P-01.14 Intracellular and extracellular biochemical functions of V-domain Ig-containing suppressor of T cell activation are regulated by crucial factors of tumour microenvironment

<h3>Background</h3> V-domain Ig-containing suppressor of T cell activation (VISTA) is a unique multifunctional protein displaying suppressive activities towards anti-cancer immunity. VISTA shows both receptor and ligand activities.^{1 2} However, the mechanisms of regulation of VISTA activity and functions by factors of tumour microenvironment (TME) remain unclear and understanding these processes is required to develop successful personalised cancer immunotherapeutic strategies and approaches. <h3>Materials and Methods</h3> We used human cancer and non-cancerous human cell lines including LN-18 glioblastoma cells, Calu6 lung cancer cells, PANC-1 pancreatic ductal adenocarcinoma cells, HuH7 hepatocellular carcinoma cells, BEAS-2B bronchial epithelial cells, THP-1 human acute myeloid leukaemia monocytes, Jurkat T cells, TALL-104 cytotoxic T cells and primary human CD3-positive T cells. We also used primary human lung, colorectal and breast cancer samples. Western blot analysis, ELISA set-ups, flow cytometry, on-cell Western analysis, fluorescent microscopy, quantitative RT-PCR, a wide range of biochemical assays and synchrotron radiation circular dichroism spectroscopy were used to conduct the studies. <h3>Results</h3> In this work we report for the very first time that VISTA engages in intracellular interaction with galectin-9 facilitating its interaction with TGF-β-activated kinase 1 (TAK1). This process is required for lysosome protection. TGF-β in various cancer cell types can induce expression of VISTA. While in a number of cancer and healthy cell types TGF-β downregulates VISTA expression. TGF-β-induced upregulation of VISTA expression is controlled via Smad3 pathway. While downregulation is triggered by TGF-β-induced TAK1 activation. TAK1 downstream pathway activates transcription factor ATF-2 which interacts with promoter region of VSIR (VISTA gene) repressing its expression. We found that VISTA expression can be induced by two crucial factors of tumour microenvironment - low oxygen availability (via activation of hypoxia-inducible factor 1 (HIF-1) transcription complex) and TGF-β. ATF-2 can repress both TGF-β and HIF-1-induced activation of VISTA expression. Downregulatory effects of TAK1/ATF-2 pathway are observed mainly in the cells where VISTA is located inside the cell, so that galectin-9 can be released and complexed with Tim-3 for secretion aiming to suppress cytotoxic immune attacks. If cells translocate VISTA onto their surface TGF-β-induced TAK1 does not take place. As such, VISTA expression is induced by TGF-β in these cases. On the cell surface VISTA plays a major role as immune checkpoint participating in cancer immune evasion machinery and suppressing T cell functions. <h3>Conclusions</h3> This work highlighted that some cancers (e. g. breast carcinoma, pancreatic ductal adenocarcinoma, pulmonary carcinoma) VISTA plays the role of immune checkpoint protein, while in other cancers (e. g. hepatocellular carcinoma, various colorectal cancers VISTA is mainly engaged in intracellular biochemical functions associated). These results indicate importance of understanding of differential functions of VISTA for development of personalised immunotherapies of cancer. <h3>References</h3> Schlichtner S, Yasinska IM, <i>et al. J Immunother Cancer</i> 2023;<b>11</b>:e005714. Schlichtner S, Yasinska IM, <i>et al. OncoImmunology</i> 2023;<b>12</b>:2244330. M. Abooali: None. S. Schlichtner: None. I. Yasinska: None. X. Lei: None. E. Fasler-Kan: None. V.V. Sumbayev: None.