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. Author manuscript; available in PMC: 2010 Apr 6.
Published in final edited form as: J Immunol. 2009 Sep 4;183(7):4475–4482. doi: 10.4049/jimmunol.0901283

FIGURE 1.

FIGURE 1

IL-12 induces IL-10 production from human memory CD4+ T cells in a PI3K-dependent manner. A, Cell-sorted autologous human naive (CD45RA+CD45RO−) and memory (CD45RO+CD45RA−) CD4+ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml) in the presence or absence of human IL-12p70 (500 pg/ml) and cell-free supernatants were analyzed by ELISA on day 3. B, Cell-sorted human memory CD4+ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml) in the presence or absence of human IL-23 (500 pg/ml) and cell-free supernatants were analyzed by ELISA on day 3. C, Cell-sorted human memory CD4+ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml) in the presence or absence of human IL-12p70 (500 pg/ml). Monensin was added during the last 6 h of a 72-h culture and cells were analyzed for intracellular IL-10 by flow cytometry. D, Cell-sorted human memory CD4+ T cells were stimulated with immobilized anti-CD3 (3 μg/ml) in the presence or absence of IL-12 (500 pg/ml) and analyzed for spot-forming units by ELISPOT on day 4. E, Cell-sorted human memory CD4+ T cells were pretreated with a PI3K inhibitor (wortmannin; 100 nM) or DMSO (0.1%) alone and stimulated with plate-bound anti-CD3 (3 μg/ml) in the presence or absence of human IL-12p70. IL-10 mRNA levels were analyzed by real-time PCR on day 3 and related to the endogenous housekeeping gene GAPDH. F, Cell-sorted human memory CD4+ T cells were pretreated with a PI3K inhibitor (LY294002; 25μM) or DMSO (0.1%) alone and stimulated with plate-bound anti-CD3 (3 μg/ml) in the presence or absence of human IL-12p70 and cell-free supernatants were analyzed by ELISA on day 3. Results are representative of at least three individual experiments. *, p < 0.05 determined by ANOVA and post hoc Tukey test.