Sema-3A binds T cells and inhibits T-cell proliferation. (A) Resting T cells were incubated with Sema3A/Fc or hIgG and then analyzed by flow cytometry as described in “Materials and methods.” Mean fluorescence intensity (MFI) derived from 3 independent experiments is shown (left). Alternatively, cells were first incubated with the blocking anti-NP-1 antibody or a control Ab. Then, T cells were treated with Sema3A/Fc or hIgG as described (right). (B) COS-7 cells were stably transfected with a vector that allowed a high (COS-7/C1) and intermediate (COS-7/C2 and COS-7/C3) amount of Sema-3A expression, as shown by Western blot in concentrated serum-free supernatants (CM) (insert). CM (48 hours of conditioning, 1:2 of dilution) derived from COS-7 cells and Sema-3A-expressing COS-7 cells were incubated with T cells for 18 hours in the absence (Control) or in the presence of soluble anti-CD3 (300 ng/mL), either alone or with anti-CD28 (200 ng/mL) or PMA (5 ng/mL). T-cell proliferation was then measured by [³H]thymidine incorporation. ^*P ≤ .001 versus COS-7/-, ANOVA, n = 3. (C) T cells were treated with CM derived form COS-7/- and COS-7/C1 clones, stained with PI and analyzed by flow cytometry. (D) T cells were treated as described in panel A in the presence of CM derived form COS-7/- and COS-7/C1 clones for the indicated times, and the p21^Kip1 protein expression was detected by Western blotting with anti-p27^Kip1 antibody. Expression of actin was used as loading control. Data are representative of 3 experiments. Error bars indicate SEM (n = 3).