Abstract 2994: Hypoxia blocks the presentation of tumor antigens to CD8+ T cells
Authors: Smith Matthew G. , Panchal Heena , Mailloux Adam 
Published: April 2023

Tumor hypoxia is known to enhance tumor growth and immune evasion by activating angiogenic pathways supporting tumor progression and fostering suppressive phenotypes of immune cells within the tumor-microenvironment. However, the consequence of tumor hypoxia on antigen processing and presentation to surveilling CD8+ T cells is a notable knowledge gap with direct relevance towards adaptive immune responses against cancer. Here, we present evidence using the well-established OT-I transgenic model that hypoxia decreases antigen specific T cell effector responses, and importantly, that these decreases in effector responses are not attributable to any hypoxic influence on T cell function. Murine B16 melanoma cells expressing ovalbumin (OVA; MO4) or parental B16 were grown under 20% or 2% oxygen for 48 hours. These cells were then treated with 0 or 10 ng/mL recombinant murine interferon gamma (IFN-g) for 24 hours at their respective oxygen conditions. For analysis of cytotoxicity and effector responses, these cells were labeled with a fluorescence enhancing ligand (BATDA) and then incubated with OT-I T cells recognizing OVA-peptide (SIINFEKL) presented by C57BL/6 mouse MHC I (H2kb) for 4 hours at 20% oxygen. Supernatants were then tested for BATDA release using a europium solution and subsequent fluorescent signal acquisition. OT-I T cells displayed significantly more specific lysis of target MO4 cells cultured under 20% oxygen compared to those cultured under 2% oxygen (p value &lt; 0.0001; 2-way ANOVA). Control experiments using parental B16 target cells displayed little to no specific lysis. Additionally, OT-I T cells were incubated with B16 or MO4 cells cultured as described above for 18 hours under 20% oxygen in the presence of protein transport inhibitors and analyzed for effector cytokine production. Tumor necrosis factor alpha (TNF-a) production was significantly increased in OT-I cells co-cultured with IFN-g treated MO4 target cells preconditioned in both 20% and 2% oxygen (p value &lt; 0.05; unpaired T test), however the fold change in TNF-a production for OT-I cells incubated with 20% oxygen cultured MO4 cells was approximately twice the increase observed in OT-I cells incubated with 2% oxygen cultured MO4 cells. These differences are not attributable to altered OVA expression under hypoxia. Furthermore, IFN-g dependent up-regulation of MHC-I and immunoproteasome catalytic subunits was found to be blocked by hypoxia, suggesting that hypoxic attenuation of the MHC I pathway is responsible for observed decreases in cytotoxic and effector responses in OT-I co-culture assays. Overall, these studies suggest that tumor hypoxia blunts anti-tumor immune responses irrespective of the oxygenation status of surveilling CD8+ T cells.

Citation Format: Matthew G. Smith, Heena Panchal, Adam Mailloux. Hypoxia blocks the presentation of tumor antigens to CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2994.