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GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma

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Abstract

Amino acid deprivation is a strategy that malignancies utilize to blunt anti-tumor T-cell immune responses. It has been proposed that amino acid insufficiency in T-cells is detected by GCN2 kinase, which through phosphorylation of EIF2α, shuts down global protein synthesis leading to T-cell arrest. The role of this amino acid stress sensor in the context of malignant brain tumors has not yet been studied, and may elucidate important insights into the mechanisms of T-cell survival in this harsh environment. Using animal models of glioblastoma and animals with deficiency in GCN2, we explored the importance of this pathway in T-cell function within brain tumors. Our results show that GCN2 deficiency limited CD8+ T-cell activation and expression of cytotoxic markers in two separate murine models of glioblastoma in vivo. Importantly, adoptive transfer of antigen-specific T-cells from GCN2 KO mice did not control tumor burden as well as wild-type CD8+ T-cells. Our in vitro and in vivo data demonstrated that reduction in amino acid availability caused GCN2 deficient CD8+ T-cells to become rapidly necrotic. Mechanistically, reduced CD8+ T-cell activation and necrosis was due to a disruption in TCR signaling, as we observed reductions in PKCθ and phoshpo-PKCθ on CD8+ T-cells from GCN2 KO mice in the absence of tryptophan. Validating these observations, treatment of wild-type CD8+ T-cells with a downstream inhibitor of GCN2 activation also triggered necrosis of CD8+ T-cells in the absence of tryptophan. In conclusion, our data demonstrate the vital importance of intact GCN2 signaling on CD8+ T-cell function and survival in glioblastoma.

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Abbreviations

ATF4:

Activating transcription factor 4

CCR7:

C–C Chemokine receptor type 7

CNS:

Central nervous system

EAE:

Experimental autoimmune encephalomyelitis

EIF2α:

Eukaryotic translation initiation factor 2α

ER:

Endoplasmic reticular

GCN2:

General control nonderepressible 2

GZMB:

Granzyme B

i.c.:

Intracranial

ISRIB:

Integrated stress response inhibitor

ISR:

Integrated stress response

KO:

Knockout

KYN:

Kynurenine

LC-MS:

Liquid chromatography–mass spectrometry

NCI:

National Cancer Institute

PKC-ϴ:

Protein kinase C-theta

+ TRP:

Tryptophan added

− TRP:

Tryptophan free

WT:

Wild type

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Acknowledgements

We would like to thank Peng Gao at the Metabolomics Core Facility at Northwestern for performing and analyzing bulk metabolomics included in this study.

Funding

Financial support comes from an Outstanding Investigator Award from the National Cancer Institute (NCI) to Maciej S Lesniak (R35CA197725) and a grant from the National Institute of Neurological Disorders and Stroke (NINDS) to Maciej S Lesniak (R01NS093903). Jason Miska received a fellowship from the NINDS (1F32NS098737-01A1). This work was supported by the Northwestern University Robert H. Lurie Cancer Center Flow Cytometry Facility Support Grant (NCI CA060553).

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Author notes

  1. Aida Rashidi and Jason Miska contributed equally.

Authors and Affiliations

  1. Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, 676 N St. Clair, Suite 2210, Chicago, IL, 60611, USA

    Aida Rashidi, Jason Miska, Catalina Lee-Chang, Deepak Kanojia, Wojciech K. Panek, Aurora Lopez-Rosas, Peng Zhang, Yu Han, Ting Xiao, Katarzyna C. Pituch, Julius W. Kim, Mahsa Talebian, Jawad Fares & Maciej S. Lesniak

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  1. Aida Rashidi
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Contributions

AR, JM, and MSL conceived the study. AR, JM, CLC, PZ, DK, and JWK designed and performed the experiments. ALR maintained animal colonies, performed and analyzed the GCN2 backcrosses, and validated all mice used in this study via genotyping. YH and WKP performed all animal surgeries and monitoring of endpoint analyses. KCP, JF, and MT assisted with the construction and provided critical feedback for the manuscript. MSL and JM oversaw the research program and assisted in the preparation of this manuscript. TX performed all statistical analyses and provided feedback regarding animal numbers. All authors have reviewed the manuscript for accuracy and provided feedback during the writing and revision process.

Corresponding author

Correspondence to Maciej S. Lesniak.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

The IACUC committee approved all animal work within the Center for Comparative Medicine (CCM) at Northwestern University. Northwestern University has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (A3283-01). Northwestern University conducts its reviews following United States Public Health Service (USPHS) regulations and applicable federal and local laws. The composition of the IACUC meets the requirements of the USPHS policy and the Animal Welfare Act Regulations. The animal protocol number is IS00002459.

Animal source

C57BL/6 (WT), GCN2 KO, Rag1 KO, OT-1, and Foxp3-IRES-GFP C57/B6 mice were purchased directly from The Jackson Laboratory (Bar Harbor, ME).

Cell line authentication

GL-261 was purchased directly from the National Cancer Institute (NCI) Frederick National Tumor Repository Laboratory (Frederick, MD. USA). CT2A was acquired from the Balyasnikova laboratory at Northwestern University, Feinberg School of Medicine (Chicago, IL. USA). All cell lines were used in Northwestern’s Neuro-oncology research. All cell lines are routinely tested for Mycoplasma contamination every 2 months using the Universal Mycoplasma Detection Kit (ATCC® 30-1012 K™). The identity and purity of cell lines were determined using short tandem repeats (STR) profiling performed by the Northwestern sequencing facility.

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Rashidi, A., Miska, J., Lee-Chang, C. et al. GCN2 is essential for CD8+ T cell survival and function in murine models of malignant glioma. Cancer Immunol Immunother 69, 81–94 (2020). https://doi.org/10.1007/s00262-019-02441-6

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