IL-21 indirectly affects Treg homeostasis. (A) BALB/c CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3 and CD19⁺ B cells (5 × 10⁴) alone or in the presence of IL-21. Plots show secreted IL-2 levels for gated CD4⁺Foxp3⁻ cells at day 3. (B) IL-21R–deficient CD4⁺CD25⁻ Tconv were cultured with anti-CD3 and CD19⁺ B cells. Plots show intracellular IL-2 staining at day 3 for gated CD4⁺Foxp3⁻ cells. (C) BALB/c CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3, CD19⁺ B cells (5 × 10⁴), and CD4⁺CD25⁺ Tregs (1.25 × 10⁴) alone or in the presence of IL-21. The percentage of CD4⁺Foxp3⁻ cells staining for intracellular IL-2 at day 3, and representative contour plots, are shown. (D) BALB/c CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3, CD19⁺ B cells (5 × 10⁴), and CD4⁺CD25⁺ Tregs (1.25 × 10⁴) alone or in the presence of either IL-21 or anti–IL-2 Ab. The relative number of CD4⁺Foxp3⁺ Tregs is shown at day 3. (E) BALB/c IL-21R^−/−CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3, CD19⁺ B cells (5 × 10⁴), and IL-21R^+/+CD4⁺CD25⁺ Tregs (1.25 × 10⁴) in the presence or absence of IL-21. The relative number of CD4⁺Foxp3⁺ Tregs is shown at day 3. (F) CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3, CD19⁺ B cells (5 × 10⁴) and CD4⁺CD25⁺ Tregs (1.25 × 10⁴) alone or in the presence of IL-21. Histograms represent pSTAT5 staining for gated CD4⁺Foxp3⁺ cells at day 3 in the presence or absence of IL-21 compared with levels in unstimulated control T cells. (G) CD4⁺CD25⁻ Tconv (2.5 × 10⁴) were cultured with anti-CD3 and CD19⁺ B cells (5 × 10⁴) alone or with CD4⁺CD25⁺ Tregs (2.5 × 10⁴) in the presence of IL-21 where indicated. Tregs were preincubated alone or with 20 ng/mL IL-2. At day 3, cells were analyzed by flow cytometry, and the relative Tconv cell count is shown. The average Tconv cell number in the absence of cytokine or Treg was 111 995 ± 22 306. Data are representative of at least 3 independent experiments. *P < .05; **P < .01. ***P < .001. ns indicates not significant.