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ER Dysmorphia Underlies CD8+ TIL Dysfunction in Solid Cancers
Elizabeth G. Hunt1,2, Andrew S. Kennedy1,2, Brian P. Riesenberg1, Katie E. Hurst1, E. Diane Wallace3, Justin J. Milner1,4, Barbara Savoldo, R. Luke Wiseman5, Jessica E. Thaxton1,3
1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA
2 Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, USA
3 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, USA
4 Department of Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, USA
5 Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, USA
6 Department of Molecular Medicine, Scripps Research Institute, La Jolla, USA
Background: The success of tumor immunotherapies is limited by mechanisms of immune suppression posed by the solid tumor microenvironment (TME), such as persistent antigen encounter, nutrient deprivation, and hypoxic stress. The endoplasmic reticulum (ER) plays a critical role in sensing and responding to cell stress, while simultaneously controlling protein and lipid biosynthesis, proving ER function vital for T cells fighting cancers. T cells in solid tumors experience persistent protein stress, mediated by chronic signaling of the unfolded protein response (UPR) that undermines T cell immunity in tumors. Given that the major ER stress sensor axis, IRE1a-XBP1s, regulates ER structure in multiple cell types, we hypothesized that chronic activation of the IRE1a-XBP1s axis in CD8 tumor-infiltrating T cells (TILs) could dysregulate ER structural homeostasis, leading to poor ER function and impaired tumor immunity.
Aims: Here we aimed to discern whether ER structure is altered in CD8+ TILs traversing patient cancers and in multiple solid cancer mouse models. Using genetic and pharmacological approaches, we studied the contribution of IRE1a -XBP1s, and downstream structural targets, to regulate ER morphology and subsequent functional tumor immunity in CD8+ TILs.
Methods:We used confocal microscopy and flow cytometry to assess ER morphology in CD8+ TILs infiltrating patient cancers, MCA-205 fibrosarcomas, MC-38 adenocarcinomas, and B16F1 melanoma tumors relative to ER structure in CD8+ T cells isolated from patient peripheral blood or autologous non-tumor organs, respectively. Next, we utilized shRNA and pharmacological approaches coupled with imagining and spectral flow cytometry to test the effect of IRE1a -XBP1s on the regulation of ER structure and immune efficacy in CD8+ TILs. Finally, using shRNA and CRISPR/Cas9-mediated gene editing of ER sheet proliferation regulated by Ckap4, we tested whether direct modulation of ER structure repairs dysregulated T cell immunity against solid cancer.
Results:Across multiple tumor models and patient samples, we observed that CD8+ TILs undergo radical ER distention relative to autologous T cells in peripheral non-tumor organs. We observed that ER distention correlated with CD8+ TIL dysfunction measured by terminal exhaustion was associated with expression of IRE1a-regulated transcription factor XBP1s. Using in vivo models of acute and chronic viral infection mediated by antigen-specific T cell responses coupled with an assessment of T cells responding to melanomas bearing the viral antigen, we identified that ER structural dysmorphia was directly induced by tumor hypoxia. Inhibition of gene deletions within the IRE1a-XBP1s axis rescued ER structural dysmorphia and tumor control. Using RNA-sequencing validated by western blotting and confocal imaging, we observed that CD8+ TILs undergo robust ER sheet proliferation in tumors regulated by hypoxic stress. Thus, genetic ablation of ER sheet proliferation mediated by Ckap4 restored ER structural homeostasis in tumor stress and augmented control of solid cancers.
Conclusions: We discovered that ER morphology is regulated by chronic IRE1a-XBP1s signaling in CD8+ TILs in solid cancers. We identified that ER structural dysmorphia was potentiated by Ckap4-mediated ER sheet proliferation that could be genetically ablated to restore ER structural homeostasis and T-cell immunity against solid cancers. Our data unveil regulation of ER structural homeostasis as a completely new avenue to increase the potency of cancer immunotherapies.