New tricks for an old dog: Nuclear localization of PD-L1

Introduction: PD-L1 is a cell membrane immune checkpoint inducing immunesuppression, serving as target in cancer treatment. Recently we have identified immune-independent roles for PD-L1, promoting in cell-autonomous manners the pro-metastatic functions of human breast cancer (BC) cells, in vitro and in vivo. Also, we found by in-culture and animal studies that an intact S283 intracellular residue of PD-L1, PD-L1 N-linked glycosylation and activation of STAT1/STAT3 are required for such activities of PD-L1. Following emerging publications describing pro-malignancy roles for NUCLEAR PD-L1 (nPD-L1), we aim in this study to determine the patterns and regulation of PD-L1 nuclear localization in breast tumor cells, to be followed by studying the transcriptional roles of PD-L1, possibly leading to its cell-autonomous pro-metastatic activities in human BC cells. Methods and Results: Our current findings demonstrate that PD-L1 is localized in the nucleus of several subtypes of breast tumor cells. Confocal microscopy immunoflouresnce analyses revealed the localization of PD-L1 in the nucleus of three subtypes of BC cell lines, with differences in the nuclear pattern between them: A punctuate nuclear pattern of PD-L1 was noted in human TNBC MDA-MB-231 cells (MDA), whereas a dispersed nuclear localization was observed in the human luminal-A MCF-7 and T47D cells. Fractionation of cell lysates, followed by Western blotting (WB) confirmed PD-L1 presence in the nuclear fractions of MDA and MCF-7 cells. Nuclear localization of PD-L1 was also noted in N-linked glycosylation mutants of PD-L1, N35A and N219A. However, the N35A-mutated PD-L1 presented a different glycosylation pattern (based on molecular weight distribution) in the nucleus compared to its WB distribution layout in the entire cell. Blocking nuclear export led to PD-L1 enrichment in the nucleus and in parallel up-regulated the expression of membrane PD-L1. Down-regulation of STAT3 expression resulted in reduced PD-L1 levels in the nucleus. Conclusions: In breast tumor cells, PD-L1 is localized in the cell nucleus in a STAT3-meidated process. The actual localization of PD-L1 in the nucleus is not dependent on its glycosylation; however the modified pattern of N35A glycosylation in the nucleus may lead to its reduced ability to exert pro-metastatic functions in BC cells.