Figure 5.

DC and culture supernatants of DC stimulate the proliferative responses and IFN-γ production by CD4⁺ T cells from KLH-immunized GM-CSF −/− mice. (a) CD4⁺ T cells from KLH-immunized or naive mice were stimulated with autologous spleen cells or DC pulsed with KLH as indicated. (b) To analyze the effect of supernatants from DC, 100 μl supernatants were added to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from immunized GM-CSF −/− mice at a final volume of 200 μl. Two sources of DC supernatants were used; (i) cocultures of immunized GM-CSF +/+ or GM-CSF −/− CD4⁺ T cells and KLH-pulsed autologous DC (mitomycin C-untreated) for 2 days, and (ii) cultures of DC from LPS-treated GM-CSF +/+ or GM-CSF −/− mice with 100 ng/ml rGM-CSF for 3 days. (iii) For control purposes, the effects of rGM-CSF or IL-12 added to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from immunized GM-CSF −/− mice were also tested. Proliferation was determined by incorporation of [methyl-³H]thymidine and levels of IFN-γ in the culture supernatants were measured by ELISA.