Decitabine priming increases anti–PD-1 antitumor efficacy by promoting CD8+ progenitor exhausted T cell expansion in tumor models

CD8+ exhausted T cells (Tex) are heterogeneous. PD-1 inhibitors reinvigorate progenitor Tex, which subsequently differentiate into irresponsive terminal Tex. The ability to maintain a capacity for durable proliferation of progenitor Tex is important, but the mechanism remains unclear. Here, we showed CD8+ progenitor Tex pretreated with decitabine, a low-dose DNA demethylating agent, had enhanced proliferation and effector function against tumors after anti–PD-1 treatment in vitro. Treatment with decitabine plus anti–PD-1 promoted the activation and expansion of tumor-infiltrated CD8+ progenitor Tex and efficiently suppressed tumor growth in multiple tumor models. Transcriptional and epigenetic profiling of tumor-infiltrated T cells demonstrated that the combination of decitabine plus anti–PD-1 markedly elevated the clonal expansion and cytolytic activity of progenitor Tex compared with anti–PD-1 monotherapy and restrained CD8+ T cell terminal differentiation. Strikingly, decitabine plus anti–PD-1 sustained the expression and activity of the AP-1 transcription factor JunD, which was reduced following PD-1 blockade therapy. Downregulation of JunD repressed T cell proliferation, and activation of JNK/AP-1 signaling in CD8+ T cells enhanced the antitumor capacity of PD-1 inhibitors. Together, epigenetic agents remodel CD8+ progenitor Tex populations and improve responsiveness to anti–PD-1 therapy.


Supplemental Methods
Clinical study. To investigate the safety and efficacy of decitabine-plus-anti-PD-1 combination treatment, we conducted a phase II study at the Chinese People's Liberation Army General Hospital in patients with advanced solid tumors and lymphoma (NCT02961101). In this article, we report clinical results from patients with measurable histologically confirmed advanced solid tumors, who received decitabine (Jiangsu Chia Tai-Tianqing Pharmaceuticals Co. Ltd., 10 mg/d, days 1 to 5) plus anti-PD-1 antibody camrelizumab (Jiangsu Hengrui Pharmaceuticals Co. Ltd., 200 mg, day 8) combination therapy every 3 weeks until disease progression or unacceptable toxicity occurred, and appropriate chemotherapy (days 6 to 7) was used for patients with large tumor burdens. Patients provided written informed consent and study procedures were approved by the Institutional Review Board of the Chinese People's Liberation Army General Hospital (S2016-127-01). The primary efficacy end point was objective response rate (ORR), according to the Response Evaluation Criteria in Solid Tumors (RECIST1.1). Tumor assessment was performed at baseline and after every 6 weeks by CT/MRI. Adverse events were evaluated according to the NCI CTCAE v4.0.
ATAC-seq data processing and analysis. T-cells from the indicated conditions were collected and chromatin accessibility mapping performed by ATAC-seq, as described previously (40). The quality control assessment was performed by FastQC v0.11.9. Raw reads were trimmed using trim_galore v0.6.6 and aligned to the mm10 mouse reference genome using Bowtie2 v2.3.5.1. Duplicate reads were removed using picard MarkDuplicates v2.25.5 and only primary alignments with mapping quality greater than 30 were retained. Deeptools v3.5.1 computed the ATAC-seq signal in promoter regions ( ± 1 kb around the transcription start site (TSS)) and then the pearson correlation among samples was calculated. ATAC-seq peaks were called using MACS2 v 2.2.7.1 on each individual sample. Peaks that present in both replicates are preserved for downstream analysis. If the peak in one sample does not overlap in another sample, then it is identified as "gain" peak, and vice versa as "loss" peak. Homer v4.11 findMotifsGenome.pl was used to find enriched motifs in "gain" or "loss" peaks.
JunD ChIP-seq Data Processing and Analysis. JunD ChIP-seq data were from Gene Expression Omnibus database under accession codes GSE77857. Raw reads were aligned to the mm10 mouse reference genome using Bowtie2 v2.3.5.1. Duplicate reads were removed using picard MarkDuplicates v2.25.5 and only primary alignments with mapping quality greater than 30 were retained. ChIP-seq peaks were called using MACS2 v 2.2.7.1 on each individual sample. There were 342 JunD ChIP-seq peaks overlapping with DP-gained peaks (versus P group) and 326 genes were assigned to these peaks using clusterProfiler v4.0.5.

Con-T cells+anti-P (P) DAC-T cells+anti-P (DP)
DP P DP P DP P DP P DP P DP P DP P

Figure S3. Analysis of the transferred CD8 + T-cells by scRNA-sequencing.
(    Table S4. TCR information of CD8 + T-cells.   Table S7. ATAC-seq peaks information, genes assigned to changed peaks and enriched TF of changed peaks. Table S8. JunD ChIP-seq peaks information, related genes and GO results.