Investigation of VAMP-3 as a potential target of PD-1-mediated T cell inhibition

T cells are crucial for anticancer immunosurveillance. Engagement of the inhibitory receptor PD-1 on T cells by its ligand, PD-L1, expressed on tumor cells, contributes to suppression of tumor infiltrating lymphocytes (TILs) and immune escape of cancer cells. The PD-1/PD-L1 axis has thus been targeted with specific monoclonal antibodies, yielding significant clinical efficacy in several tumors. Still, these therapies benefit only a subset of patients that is difficult to identify, they can cause immune-related adverse events, and resistance development is observed. Gaining additional insights into biochemical events triggered by PD-1 could help to overcome these issues. Previous work of the host laboratory had identified VAMP-3 as a protein whose phosphorylation was significantly decreased upon PD-1 engagement. VAMP-3 is a vesicle-associated SNARE protein that mediates nearing and fusion of vesicles with the plasma membrane. In T cells, VAMP-3 is crucial for polarized recycling of the T-cell receptor (TCR) to the immune synapse (IS), an essential process for continuous supply of TCRs at this site and, therefore, for T-cell activation. We hypothesized that, by regulating VAMP-3 phosphorylation PD-1 could inhibit TCR recycling, IS formation and, ultimately, the T-cell immune response. We started to test this hypothesis by confirming PD-1-mediated reduction of VAMP-3 phosphorylation in primary T cells. Then, we generated Jurkat T-cell lines stably overexpressing either a wildtype (WT) or a phosphorylation-deficient mutant of a fluorescent GFP-VAMP-3 fusion protein. Using these models, we observed that PD-1 signaling did not prevent recruitment of GFP-VAMP-3 WT or its mutant to the IS, nor did it alter VAMP-3 colocalization with the CD3ζ cargo. Transcriptional and cytokine responses were also comparable in these cells under stimulatory and inhibitory conditions. Finally, we observed a PD-1-mediated reduction in the stimulation-induced downmodulation of TCRs from the T-cell surface, but the effect was again comparable in cells expressing WT or mutant GFP-VAMP-3. In conclusion, our findings do not support a role for PD-1-mediated VAMP3 dephosphorylation in IS formation and T-cell activation. However, our experiments were performed in Jurkat cells overexpressing WT or mutant GFP-VAMP3 and should be repeated upon silencing of endogenous VAMP-3, which might mask possible effects of the VAMP3 mutant. Further experiments are, thus, needed to assess whether modulation of VAMP-3 phosphorylation might provide a new mode for targeting the PD-1 pathway or allow the development of a novel predictive biomarker for better patient stratification.