ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression

Understanding the intrinsic mediators that render CD8+ T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8+ T cells. Chop expression is increased in tumor-infiltrating CD8+ T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8+ T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8+ T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8+ T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.

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No customized software was used. Applied Biosystems StepOnePlus real-time PCR system and associated software were used to collect real-time PCR data. Western blot data were collected in a ChemiDoc™ Imaging System (Bio-Rad) and densitometry analyses completed using the Image Lab™ Software (Bio Rad). FACS acquisition was performed in a CytoFLEX II (Beckman Coulter), LSRII (BD) or FACSAria II unit (BD) and analyzed using FlowJo 10.3. Samples were processed for RNA-sequencing using the NuGen Ovation Mouse RNA-Seq Multiplex System (NuGEN Technologies). The libraries were then sequenced in an Illumina NextSeq 500 v2 sequencer with 75-base single-end run. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using a XF96 extracellular flux analyzer (Seahorse Bioscience). Immunofluorescence of the tissue microarray histology slides were visualized in a Leica SP8 Confocal microscope, and then scanned using the APERIO ScanScope FL (Leica Biosystems, Wetzlar, Germany). Images were stored in Aperio's Spectrum software, segmented into individual cores using the software's TMA lab module, and individual core images analyzed using the Definiens Tissue Studio v4.2 suite cellular analysis. Additional information about used software has been described in the manuscript or is available upon reasonable request.

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No custom-made software was used for data analysis. TopHat v2.0.13 was used for RNA-Seq reads alignment; HTSeq v0.6.1 was used to quantify the aligned RNA-Seq reads; DESeq2 v1.6.3 was used for read counts normalization. Gsea-3.0 was used for gene set enrichment analysis. FACS data was analyzed using nalyzed using FlowJo 10.3. Graphpad Prism 7.03 was used for generating graphs and performing statistical studies.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The RNA-Seq raw data that support the findings of the study have been deposited in Gene Expression Omnibus database, the GEO accession number is GSE112823. The authors declare that the all data supporting the findings of this study are available within the paper and its supplementary figures. The supporting raw data of this study are available from the corresponding author upon reasonable request.

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Most of our experiments are in vitro experiments using primary cells from mice or donors. Sample sizes were chosen on the basis of our previous studies or publications, as well as the availability of mice and donors. For most of the in vitro studies, 3 independently developed repeats were used. Power analysis at 80% power and type I error controlled at 0.05 were used for most of the animal experiments. For simplicity, t test was used for the power analysis.
Data exclusions We excluded one set of data from RNA-Seq dataset. Based on the PCA study, we found one sample in the Ddit3-/-group was clustered very differently from the other two replicates, we then identified it as an outlier and ruled it out from the analysis.

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Randomization In the tissue micro-array studies, the ovarian cancer samples were collected randomly by Moffitt Cancer Center Tissue Core prior to this study. Commercially available buffy coats (One-blood) were used to isolate human CD8+ T cells and were from de-identified and randomly picked up healthy donors.

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The statistical studies for tissue micro-array results and RNA-Seq results have been done by Moffitt's cancer bio-informatics and statistics group in a blinded way. For the rest of the experiments, considering appropriate handling and data acquisition, investigators were not blinded to the studies. Moreover, because samples were treated equally and data collection and/or analysis were mainly performed by computerbased methods (such as flow cytometric analysis), we believe the blinding was not necessary to our study.

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