expression to validate our speculation with the change of autophagy activity. Results: ATR inhibitor (VX970) had been shown to enhance the cytotoxic effect of CD8+ T cells on tumor cells. RNA-seq analysis revealed the enrichment of pathways related to autophagy and its regulation, as well as inflammatory factor pathways. Accumulation of p62/SQSTM1 and an increased LC3II/I ratio had been found, indicating the involvement of autophagy after the inhibition of ATR. No further increase in autophagy proteins was observed upon treatment with autophagy inhibitors, suggesting the late stage of autophagy inhibition was affected. In addition, increased MHC-I molecular expression has been found in accordance with autophagy inhibition, which could further explain the reason ATR inhibition could induce the accumulation of CD8+ T cells. Conclusions: Targeting DDR pathway leads to stimulated CD8+ T cells accumulation probably by increased MHC-I surface expression through inhibition of late-phase autophagy, which may in turn enhance the efficiency of immunotherapy.