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

Abstract
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.