3), indicating that in these coculture assays, inhibition of responder cell proliferation by CD8+CD39+ T cells is not the result of cytotoxicity. In this study, we describe for the first time the expression of, and a functional role for, CD39 on human pathogen activated CD8+ Treg cells. CD8+CD39+ T cells from

PPD-responsive individuals specifically co-expressed the known classical Treg-cell markers CD25, Foxp3, LAG-3, and CCL4. To assess if CD39 expression was merely a marker of CD8+ Treg cells or was directly involved in the CD8+CD39+ T cell’s suppressive activity, we purified CD8+CD39+ T cells, and showed that they were Ridaforolimus mouse strongly enriched for suppressive activity and the expression of Treg markers, and that both the chemical CD39 antagonist, ARL, as well as a blocking anti-CD39 antibody were able to partly inhibit Nutlin 3a the suppressive activity of CD8+CD39+ T cells. Altogether these data indicate that CD39 is a marker for regulatory CD8+ T cells

and that CD39 contributes functionally to the suppression mediated by human CD8+CD39+ T cells. Both ARL as well as the blocking anti-CD39 antibody only partly inhibited suppressive activity, indicating that also other mechanisms may contribute to suppression. We previously demonstrated the expression of LAG-3 and the functional involvement of CCL4 in immune regulation by BCG-activated CD8+ Treg cells. In the current study, ≥43% of CD8+CD39+ T cells also expressed CCL4, while we did not find any expression of IL-10 on these T cells. CD8+ Treg cells have been described in human Mycobacterium-infected LNs [8] and lepromatous lesions [9, 10], demonstrating that CD8+ Treg cells are present at the site of disease and suggesting a potential role for these cells in disease pathogenesis. In line with our previous studies showing that BCG activated CD8+ Treg cells in PPD-responsive individuals, but not in donors

that Sulfite dehydrogenase did not recognize PPD in vitro [10], also in the current study CD8+CD39+ Treg cells were confined to PPD responders, suggesting that these cells originated from preexistent antigen-specific memory T cells. We have previously hypothesized that Treg cells could contribute to the relative failure of BCG vaccination in conferring protection against pulmonary TB in adults [6]. In TB, recent results have suggested a role for Th17 cells both in protection and pathology. IL-17 producing CD4+ T cells in the lung, induced by BCG vaccination, were associated with protective immunity to TB in mice [2, 38]; interestingly, in human tuberculous pleural effusions, the number of CD4+CD39+ Treg cells was inversely related to the number of Th17 cells, and CD39+ Treg cells suppressed the differentiation of naïve CD4+ cells into Th17 cells [39]. Frequencies of CD4+CD39+ T cells correlated negatively with IL17A responses in stimulated PBMCs after MVA85A vaccination [40].