IL-10 Production by Th1 Cells Requires IL-12-Induced STAT4 Activation
CD4⁺ T cells from DO11.10 animals on a (A, B, and C) WT, Stat4^−/−, or Stat6^−/− background, (D) Ifnγ^−/− background, or (E and F) Il4^−/− background were isolated and differentiated as in Figure 1. As shown in (B), Th2-driven cells were differentiated for 1 week in medium containing IL-4 (10 ng/ml) and anti-IL-12 (10 μg/ml) plus 1 μM OVA; or (C and F) 0.05 μM OVA alone (low-antigen-dose-driven Th2 cells). On day 7, cells were restimulated and IL-10, IFN-γ, and IL-4 production were detected by ICS or by immunoassay as in Figure 1. Represented is one of three experiments performed. Detection of cytokines by ELISA in the supernatants of cultures restimulated for 48 hr (data not shown) confirmed the results observed for ICS in (A) and (B). Error bars refer to the standard deviation of replicate values of three independent cultures.

High Antigen Dose and IL-12 Are Required for Maximal Production of IL-10 by Th1 Cells
CD4⁺ T cells from DO11.10 TCR transgenic animals were cultured for 1 week with a high (0.7 μM) dose of OVA and DC in the (A and B) presence or (C) absence of IL-12 and subsequently washed, counted, and recultured with a high (0.7 μM; B, D, and E) or low (0.01 μM; F) dose of OVA and DC in the absence or presence of IL-12 as indicated. CD4⁺ T cells from DO11.10 TCR transgenic animals were cultured with a high (0.7 μM) dose of OVA and WT or Il12b^−/− DCs in the absence of added IL-12 for 2 weeks (E). At the end of the first (A, C, and E) and the second week (B, D, E, and F), cells were restimulated with anti-CD3 and anti-CD28, and IFN-γ and IL-10 cytokine expression were detected by ICS. Represented is one of three experiments performed.

c-maf Is Expressed in IL-10-Producing Th1, Th2, and Th17 Cells
CD4⁺ T cells from DO11.10 TCR transgenic animals were differentiated for 1 week in medium containing increasing doses of OVA plus DC in the absence (Med) or presence of IL-12, as indicated. On day 7, cells were (A) restimulated for cytokine expression or (B) not restimulated for transcription factor expression as in Figure 1. CD4⁺ T cells from C57Bl/6 animals were isolated and cultured for 5 days in the presence of plate-bound anti-CD3 (50 ng/ml), soluble anti-CD28 (50 ng/ml), IL-6 (50 ng/ml), TGF-β (1 ng/ml), and IL-1 (10 ng/ml); Th17 cells were restimulated on day 5 (for cytokine expression) or not (for transcription factor expression) as in Figure 5C. Th1 and Th17 cells were cultured as in Figure, all in the absence (−; Control), with DMSO, or presence (+) of the PD184352 MEK inhibitor. Transcription factor expression is shown in (D). Similar results were obtained from three (A, B, and C) or two experiments (D, with two doses of inhibitor or control). Cytokine and transcription factor expression were analyzed from RNA isolated from all populations by RT real-time PCR.

IL-12 Enhances IL-10 Production at High Antigen Dose Only
Naive CD4⁺ T cells from DO11.10 TCR transgenic animals were isolated and differentiated for 1 week with splenic DCs in medium containing increasing doses of OVA in the absence (medium) or presence of IL-12, as indicated. On day 7, cells were restimulated for 4 hr with plate-bound anti-CD3 and soluble anti-CD28 and BrefeldinA and (A) stained at the single-cell level for IL-10, IFN-γ, and IL-4, or (B) stimulated similarly for 48 hr in the absence of BrefeldinA, and the culture supernatants analyzed by ELISA. Data are representative of six experiments performed; error bars in (B) refer to the standard deviation of three independent cultures.

In Vivo Generation of IL-10-Producing Th1 Cells Requires High Antigen Dose, Innate Immune Signals and STAT4
(A) Spleen cell suspensions from DO11.10 animals were injected into BALB/c recipients. Cells were isolated from the inguinal lymph nodes of mice injected with PBS, OVA protein (5 mg) plus or minus LPS (5 μg) and restimulated in vitro with OVA for 48 hr. IL-10 and IFN-γ production was analyzed by ELISA. Represented is the mean ± SD of groups of three animals each. Spleen cell suspensions from DO11.10 WT (ten animals), Stat4^−/− (nine animals), or Stat6^−/− (ten animals) were transferred into BALB/c recipients, which were then injected with OVA protein and LPS (5 mg and 5 μg, respectively). Cells recovered as above were restimulated in vitro, for 24–48 hr with the last 6 hr in the presence (for ICS, B) or absence (for ELISA, C) of BrefeldinA. IL-10 and IFN-γ expression was detected by ICS and represented within the CD4⁺ KJ1-26⁺ T cells population.
(B) We obtained the plots represented by merging the plots from each mouse within one group. Two independent experiments confirmed the result showed and the mean percentage of IL-10/IFN-γ producers ± SD was 3.95 ± 1.15 and 2.25 ± 0.68; 4.29 ± 1.36 and 7.11 ± 1.86; and 3.82 ± 0.96 and 10.19 ± 2.25 for DO11.10 WT, Stat4^−/−, and Stat6^−/−, respectively.
(C) Error bars refer to the standard deviation of replicate values on two independent experiments.

IL-10 Production by Th1, Th2, and Th17 Cells Requires ERK1 and ERK2 Activation
CD4⁺ T cells from DO11.10 TCR transgenic animals were cultured with a high (0.7 μM) or low (0.01 μM) dose of OVA and DC in the absence or presence of IL-12. On day 3, cells were harvested, rested for 5 hr in low (1%) serum medium, and restimulated with high (0.7 μM) or low (0.01 μM) antigen dose, in the (A and B) absence or in the (B) presence of IL-12, for the indicated time points. Cellular lysates were prepared, proteins were separated by SDS-PAGE, and phospho(p)- or total(t)-ERK1 and ERK2 were detected by immunoblotting. Actin was included as a loading control (A and B). CD4⁺ T cells from BALB/c animals were isolated and cultured for 7 days in the presence of increasing doses of plate-bound anti-CD3, soluble anti-CD28, and IL-12 (3 ng/ml), in the absence (control, with DMSO) or presence of U0126 (2.5 μM) (MEK Inhibitor). As shown in (C), on day 7, cells were restimulated as above and the expression of IL-10 and IFN-γ was detected by ICS. Cytokine production in the supernatants of 48 hr restimulated cultures was analyzed by ELISA and confirmed the ICS results (data not shown). As shown in (D), CD4⁺ T cells from BALB/c animals were isolated and cultured for 5 days in the presence of plate-bound anti-CD3, soluble anti-CD28 and one of the following combinations: IL-12 (3 ng/ml) plus anti-IL-4 (20 μg/ml), for Th1; IL-4 (10 ng/ml) plus anti-IL-12 (10 μg/ml), for Th2; or IL-6 (50 ng/ml), TGF-β (1 ng/ml) and IL-1 (10 ng/ml), for Th17, all in the absence (control, with DMSO) or presence of PD184352 MEK Inhibitor, as in (C). On day 5, cells were restimulated with PdBU and ionomycin, and the expression of IL-10, IFN-γ, IL-4, and IL-17 was detected by ICS. Represented is one of three (A, B, C, and F) or one of four (D and E) experiments performed.