CD161+FoxP3+CD4+ T cells are suppressive and anergic in vitro. Treg were sorted by flow cytometry. (A) Sorting strategy for subpopulations of Treg defined by CD25^hiCD127^lo, and CD161 expression (a, CD161–; b, CD161+), with representative sort purity of ≥90% purity for both subsets for FoxP3 and CD25 (dot plots) and CD161 staining (overlay histogram). (B) Both CD161+ and CD161– Treg are anergic in vitro. Cells were sorted, labeled with CFSE, and stimulated in vitro for 5 days with anti-CD3 antibody. Representative histograms showing CFSE dilution of sorted CD161– Treg (left), CD161+ Treg (middle), and Tconv (CD25–CD127+, right) from healthy adult (1 of 2). Numbers above the bars represent the percentage of divided cells. (C) CD161+ Treg suppress proliferation and cytokine production. Sorted Tconv were labeled with CFSE, mixed with unlabeled Treg subpopulations as shown, and stimulated for 5 days as described, before analysis by flow cytometry. Representative plots show CFSE dilution, and the production of IFN-γ (upper panels) or IL-17A (lower panels) by Tconv; summary plots at day 5 for the % suppression of division of Tconv in the various cultures (n = 4; statistical analysis, 1-way ANOVA). (D) IFN-γ and IL-17A protein levels were assessed in the supernatants from cocultures by Luminex assay. Data in dot plots and histograms are gated on CD4+ lymphocytes; all summary plots show mean ± SEM. ***P < .001; ns, not significant.