CD4+ T cells are crucial orchestrators of immune responses against pathogens and cancer. Environmental cues such as metabolite availability are intimately linked to CD4+ T cell differentiation, as metabolic activity determines the metabolic intermediates and ATP levels available to support their functional state. The short chain fatty acid butyrate is a metabolite generated as a by-product of dietary fibre fermentation by the intestinal microbiota. While butyrate’s effects under Treg and Th17-polarising conditions have been intensively studied, its impact on Th0 cell differentiation and function is poorly understood. We aimed to investigate how butyrate influences the capacity of CD4+ T cells to differentiate into distinct subsets, and its mechanisms of action in anti-viral and anti-tumour immunity.
We demonstrated that butyrate promoted CD4+ T cell mitochondrial respiration. Under non-polarising conditions, this correlated with significantly increased expression of the Th1 master transcription factor T-bet. Eomes co-expression suggested differentiation into CD4+ cytotoxic lymphocytes – an underappreciated but important subset that can directly eliminate target cells. In line with this, butyrate drastically improved CD4+ T cell-mediated killing of B16 melanoma cells. This was accompanied by significantly upregulated expression of cytotoxic mediators IFN-g, granzyme B, and Fas ligand, suggesting that butyrate indeed induced Th1 polarisation to an extent which was comparable to Th1 polarising conditions. This greater Th1 polarisation of butyrate-treated CD4+ T cells was maintained long-term after adoptive transfer and HSV-1 challenge in vivo. Finally, we showed that butyrate likely promoted Th1 characteristics in vitro by mediating epigenetic changes rather than receptor-mediated mechanisms. Together, these findings underline the importance of metabolite availability, such as butyrate, in the microenvironment for immune responses against infection and cancer. Understanding butyrate’s ability to skew CD4+ T cell differentiation towards cytotoxic Th1 cells could lead to its application in improving current immunotherapy, such as adoptive cell therapy in melanoma treatment.