FIGURE 6.

TNFR2⁺ Tregs display superior suppressive function and survival in vitro. (A, B) TNFR2⁺ and TNFR2⁻ Tregs were sorted from the spleens of Foxp3 ^{eGFP‐Cre‐ERT2} mice, and their phenotype was freshly analyzed through flow cytometry. The plots display histogram overlays (A) and a heatmap of the z‐score (B), calculated from means and SD of gMFI of each marker in Tconvs, TNFR2⁺, and TNFR2⁻ Tregs (n = 4). Data are from one experiment representative of two. *p < 0.05, by Mann–Whitney test. (C–F) Tconv were sorted as YFP⁻ cells, labeled with the CTV proliferation dye, and cultured in vitro either alone or at scaled ratios with eF670‐labeled TNFR2⁺ or TNFR2⁻ Tregs. (C) Histogram overlays showing CTV dilution and respective gMFI in gated Tconv cultured alone (black) or cocultured with TNFR2⁺ (red) or TNFR2⁻ (blue) Tregs in the indicated conditions. (D) Analysis of the gMFI of CTV and of the percentage of proliferating cells in gated Tconv in the indicated conditions. (E, F) Representative cytograms and cumulative analysis showing the percentages and the counts of Treg in each coculture condition. Data are from one representative of two independent experiments. Each condition was tested in 2–6 replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by unpaired t‐test with Holm–Sidak correction for multiple comparisons.