Tumor growth suppression in adoptive T cell therapy via IFN-γ targeting of tumor vascular endothelial cells

<b>Rationale:</b> In adoptive T cell therapy (ACT), the direct cytotoxic effects of CD8 T cells on tumor cells, including the release of interferon-gamma (IFN-γ), are considered the primary mechanism for tumor eradication. Cancer antigen escape diminishes the T cell responses, thereby limiting the therapeutic success. The impacts of IFN-γ targeting non-tumor cells in ACT, on the other hand, remains under-investigated. We hypothesized that IFN-γ action on non-tumor cells, particularly tumor vascular endothelial cells within the physiological tumor microenvironment, could influence therapeutic efficacy. <b>Methods:</b> ACT was performed against ovalbumin (OVA)- or OVA-peptide SIINFEKL-expressing syngeneic mouse tumors, MCA-205-OVA-GFP fibrosarcoma or MOC2-SIINFEKL oral squamous cell carcinoma, using <i>ex vivo</i>-activated OT-1 CD8 T cells expressing the T cell receptor against OVA. Efficacy was examined in wild-type mice, mice deficient for IFN-γ receptor 1 (IFN-γR1KO), and bone marrow chimeras lacking IFN-γR1 expression in endothelial cells. To exclude direct IFN-γ action against tumor cells, IFN-γR1KO-MCA-205-OVA-GFP tumors were used. IFN-γ production, STAT1 induction in its targets, and subsequent changes, especially in vasculatures in the tumor, were examined. <b>Results:</b> ACT suppressed the growth of MCA-205-OVA-GFP and MOC2-SIINFEKL tumors in wild-type mice but failed in IFNγR1KO mice. Furthermore, in the bone marrow chimeras lacking endothelial cell IFN-γR1, ACT efficacy was lost, thus implicating a vital role of IFN-γ action on the endothelium. IFN-γR1KO-MCA-205-OVA-GFP tumor growth was successfully suppressed by ACT in wild-type mice, suggesting that IFN-γ targeting of tumor cells may not be essential for ACT efficacy. OT-1 CD8 T cells interacted with endothelial cells or localized in proximity to the vessels on Day 1.5 after transfer, as observed by intravital microscopy. The OT-1 T cells found in tumors were limited in number but produced high levels of IFN-γ on Day 1.5, while their number peaked on Day 5.5 with negligible IFN-γ production. Together with IFN-γ production by endogenous lymphocytes, IFN-γ levels in the whole tumor peaked on Day 1.5, inducing IFN-γ/STAT1 signaling in endothelial cells. Early targeting of tumor vascular endothelial cells by IFN-γ led to endothelial regression, reduced perfusion, and tumor hypoxia/necrosis (Day 4.5-7). <b>Conclusions:</b> These findings highlight the critical role of T cell-derived IFN-γ action on endothelial cells early in ACT, emphasizing its dynamic influence on the tumor microenvironment, and offering insights into addressing antigen escape.