Irradiation Suppresses IFN gamma-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8(+) T Cells Cytotoxicity

Tumor cells express immune checkpoints to exhaust CD8(+) T cells. Irradiation damages tumor cells and augments tumor immunotherapy in clinical applications. However, the radiotherapy-mediated molecular mechanism affecting CD8(+) T cell activity remains elusive. We aimed to uncover the mechanism of radiotherapy augmenting cytotoxic CD8(+) T cells in non-small-cell lung cancer (NSCLC). EGFR-positive NSCLC cell lines were co-cultured with CD8(+) T cells from healthy volunteers. Tumor cell viability and apoptosis were consequently measured. IFN gamma was identified secreted by CD8(+) T cells and PBMCs. Therefore, RNAseq was used to screen the IFN gamma-mediated gene expression in A549 cells. The irradiation effect to IFN gamma-mediated gene expression was investigated using qPCR and western blots. We found that the co-culture of tumor cells stimulated the increase of granzyme B and IFN gamma in CD8(+) T, but A549 exhibited resistance against CD8(+) T cytotoxicity compared to HCC827. Irradiation inhibited A549 proliferation and enhanced apoptosis, augmenting PBMCs-mediated cytotoxicity against A549. We found that IFN gamma simultaneously increased phosphorylation on STAT1 and STAT3 in EGFR-positive lung cancer, resulting in overexpression of PD-L1 (p < 0.05). In RNAseq analysis, MCL1 was identified and increased by the IFN gamma-STAT3 axis (p < 0.05). We demonstrated that irradiation specifically inhibited phosphorylation on STAT1 and STAT3 in IFN gamma-treated A549, resulting in reductions of PD-L1 and MCL1 (both p < 0.05). Moreover, knockdowns of STAT3 and MCL1 increased the PBMCs-mediated anti-A549 effect. This study demonstrated that A549 expressed MCL1 to resist CD8(+) T cell-mediated tumor apoptosis. In addition, we found that irradiation suppressed IFN gamma-mediated STAT3 phosphorylation and PD-L1 and MCL1 expression, revealing a potential mechanism of radiotherapy augmenting immune surveillance.