Natural Treg express the transcription factor FOXP3, but isolation using FOXP3 is not tractable for functional assays or for cell enrichment. A cell surface surrogate for FOXP3 is hence required, and such biomarkers are critical for understanding immune homeostasis and its breakdown in disease. To discover novel human Treg cell surface molecules we performed a genome wide analysis of differential gene expression (Sadlon et al JI, 185(2):1071-81). This revealed significant enrichment for surface molecules, (42.6% Treg cell surface vs. 23.8% in control groups, p=3.36E-16). In search of a biomarker surrogate for FOXP3, we mined for up-regulated novel surface proteins, and Peptidase Inhibitor 16 (PI16) was identified. Analysis of resting and stimulated Treg and Thelper cells demonstrated that PI16 was readily detectable on the surface of resting nTreg. In conjunction with CD25+, PI16 is able to identify 20-80% of both resting and stimulated FOXP3+ cells, and a similar proportion of resting CD25- cells. Detailed characterisation of PI16+ve Treg cells reveals an antigen experienced memory phenotype. PI16+ CD25+ cells are highly suppressive in vitro and preliminary data suggest that they may be more potent than the CD25+ Treg pool. Importantly, stimulation of CD25-Thelper cells, which substantially up regulates CD25 expression and transiently induces FOXP3, does not induce detectable expression of PI16, suggesting that PI16 is able to segregate Treg subsets, as it is not expressed on CD25- derived iTreg. PI16+ Treg are also resistant to effector conversion when exposed to proinflammatory cytokines, whereas iTreg are not. When a cohort of 30 type 1 diabetes samples were analysed for PI16+ Treg FOXP3 expression, we observed a significant decrease in FOXP3 expression levels in t1d, suggesting a loss of functional fitness in these Treg. We propose that PI16 identifies functional FOXP3+ve Treg and FOXP3-ve Thelper subsets with clinically relevant functional capacity in humans.