Deciphering the interplay between tumour metabolites and T regulatory cells epigenome

Metabolic restrictions and suppressive cells in the tumor microenvironment (TME) limit immunotherapy effectiveness. FOXP3+ regulatory T cells (Treg) are key players and promising therapeutic targets. They adapt their metabolism to the hypoxic, acidic and nutrient-deprived TME and exploit lactic acid, a metabolic waste product, to sustain their suppressiveness. Since metabolites are the substrates used to generate protein modifications, including histone marks, cellular metabolism and the epigenetic regulation are strictly linked. We want to explore how the peculiar TME’s metabolite cocktail shapes Treg chromatin landscape, enabling their suppressive function despite the hostile tumor milieu. In particular, considering that Tumor Infiltrating (TI)-Tregs uptake and metabolize lactic acid, we are investigating the relevance of lactate-derived histone lactylation for their adaptation to the TME. Understanding the link between lactate metabolism and histone lactylation deposition could reveal targetable gene regulatory networks to weaken TI-Treg in the TME. We first quantified the overall extent and modulation of global lactylation with a flow-cytometry based approach and observed a slight increase of lactylation in Treg treated with lactic acid at ambient oxygen levels; that was amplified by the simultaneous exposure to a hypoxic environment; and concordant with intracellular lactate levels. Then, immunofluorescence analysis revealed predominant nuclear lactylation in Treg. Antibodies are available for a fraction of lactylated residues; thus, we performed post-translational modification (PTM) Mass Spectrometry (MS) of lactic acid and hypoxia-treated healthy donor Tregs and we singled out key lactylated histone residues for further study. Moreover, we previously generated a multi-omic profile of TI-Treg epigenome by combining chromatin accessibility and different histone modification patterns. Thus, the integration of the genomic distribution of MS- defined histone lactylation residues with our in-house dataset will clarify its function in TI-Treg specific gene regulatory networks. Identifying lactylation as a means of TI-Treg adaptation to the TME could highlight novel therapeutic targets to dampen their suppressive activity via metabolic and epigenetic pathways.