Hypoxia hinders tumor responses

Immunotherapy using immune checkpoint molecule blockade has limited effectiveness in some tumors due to the presence of exhausted CD8+ T. Ford et al. examined underlying mechanisms associated with accumulation of terminally differentiated exhausted CD8+ T cells in tumors. They profiled histone modifications in tumor-infiltrating CD8+ T cells using the low-input chromatin immunoprecipitation method (CUT&RUN), and they observed that terminally exhausted T cells in tumors had chromatin modifications that limited transcriptional potential but could be restored with sufficient costimulatory signaling. Hypoxia associated with the tumor microenvironment was linked directly to increased histone bivalency, which also limited transcriptional potential, but forced expression of the hypoxia insensitive histone demethylase Kdm6b overcame these defects and promoted antitumor T cell responses. These findings indicate that terminally exhausted T cells can become transcriptionally active with reversal of hypoxia and appropriate costimulation.


Response rates to immunotherapy in solid tumors remain low due in part to the elevated prevalence of terminally exhausted T cells, a hypofunctional differentiation state induced through persistent antigen and stress signaling. However, the mechanisms promoting progression to terminal exhaustion in the tumor remain undefined. Using the low-input chromatin immunoprecipitation sequencing method CUT&RUN, we profiled the histone modification landscape of tumor-infiltrating CD8+ T cells throughout differentiation. We found that terminally exhausted T cells had unexpected chromatin features that limit their transcriptional potential. Terminally exhausted T cells had a substantial fraction of active chromatin, including active enhancers enriched for bZIP/AP-1 transcription factor motifs that lacked correlated gene expression, which was restored by immunotherapeutic costimulatory signaling. Reduced transcriptional potential was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Enforced expression of the hypoxia-insensitive histone demethylase Kdm6b was sufficient to overcome hypoxia, increase function, and promote antitumor immunity. Our study reveals the specific epigenetic changes mediated by histone modifications during T cell differentiation that support exhaustion in cancer, highlighting that their altered function is driven by improper costimulatory signals and environmental factors. These data suggest that even terminally exhausted T cells may remain competent for transcription in settings of increased costimulatory signaling and reduced hypoxia.

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Volume 7 | Issue 74
August 2022

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We thank S. Henikoff (Fred Hutch Cancer Center) for pA-MNase reagent and S. Hainer (U. Pittsburgh) for pA-MNase plasmid and associated CUT&RUN technical advice. We thank the UPMC Hillman Cancer Center Cytometry Flow core and the UPMC Children’s Hospital of Pittsburgh Flow Core for help with cell sorting and analysis. We thank the Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh for Next Generation Sequencing Services and the University of Pittsburgh Center for Research Computing for computing cluster access and support.
Funding: B.R.F. was supported by T32 CA082084; N.E.S. was supported by the NCI Predoctoral to Postdoctoral Fellow Transition Award (F99/K00) (no. F99CA222711). P.D.A.V. was supported by the National Cancer Institute of the National Institutes of Health (NIH) Ruth L. Kirschstein National Research Service Award 1F30CA247034-01 and T32CA082084 and the National Institute of General Medical Sciences of the NIH (T32GM008208). R.P. was supported by T32AI089443. A.C.P. was supported by R01AI153104 and R21AI135027. G.M.D. was supported by an NIH New Innovator Award (DP2AI136598) and R21AI135367, the UPMC Hillman Cancer Center Melanoma/Skin Cancer (P50CA121973), Head and Neck Cancer SPORE (P50CA097190), the Alliance for Cancer Gene Therapy, the Mark Foundation for Cancer Research Emerging Leader Award, the Cancer Research Institute Lloyd J. Old STAR Award, and the Sy Holzer Endowed Cancer Immunotherapy Fund. This work used flow cytometry and animal facilities at UPMC Hillman Cancer Center, supported by P30CA047904. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Author contributions: B.R.F., P.D.A.V., N.L.R., N.E.S., G.M.D., and A.C.P. contributed to concept and experimental design. N.E.S., P.D.A.V., B.R.F., N.L.R., A.T.F., R.P., and K.L. performed all experiments. Genomic data analysis, data curation, and validation were performed by N.L.R. and B.R.F. Data analysis was performed by B.R.F., N.L.R., N.E.S., P.D.A.V., and A.C.P. The manuscript was written by B.R.F., A.C.P., and G.M.D. and edited by B.R.F., P.D.A.V., N.E.S., N.L.R., A.C.P., and G.M.D. The project was supervised and resources and funding were acquired by A.C.P. and G.M.D.
Competing interests: G.M.D. declares competing financial interests and has submitted patents covering the use of metabolic reprogramming in cell therapies that are licensed or pending and is entitled to a share in net income generated from licensing of these patent rights for commercial development. G.M.D. consults for and/or is on the scientific advisory board of BlueSphere Bio, Century Therapeutics, Nanna Therapeutics, Novasenta, Pieris Pharmaceuticals, and Western Oncolytics/Kalivir; has grants from bluebird bio, Nanna Therapeutics, Novasenta, Pfizer, Pieris Pharmaceuticals, TCR2, and Western Oncolytics/Kalivir; and owns stock in Novasenta. The other authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. The datasets generated during this study are available at NCBI GEO repository GSE175443. Additional datasets not generated by this study are available at NCBI GEO repository under accession numbers GSE155192, GSE123235, GSE123236, GSE95237, and GSE54191. All materials used in this study are available upon request addressed to G.M.D. or A.C.P.



Division of Pediatric Rheumatology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Software, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Division of Pediatric Rheumatology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, and Writing - review & editing.
Present address: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, and Writing - review & editing.
Present address: Division of Biological Sciences, Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Formal analysis, Investigation, and Methodology.
Konstantinos Lontos
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Department of Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA 15260, USA.
Roles: Investigation and Resources.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Formal analysis, Investigation, Methodology, and Validation.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Tumor Microenvironment Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.
Division of Pediatric Rheumatology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Roles: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, and Writing - review & editing.

Funding Information


Corresponding author. Email: [email protected] (G.M.D.); [email protected] (A.C.P.)
These authors contributed equally to this work.

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