The IL-4 Receptor α-Chain-Binding Cytokines, IL-4 and IL-13, Induce Forkhead Box P3-Expressing CD25⁺CD4⁺ Regulatory T Cells from CD25⁻CD4⁺ Precursors

Characterization of IL-4-generated CD25⁺ Tregs. Freshly isolated human naive CD4 T cells either unfractionated or depleted of CD25⁺ T cells where indicated were cultured in the presence of autologous APC and in the presence or the absence of IL-4. After 10 days of culture, CD4 T cells were sorted into CD25⁻ and CD25⁺ T cells using a magnetic bead CD25⁺ cell depletion system. A, CD25⁺ and CD25⁻ T cells isolated from IL-4-treated cultures of naive CD4 T cells either initially depleted of CD25⁺ cells (depleted) or unfractionated (unseparated) were analyzed for their proliferative capacity in response to anti-CD3 and for the expression of Foxp3 mRNA. The left panel demonstrates proliferation of 25 × 10³ CD25⁻ T cells in the absence or the presence of increasing numbers of CD25⁺ T cells in response to anti-CD3. The right panel shows Foxp3 mRNA expression by CD25⁺ and CD25⁻ T cells relative to expression by CD25⁻ T cells. The results of one representative experiment of six performed for proliferative capacity and five performed for Foxp3 mRNA levels are shown. B, Expression of CTLA-4 and GITR by CD25⁺ and CD25⁻ T cells isolated from IL-4-treated cultures either directly after purification (nonactivated) or after 24-h stimulation with anti-CD3 (αCD3-activated). C, CD25⁺ and CD25⁻ T cells isolated from cells cultured in the presence or the absence of IL-4 were stimulated with PMA/ionomycin for 24 h. Levels of IL-10, IL-4, IFN-γ, and TNF were determined in supernatants by ELISA. The results of 12 independent experiments using cells from different donors (demonstrated by circles) are shown. n.s., nonsignificant. D, The proliferative capacity of CD25⁺ and CD25⁻ T cells isolated from cultures of IL-4-stimulated cells in response to anti-CD3 was assessed. The left panel shows inhibition of the proliferation of CD25⁻ T cells by CD25⁺ T cells in the presence of neutralizing Abs to IL-4, IL-10, and TGF-β. The right panel demonstrates proliferation of CD25⁻ cells when CD25⁺ cells were kept in a Transwell. A representative experiment of five (left panel) and four (right panel) performed is shown.

Endogenously produced cytokines in the IL-4-induced differentiation of CD25⁺ Tregs. Freshly isolated human naive CD4 T cells were cultured in the presence of autologous APC, IL-4, and neutralizing Abs to TGF-β, IL-10, or IL-2 or the appropriate isotype-matched Abs. After 10 days of culture, CD4 T cells were analyzed for the extent of expansion, as calculated by comparison of cell numbers before and after the 10-day culture, for the frequency of CD25⁺ cells, for the absolute number of CD25⁺ cells generated from an initial 1 × 10⁶ CD4 T cells, and for the proliferative capacity of CD25⁺ and CD25⁻ T cells in response to anti-CD3. A, The effects of endogenous TGF-β and IL-10 on the expansion of CD4 T cells (a), the frequencies (b) and absolute numbers (c) of recovered CD25⁺CD4 T cells, and the proliferation of recovered CD25⁺ and CD25⁻ cells isolated from cultured cells in response to anti-CD3 (d) were investigated by addition of neutralizing anti-TGF-β (αTGFβ), anti-IL-10 (αIL-10) or isotype-matched (IgG2b) Abs. The results of seven independent experiments using cells from different donors (demonstrated by circles or as the mean ± SD) are shown. B, The effects of endogenous IL-2 on the expansion of CD4 T cells (a), the frequencies (b) and the absolute numbers (c) of recovered CD25⁺CD4 T cells, and the proliferation of recovered CD25⁺ and CD25⁻ cells isolated from cultured cells in response to anti-CD3 (d) were investigated by addition of neutralizing anti-IL-2 (αIL-2) or isotype-matched (IgG2a) Abs. The results of 14 independent experiments using cells from different donors (demonstrated by circles or as the mean ± SD) are shown. n.s., Nonsignificant.

Effect of Th2 cytokines on the induction of CD25⁺ Tregs. Freshly isolated human naive CD4 T cells were cultured in the presence of autologous APC and in the presence of IL-4, IL-5, IL-9, or IL-13. After 10 days of culture, CD4 T cells were analyzed for the extent of expansion, as calculated by comparison of the cell numbers before and after the culture (A), for the frequency (B) and the absolute numbers (C) of recovered CD25⁺ cells, and for the proliferative capacity of CD25⁺ and CD25⁻ T cells isolated from cultured cells in response to anti-CD3 (D). The results of nine independent experiments using cells from different donors (demonstrated by circles or as the mean ± SD) are shown. n.s., Nonsignificant.

Analysis of the Ag specificity of IL-4-induced CD25⁺ Tregs. Freshly isolated human naive CD4 T cells were cultured for 10 days in the presence of autologous APC and in the presence or the absence of IL-4. A, The requirement for TCR and costimulatory signaling in the IL-4-mediated generation of CD25⁺ T cells was investigated by the addition of neutralizing anti-HLA-DR (αHLA-DR), CTLA-4-Ig (CTLA-4-Ig), or irrelevant mouse IgG2a (IgG2a). After 10 days of culture, CD4 T cells were analyzed for the extent of expansion, as calculated by comparison of cell numbers before and after culture (a), for the frequency of CD25⁺ cells (b), for the absolute numbers of CD25⁺ cells generated from an initial 1 × 10⁶ CD4 T cells (c), and for the proliferative capacity of CD25⁺ and CD25⁻ T cells isolated from cultured cells in response to anti-CD3 (d). The results of seven independent experiments using cells from different donors (demonstrated by circles or as the mean ± SD) are shown. n.s., nonsignificant. B, The Ag specificity of IL-4-induced CD25⁺ Tregs was tested by assessing the proliferative capacity of CD25⁺ T cells generated in the absence of IL-4 (CD25+ no IL-4) and the presence of IL-4 (CD25+ IL-4) in response to autologous (auto DC) or allogeneic (allo DC) irradiated DC, alone or in coculture. The results of one representative experiment performed in triplicate of 11 independent experiments using cells from different donors are shown.