Down-regulation of Sema-3A expression in tumor cells sensitizes T-cell activation. (A) Caki-1 and H28 cells were transfected with C-siRNA or Sema-3A siRNA (600 nM). Then, immunoprecipitated whole-cell lysates and TSNs were prepared 72 hours after transfection and analyzed as reported in Figure 1. (B) TSNs of transfected H28 cells were collected, and IL-6, TGF-β, and VEGF concentrations were assessed by ELISA. (C-D) T cells were cultured for 24 hours with either anti-CD3 + anti-CD28 alone or with the indicated TSNs (1:2) from Caki-1 cells in the absence or presence of increasing concentrations of Sema3A/Fc or Sema6A/Fc. T-cell proliferation was then assessed by [³H]-thymidine uptake (C). In parallel, supernatants of T cells were collected, and IL-2 and IFN-γ concentrations were assessed by ELISA (D). ^*P < .001, ANOVA, n = 4. Error bars indicate SEM.