PurposeThe study aimed to determine the expression of VISTA and TOX within venous tumor thrombus and primary clear cell renal cell carcinoma (ccRCC) and to assess their prognostic value.MethodsThe study enrolled 82 patients with ccRCC and coexisting venous tumor thrombus treated radically from 2012 to 2019 in two tertiary centers. Tissue microarrays were prepared and stained with respective antibodies. The expression of markers was assessed separately on tumor cells (TCs) and/or tumor-associated immune cells (TAICs).ResultsTOX expression was positively correlated with the percentage of VISTA-positive TAICs in venous thrombus (p= 0.011), but not in the primary tumor (p= 0.674). High TOX expression was associated with a higher percentage of PD-L1-positive TAICs in both compartments (p= 0.001,p= 0.011, respectively). Positive expression of VISTA on TAICs was associated with PD-L1 expression on TCs (p= 0.005) and TAICs (p= 0.004) in the primary tumor, and only with PD-L1 on TAICs in thrombus (p= 0.006). The presence of VISTA-positive TAICs in venous thrombus was significantly more common in females (p= 0.034), and positively correlated with metastases (p= 0.028), and tumor necrosis (p= 0.013). The cases with VISTA-positive TAICs in venous tumor thrombi had significantly shorter OS than VISTA-negative cases (p= 0.041).ConclusionFor the first time, we demonstrated the expression of VISTA- and TOX-positive TAICs in the venous tumor thrombus. We found the association between immune checkpoint receptors andTcell exhaustion markers in both tumor mass and venous thrombus. Finally, we demonstrated that abundance of VISTA-positive TAICs in venous tumor thrombus correlates with worse outcomes in ccRCC.