Background Despite the transformative impact of current immune checkpoint inhibitors (ICIs) targeting the PD-1 and CTLA-4 pathways on the management of advanced renal cell carcinoma (RCC), many patients still do not receive long-term benefit and often suffer from adverse events. Therefore, we aim to validate the efficacy of novel immunomodulatory therapies by focusing on the functional assessment of T cells and inhibiting the T-cell immunoreceptor with Ig and ITIM domains (TIGIT). In this study, we established an antigen-specific killing assay to directly evaluate human T-cell cytotoxic effects on target RCC cells. Furthermore, we assessed the change in the cytotoxic activity of exhausted T cells using an anti-TIGIT antibody for the first time in the RCC area.

Methods T cells were isolated from healthy donor peripheral blood. To assess the cytotoxicity of T cells, we designed an antigen-specific killing system utilizing NY-ESO-1 (CTAG1B) as a model antigen. Human RCC cell lines A498 (HLA-A*02:01+), UOK127 (A*02:01+), and 769P (A*02:01−) were used as target cells. The expression of the TIGIT ligands PVR and CD112 were confirmed on these tumor cells. CTAG1B (encoding NY-ESO-1) and luciferase genes were introduced into these cell lines, and T cells were engineered to express the NY-ESO-1 T cell receptor restricted by HLA-A*02:01 (NY-ESO-1 T cells). Genetically-engineered RCC cell lines and T cells were co-cultured at varying ratios of effector and target cells. To investigate the efficacy of TIGIT pathway blockade, TIGIT gene was introduced into the effector T cells (NY-ESO-1-TIGIT T cells) and tiragolumab (anti-TIGIT monoclonal antibody) was added at a concentration of 10 µg/mL during co-culture with tumor cells. Luminescence was quantified using Bright-Glo and SpectraMax. Specific lysis was determined as (1 – (RLUsample)/(RLUmax)) × 100.

Results In our engineered system to evaluate antigen-specific cytotoxicity, NY-ESO-1 T cells exhibited significant cytotoxicity against A498 and UOK127 with the NY-ESO-1 antigen, compared to corresponding parent cell lines. No difference was observed with control 769P (A*02:01−), with or without the NY-ESO-1 antigen. Specific lysis against A498 with NY-ESO-1 by NY-ESO-1-TIGIT T cells decreased by 22.0% compared to NY-ESO-1 T cells, and increased by 10.4% upon tiragolumab compared to no tiragolumab.

Conclusions Our engineered system for assessing T-cell cytotoxicity successfully demonstrated antigen-specific killing of RCC cells. This novel system has extensive applications in RCC research, including the evaluation of the impact of ICIs on T-cell function. Initial pilot studies suggested that blockade of the TIGIT pathway in RCC may enhance cytotoxicity of CD8⁺ T cells.

Ethics Approval This research has been approved by the Human Investigation Committee of the Yale Institutional Review Board after reviewing the research protocol, ethics application, and consent form (Approved September 10, 2021, accession number 0805003787). All investigators involved in this study have stored peripheral blood specimens in compliance with the Declaration of Helsinki.