Integrative Epigenetic Analysis Reveals AP-1 Promotes Activation Of Tumor-infiltrating Regulatory T Cells

BackgroundSeveral studies have confirmed that the existence of an immunosuppressive tumor microenvironment(TME)caused mainly by the accumulation of regulatory T(Treg)cells.Treg cells that express the master transcription factor FOXP3 in peripheral lymphoid and non-lymphoid organs play an essential role in negative regulation of the immune response in mice and humans.These cells are able to migrate into inflammatory sites throughout the body,restrain inflammatory responses to self-antigens,and exert immunosuppressive functions via various mechanisms.Abundant Treg cells have been observed in different experimental mouse tumor models and human cancers,constituting 10～50%of CD4+T cells in tumor tissues compared with 2-5%of CD4+T cells in the peripheral blood of healthy individuals.Several studies have revealed that the phenotype of TITRs is differed from the phenotype of Treg cells in peripheral blood.These TME-localized Treg cells,which are predominantly FOXP3high effector Treg cells and have a strong immunosuppressive activity,exhibit highly activated features characterized by high expression of T cell activation-associated markers,such as CTLA-4,OX40,GITR and TIM3.Transcriptome analysis studies have shown that tumor-infiltrating Treg cells(TITRs)are different from those in peripheral blood and matched non-cancerous tissues.Recent single-cell transcriptome analyses have revealed the preferential enrichment for CTLA-4high Treg cells in HCC and NSCLC,and the epigenetic landscapes of Treg cells in blood have been well investigated.However,the epigenetic profiles of Treg cells localized in human tumors remain largely unexplored,which might provide useful insights into the mechanisms of gene transcriptional regulation in TITRs.MethodsWe sorted TITRs and peripheral blood Treg cells(PBTRs)by flow cytometry based on the high expression of CD25 and low expression of CD 127 from treatment-naive primary HCC patients.ATAC-seq was used to determine the differential chromatin accessibility regions between TITRs and PBTRs.RNA-seq was used to determine the expression of genes and examine the differentially expressed genes between TITRs and PBTRs.In order to comprehensively evaluate chromatin state changes in TITRs and systematically identify putative enhancers,we performed CUT&Tag using antibodies to H3K4me1 and H3K27ac in the sorted Treg cells.Computational analysis of footprints was performed to infer multiple TF occupancy in TITRs and PBTRs.CUT&Tag assay for c-Jun on tumor-infiltrating Treg cells were conducted to identify c-Jun binding sites genome-wide.Transcriptome profiling assays were performed to identify differentially expressed genes in Treg cells after c-Jun inhibition.ResultsHere,we found TITRs not only have specific gene expression patterns but also have distinct chromatin accessibility features compared to PBTRs.Functional annotation analysis revealed that the hyper-accessible regions in TITRs were largely gene distal,which often contains enhancer elements.Significantly,we found that the changes in gene expression were positively correlated with changes of chromatin accessibility.We provided comprehensive putative enhancer profiles using H3K4me1 in the TITRs and PBTRs.Massive remodeling of the enhancer landscape between TITRs and PBTRs.Following integration of ATAC-seq and CUT&Tag data,we identified "de novo enhancer activation"(DNEA)and "poised enhancer activation"(PEA)regions in TITRs.Significantly,H3K4me1-predetermined enhancers give the TITRs a set of transcriptional options to rapidly deploy in response to tumor microenvironmental stimuli.We found that AP-1 family motifs are enriched at the enhancer regions of TITRs.We predicted a series of TFs that might be involved in establishing tumor-infiltrating Treg cell-specific gene expression programs.Finally,we validated that c-Jun binds at regulatory regions to regulate signature genes of TITRs and c-Jun is required for Treg cells activation in vitro.ConclusionIn conclusion,the epigenetic landscape of TITRs is clearly distinct from that of PBTRs,including chromatin accessibility and enhancer landscapes.Transcriptional changes of TITRs are reflected at the epigenetic level.Integration of multi-omics data analysis revealed AP-1 is required for the activation of TITRs.Overall,our results provide insights into the mechanism of AP-1-mediated activation of TITRs and can hopefully be used to develop new therapeutic strategies targeting TITRs in liver cancer treatment.