Intracellular and exosomal localization of the negative checkpoint regulator VISTA in immune cells

Strategies in immunotherapy often target the immunosuppressive environment of tumor cells. One route of therapeutic interference could involve negative checkpoint regulators of which V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA) has raised more interest recently. The protein is expressed on the surface of tumor cells, T-lymphocytes, and antigen-presenting cells (APCs), but its intracellular expression pattern has not been investigated yet. We examined the intracellular distribution of VISTA and its possible role in translocation processes by immunofluorescence and Western blots. We analyzed the expression and localization of VISTA in murine bone marrow-derived macrophages (BMDMs), human monocyte-derived macrophages, and human T lymphocytes (Jurkat). We obtained different cell fractions and organelles of various cell types and analyzed for the presence of VISTA. Monitoring a VISTA-GFP fusion construct in transfected cell lines HL-60 and THP-1 confirmed VISTA localization in these cell lines. All used cell lines showed the colocalization of VISTA and several vesicle markers together with VISTA staining along microtubule fibers. Additionally, we found VISTA in secreted exosomes and have the first hints for nucleic expression in all tested cell lines. Therefore, the storage of VISTA in vesicles and its potential presence in nuclei resembles two other well-described checkpoint regulators, CTLA-4 and PD-L1, respectively. We conclude that VISTA storage in vesicles enables a fast response to immunogenic stimuli, which needs to be considered for inhibitory experiments. The localization of VISTA in exosomes suggests a signaling function to facilitate cell-cell communication. Furthermore, the VISTA expression in the nucleus proposes a transcriptional role.