Biomarker discovery can identify new molecules which refine immune cell classification . This can potentially lead to new diagnostic tests and treatment opportunities including cell therapy. As previously reported, peptidase inhibitor 16 (PI16) identifies a functionally important subset of CD4 T cells in both CD25+ regulatory and CD25- helper compartments. Our initial comprehensive screening experiments revealed that PI16+ Th cells uniformly express CD45RO, have better migratory properties, have a pro-inflammatory phenotype and have a distinct gene signature. Here, we further investigate CD45RO (memory) matched T helper subsets by comparing PI16+ and PI16- memory Th cells. Our results indicate PI16+ mem Th cells have elevated expression of Integrin β1 (CD29), Hepatic leukaemia factor and Clusterin and reduced expression of Tyrosine kinase, CCL5 and CXCR5 relative to PI16- Th cells, which is consistent with the phenotype of antigen-experienced resting memory T cells. In addition, high expression of CCR4, CCR6, histamine H4 receptor, Angiotensin converting enzyme 1 and Galectin-1 in PI16+ cells suggest an active role in inflammation. When cultured with IFN-α, IL-2, IL-4 or IL-6 these cells show increased STAT signalling activity suggesting a more rapid response to extracellular signals. When activated with anti-CD3/ CD28 beads, PI16+ cells secrete both anti-inflammatory IL-10, and pro-inflammatory TNF and IL-17A along with IL-2. We also observed that PI16+ cells proliferate more rapidly than PI16- cells,are more apoptotic in vitro, and suppression assays showed that PI16+ mem Th cells are more resistant to suppression by Treg compared with PI16- mem Th cells. Finally we used chemokine receptor profiling to segregate Th subsets based on effector function, and although Th1, Th2, Th17, Th1/Th17 subsets express PI16 at varying proportions, the Th22 subset uniformly express PI16. In conclusion, in healthy adults PI16 segregates antigen-experienced resting memory Th cells in all Th lineages, and when activated these have a potent effector function.