Figure 3.

Tpres cells are enriched in Treg and Tresp cells in Th17

(A) Experimental setup. After purification by cell sorting, AND Tpres cells are incubated for 3–6 days in the absence or presence of naive AND Tresp cells.

(B) ELISA measurement of IL-2 and TNF-α concentrations in supernatants of Tpres cells cultured for 3 days in the presence or absence of Tresp cells. The bar graphs show the means ± SEMs of triplicates.^∗∗p < 0.01 (2-tailed unpaired Student’s t test).

(C) Expression of CCR6, CD25, and Foxp3 by Tpres and Tresp cells from the experiment in (B), measured by flow cytometry. Bar graphs represent means ± SEMs (n = 3). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 (2-tailed unpaired Student’s t test).

(D) Generation of FoxP3⁺CD25⁺ T cells within AND Tpres and Tresp cell populations. After overnight incubation with MCCp-loaded BMDCs and purification, Tpres were cultured alone (Tpres alone) or together with naive AND Tresp cells (Tpres+Tresp) for 6 days and then analyzed by flow cytometry for Foxp3 and CD25 expression. In parallel, AND T cells were cultured uninterrupted for 6 days with MCC-loaded BMDCs (DC+Tpres). Data represent the means ± SEMs of biological triplicates. ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001; ns, not significant (2-way ANOVA test).

(E) Tpres and Tresp cells were stained with surface CD25 and intracellular IL-17A, Foxp3, RORγt, or IFNγ. Two-color contour plots are on the left and quantification in the bar plots to the right. ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001 (2-tailed unpaired Student’s t test).

(F) Quantification via qRT-PCR of mRNA expression of sorted Tpres, Tresp cells, and naive AND T cells. Data are presented as the means ± SEMs of n = 2–4 biological replicates normalized to the mean values of naive cells (set as 1). ^∗p < 0.05; ^∗∗p < 0.01 (2-tailed unpaired Student’s t test).