CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an “epigenetic checkpoint” for tumor immunity.


Statistics
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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Flow cytometry data were collected on LSRFortessa (BD) instrument with FACSDiva software v8.0.1 (BD)

Data analysis
Flow cytometry data were analyzed on FlowJo v10.4 (TreeStar) and statistical analysis performed on Prism v8.0.1 (GraphPad). Genomic analyses were performed using R Studio 3.6.3 and all packages utilized were updated to most current versions. Genomic data was visualized on IGV 2.8.3 version.
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Data
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No statistical method was used to predetermine sample size. Sample sizes were based on those used in previous and preliminary studies from our lab which allow for statically valid comparisons. For single cell RNA-seq experiments, more than 30000 cells were collected from each sample, 2 replicates were used for each group.. For ATAC-seq experiment, 50000 cells were collected from each sample, 2 replicates were used for each group.
Data exclusions No data were excluded from analyses.

Replication
All data were reliably reproduced in at least two independent experiments. All presented results were repeatable.
Randomization For in vivo tumor growth experiments, mice were randomized prior treatment. Age and sex-matched animals were used for each experiment.
WT and KO mice were littermates if possible.

Blinding
Blinding was not achieved due to requirements for cage identification and labeling for treatment pourpouses.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Validation
Representative flow panels are shown in Supplementary Figure 7. Further validation is present on the manufacturer's website. Antibodies were all titrated to determine the optimal concentration. Titration stainings included additional markers in order to validate the expression patterns on know subsets (e.g. T cells). Optimal concentration was defined by comparing the expression with other validated clones of a given antibody or with other previously validated lots of the same antibody clone. Suv39h1Flox/Flox (official name is B6-Suv39h1tm1Ciphe) were originally produced at the Centre d'Immunologie de Marseille (CIPHE, B. Malissen) and were subsequently bred in the animal facility of Institut Curie. The strategy targets the exon 2 from the transcript Suv39h1-001 ENSMUST00000115638. LoxP sites were introduced in the intron 1 at 70pb in 5ʹof exon 2 and in the intron 2 at 98 pb in 3' of exon 2. The loxP site in the intron found at the 3ʹ end of exon 2 was abutted to a Frt-neoR-Frt cassette. After deletion of the Frt-neoR-Frt cassette by flipage, a residual Frt site will remain just after the loxP 3'. CD4-Cre transgenic mice were obtained from Jacques Ghysdaël (Institut Curie). Suv39h1-Flox*CD4-Cre+/-and control Suv39h1-Flox*CD4-Cre-/-mice were used for the experiment.

Wild animals
Study did not involve wild animals.
Field-collected samples Study did not involve samples collected in the field.

Ethics oversight
All animal procedures were in accordance with the guidelines and regulations of the Institut Curie veterinary department. Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee from Institut Curie, officially registered as CEEA-IC #118 and the Ministère de l'enseignement supérieur, de la recherche et de l'innovation which validated the project with the reference (APAFIS#12325_20171124123634-v2) in compliance with the international guidelines. Mice used in the experiments were age-sex-matched and were euthanized by cervical dislocation. Mice breeding were in SPF animal facilities and experimental and control animals were co-housed with housing conditions using a 12 light/12 dark cycle, with a temperature between 20-24°C with an average humidity rate between 40-70%. Humane endpoints are used for mice bearing tumors, ie. the maximal ethical size of tumors subcutaneously grafted is 2000mm3, or more than 20% of weight loss or any signs of altered mobility or eating ability, or cachexia.
Note that full information on the approval of the study protocol must also be provided in the manuscript.