Dependence of CD4 T-cell response on the C3 status of donor macrophages. (A-B) Thioglycollate-elicited macrophages were prepared from C3^+/+ and C3^-/- mice. Primed alloreactive CD3⁺, CD4⁺, and CD8⁺ T cells were prepared from BALB/c mice. Pooled macrophages (2 × 10⁵) from each group were cocultured with 2 × 10⁵ T cells for up to 5 days. Supernatants were collected for measuring the production of IL-2 (A) and IFN-γ (B) by ELISA. (C) Normal peritoneum or BM macrophages were prepared from C3^+/+ and C3^-/- mice. The pooled macrophages without further purification with CD11b beads were stimulated with LPS (100 ng/mL) for 24 hours and then cocultured with primed alloreactive CD4⁺ T cells for 3 days. Supernatants were collected for measuring the production of IL-2 and IFN-γ by ELISA. All data in panels A and B were generated from 3 independent experiments, with 5 mice/group in each experiment. Data were analyzed by variance component regression analysis. (C) The data were generated from 3 experiments, with 3 mice/group in each experiment, and analyzed by 2-way ANOVA. P values are for comparisons between C3^+/+ and C3^-/- macrophages. Six additional T-cell stimulation experiments were performed, in which CD3 or CD4 T cells were incubated for only 3 days with thioglycollate-elicited macrophages. The data are shown in Figure S2. Error bars indicate SEM.