In adoptive T cell therapy (ACT), CD8 T cells are considered cytotoxic while CD4 T cells have an ancillary role. Among a wide range of cytokines and chemokines released by activated CD8 T cells, interferon-gamma (IFN-γ) is considered crucial, exerting a direct cytotoxic effect on tumor cells. In addition, IFN-γ acts on various immune cells in the tumor microenvironment; however, its impact on stroma and blood vessels remains unclear (1,2). We hypothesized that IFN-γ acts on tumor vascular endothelial cells during ACT, thereby impairing tumor perfusion.

Using mouse ACT models against syngeneic cancers, we examined the effects of IFN-γ on the tumor microenvironment, with a focus on tumor vascular endothelial cells. ACT using 3 million ex vivo-activated OT-1 CD8 T cells expressing T cell receptor against ovalbumin (OVA) suppressed the growth of OVA- or OVA peptide-expressing subcutaneous tumors (MCA-205-OVA-GFP fibrosarcoma or MOC2-SIINFEKL oral squamous cell carcinoma) in wild type mice (n=5-7, p<0.0001 or p<0.001 by repeated-measure 2-way ANOVA, respectively) but failed in IFN-γ receptor 1 (IFN-γR1) deficient mice, implicating the importance of IFN-γ action on non-tumor cells. In bone marrow chimeras without IFN-γR1 expression in endothelial cells, ACT efficacy was abrogated, suggesting a vital role for IFN-γ on endothelium. IFN-γ in the tumor peaked on Day 1.5 after T cell transfer, measured by ELISA of tumor lysate (n=3, p<0.01 by one-way ANOVA). Flow cytometry analyses demonstrated that transferred OT-1 CD8 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 (n=3-5, p<0.05 by one-way ANOVA) but with negligible IFN-γ production. To examine the kinetics of OT-1 CD8 T cell infiltration into the tumor microenvironment, with possible interaction with the endothelial cells during extravasation, intravital microscopy imaging was performed. ACT using IFN-γ-IRES-eYFP-OT-1 CD8 T cells that exhibit YFP fluorescence signal upon IFN-γ production demonstrated strong IFN-γ production in these CD8 T cells on Day 1.5, but not on Day 5. ACT using mTmG-OT-1 CD8 T cells expressing Tdtomato fluorescence, in combination with in vivo endothelial cell labeling by Alexa Fluor 647-conjugated anti-CD31 antibody infusion, demonstrated presence of mTmG-OT-1 T cells inside and near tumor vessels on Day 1.5, while the cells infiltrated the tumor on Day 5. Quantitation of the images showed OT-1 T cell fraction localized within <20 µm of tumor vessels were significantly higher on Day 1.5 than those on Days 5 and 7 (n=2-4, 4-7 FOVs analyzed each, p<0.0001 by one-way ANOVA). Remarkably, tumor vessels regressed and became almost undetectable on Day 5, which started to recover on Day 7. Immunofluorescence histology imaging of harvested tumor showed induction of IFN-γ/STAT1 signaling in endothelial cells early after ACT (Day 1.5). Degeneration of endothelial cells on Day 4.5 was confirmed by transmission electron microscopy imaging. Consistently, whole-body optical imaging of IR800-conjugated albumin infused (50 µg) at various times after ACT showed significantly reduced perfusion in tumors on Day 5 compared to untreated tumors (p<0.0001 by one-way ANOVA). Carbonic anhydrase IX hypoxia marker immunohistochemistry and H&E staining demonstrated tumor hypoxia and necrosis on Days 4.5-7.

In conclusion, our study highlights the critical role of CD8 T cell-derived IFN-γ targeting endothelial cells in tumor suppression during ACT. Remarkably, a small fraction of exogenous tumor-specific CD8 T cells producing high levels of IFN-γ initiated the process of tumor regression at early time points, emphasizing their dynamic influence on the tumor microenvironment. Our findings could provide the basis for a strategy for overcoming the challenge of tumor antigen escape observed in clinical ACT.

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Image/Figure Caption:

A. Intravital microscopy imaging demonstrated mTmG-OT1 CD8 T cells (magenta) localized inside or near tumor vessels (red) on Day 1.5 and infiltrated into MCA-205-OVA-GFP tumor (green) on Days 5-7 after ACT. Vessel regression was observed on Day 5, which started to recover on Day 7. Arrows indicate OT-1 CD8 T cells interacting with endothelial cells.

B. Phospho-STAT1 (yellow) and CD31 (magenta) immunofluorescence histology images indicated IFN-γ/STAT1 signaling was  induced in the endothelial cells on Day 1.5.

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