Subcellular localization of PD‐L1 and cell‐cycle‐dependent expression of nuclear PD‐L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy

Abstract

The programmed cell death 1 ligand 1 (PD‐L1)/programmed cell death protein 1 (PD‐1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD‐L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD‐L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD‐L1 in various cellular compartments of six well‐characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD‐L1 in its well‐known glycosylated/deglycosylated state at 40–55 kDa. In addition, we detected previously unknown PD‐L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD‐L1 variant expression is cell‐cycle‐dependent. Immunofluorescence staining of PD‐L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD‐L1 trafficking remain less understood; however, proximity ligation assays showed a cell‐cycle‐dependent interaction of the cytoskeletal protein vimentin with PD‐L1, whereas vimentin could serve as a potential shuttle for nuclear PD‐L1 transportation. Mass spectrometry after PD‐L1 co‐immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD‐L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD‐L1 and multiple tumor cell‐intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

The immunomodulatory ballet of tumour‐derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD‐L1 pathway in cancer

“…In our investigation, subcellular protein fractionation of SCC47 unveiled the presence of PD-L1 in diverse cellular compartments, including the nucleus. Notably, PD-L1 was identified not only in its well-documented glycosylated/deglycosylated states (40-55 kDa) but also in a novel nuclear PD-L1 variant, exhibiting a higher molecular weight of approximately 70 kDa and >150 kDa, as recently reported by Schulz et al (2024). These findings were similarly observed in six thoroughly characterized HNC cell lines, including SCC9, FaDu, and PCI52.…”

“…Since the highly specific clone 28‐8 yielded the strongest mPD‐L1 and cPD‐L1 detection in this comparison, we decided to perform IHC staining in Figure 2e with clone 28‐8. Notably, the localization tends to differ in relation to the tumour's aggressiveness, with a more internal localization observed in highly malignant tumours (Schulz et al., 2024 ). This intriguing finding prompted an investigation into the potential correlation between the subcellular localization of PD‐L1 and the NLR.…”