Deciphering the interplay between tumour metabolites and CD4+ FOXP3+ 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 in this scenario and promising therapeutic targets. They adapt their metabolism to the hypoxic, acidic and nutrient-deprived TME and exploit metabolic waste products, like lactic acid, to sustain their suppressive function. Since metabolites are the substrates used to generate protein modifications, including histone marks, cellular metabolism and the epigenetic regulation are strictly correlated. We want to explore how the peculiar metabolite cocktail found in the TME impacts on the chromatin landscape of Tregs, 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. Indeed, understanding the interconnection between lactate metabolism and histone lactylation deposition could reveal targetable gene regulatory networks to hinder TI-Treg adaptation to the TME. We first quantified the overall extent and modulation of global histone lactylation with a flow-cytometry based approach and observed a slight increase of histone lactylation in Treg treated with lactic acid at ambient oxygen levels; that was amplified by the simultaneous exposure to a hypoxic environment. Antibodies are available for a fraction of lactylated residues; therefore, we are currently performing Mass Spectrometry (MS) of lactic acid and hypoxia - treated healthy donor Tregs to provide a robust quantitation of this novel histone modification in an unsupervised manner, and single 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 better elucidate its function in TI-Treg specific gene regulatory networks. Identifying lactylation as a means of TI-Treg adaptation to the TME could highlight novel targets for innovative therapeutic opportunities to dampen their suppressive activity via metabolic and epigenetic pathways.