C3^-/-macrophages have reduced potency to stimulate alloreactive T cells. Thioglycollate-elicited macrophages were prepared from C3^+/+ and C3^-/- mice. Alloreactive CD3⁺ T cells were prepared from either naive BALB/c mice or BALB/c mice that had been primed by grafting with C57BL/6 donor skin. Syngeneic CD3⁺ T cells were prepared from C57BL/6 mice. (A) Macrophages (2 × 10⁵) and T cells (2 × 10⁵) were cocultured for 3 days; supernatants were collected for measuring the production of IL-2 and IFN-γ by ELISA. (B) Macrophages (2 × 10⁵) and T cells (1 or 2 × 10⁵) were cocultured for 3 days, after which T-cell proliferation and cytokine assays were performed. T-cell proliferation was assessed using Non-Radioactive Cell Proliferation Assay Kit. The absorbance at 490 nm reflecting the amount of formazan in cell culture medium was measured. All data were generated from 3 independent experiments with 3 mice/group in each experiment and analyzed by Student t test. P values are for comparisons between C3^+/+ and C3^-/- macrophages, ^*P < .05; ^**P < .005; ^***P < .001; ns, no significant difference. Error bars indicate SEM.