Sema-3A reduces cytokines production. (A) T cells were cultured for the indicated times with anti-CD3 (300 ng/mL) plus anti-CD28 (200 ng/mL) alone or with CM derived from Sema-3A-expressing COS-7 cells as described in Figure 2A. Supernatants were collected, and IL-2, IL-10, IFN-γ, and IL-4 concentrations were assessed by ELISA. (B) T cells were treated as described in panel A for 8 hours. Total RNA was extracted, and IL-2 expression was analyzed by real-time PCR. GAPDH were used as housekeeping gene. ^*P < .001 versus COS-7/-, ANOVA, n = 3. (C) T cells were cultured for 24 hours with anti-CD3 + anti-CD28 in the presence of CM derived from COS-7/- or COS-7/C1 clones. Recombinant human IL-2, IL-4, and IL-10 (1.0 ng/mL) were added to the culture, and T-cell proliferation was measured by [³H]thymidine incorporation. ^*P < .05 versus untreated cells, paired t test, n = 3. Error bars indicate SEM.