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.

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.

Mechanical Stress Induce PG-E2 in Murine Synovial Fibroblasts Originating from the Temporomandibular Joint.

Genetic predisposition, traumatic events, or excessive mechanical exposure provoke arthritic changes in the temporomandibular joint (TMJ). We analysed the impact of mechanical stress that might be involved in the development and progression of TMJ osteoarthritis (OA) on murine synovial fibroblasts (SFs) of temporomandibular origin. SFs were subjected to different protocols of mechanical stress, either to a high-frequency tensile strain for 4 h or to a tensile strain of varying magnitude for 48 h. The TMJ OA induction was evaluated based on the gene and protein secretion of inflammatory factors (Icam-1, Cxcl-1, Cxcl-2, Il-1ß, Il-1ra, Il-6, Ptgs-2, PG-E2), subchondral bone remodelling (Rankl, Opg), and extracellular matrix components (Col1a2, Has-1, collagen and hyaluronic acid deposition) using RT-qPCR, ELISA, and HPLC. A short high-frequency tensile strain had only minor effects on inflammatory factors and no effects on the subchondral bone remodelling induction or matrix constituent production. A prolonged tensile strain of moderate and advanced magnitude increased the expression of inflammatory factors. An advanced tensile strain enhanced the Ptgs-2 and PG-E2 expression, while the expression of further inflammatory factors were decreased. The tensile strain protocols had no effects on the RANKL/OPG expression, while the advanced tensile strain significantly reduced the deposition of matrix constituent contents of collagen and hyaluronic acid. The data indicates that the application of prolonged advanced mechanical stress on SFs promote PG-E2 protein secretion, while the deposition of extracellular matrix components is decreased.