CD4+ T cells can differentiate upon activation into different effector subsets with distinct immunological roles. This important fate decision is influenced by immunometabolism due to unique bioenergetic requirements of CD4+ T cell subsets. Differentiation is thus sensitive to metabolites in the microenvironment. The short chain fatty acid butyrate is a metabolite generated as a by-product of dietary fibre fermentation by the intestinal microbiota. It is known to be metabolised by many cell types. Although the effects of butyrate on Treg and Th17 formation have been intensively studied, its impacts on Th0 and Th1 differentiation and function are poorly understood. We aimed to investigate how butyrate affects a key characteristic of CD4+ T cells: their differentiation into distinct subsets. We treated CD4+ T cells with butyrate in vitro to characterise its effects on metabolism, differentiation, and function. Additionally, we adoptively transferred butyrate-treated CD4+ T cells and challenged recipient mice with HSV-1.
We demonstrated that butyrate promotes activated CD4+ T cell mitochondrial respiration, but not glycolysis. This metabolic change was accompanied by significantly elevated T-bet expression, indicating greater polarisation into Th1 cells even under non-polarising conditions. Butyrate significantly upregulated IFN-γ, granzyme B, perforin, and Fas ligand, suggesting differentiation into CD4+ cytotoxic lymphocytes – an underappreciated but important subset that directly eliminates target cells. Cytotoxicity was hence tested using in vitro killing assays, where butyrate induced a 75% increase in CD4+ T cell-mediated direct killing of B16 melanoma cells. Greater polarisation of butyrate-treated CD4+ T cells into Th1 cells was maintained long-term after adoptive transfer and HSV-1 infection in vivo. Butyrate may therefore prime CD4+ T cells to be more effective killers. These findings highlight the importance of metabolite availability in the microenvironment for pro-inflammatory responses. Butyrate particularly has potential relevance for immunotherapy, such as improving chimeric antigen receptor (CAR) T cell therapy.