Naive CD4+CD25− T cells were collected or polarized in vitro for 3 days, split 1:2, and cultured with IL-2 alone for an additional 2 days to generate Th1, Th17, or Treg. T cell subset lysates from 3 biological replicate samples were extracted and analyzed using high-resolution LC-QE-MS for determination of cellular metabolites. Relative levels of tryptophan, serotonin (5-HT) and kynurenine are shown. Samples are shown as box plots representing 6 biological replicates per sample. * = p < .05 compared with naïve.