C3^−/− mice have impaired Ag-specific T-cell responses and C3^−/− DCs have impaired Ag-presenting function. (A) C3^−/− mice develop reduced specific T-cell response to OVA. WT and C3^−/− mice were immunized by intraperitoneal injection with 100 μg OVA protein in incomplete Freund adjuvant. After 10 days and 60 days of immunization, mice were killed. Lymphocytes from lymph nodes and spleen were used for assessing T-cell reactivity by measuring cytokine production after restimulation ex vivo with OVA protein. Each dot represents a single animal and is shown as mean of 4 replicate wells of the ex vivo culture. A representative of 2 independent experiments is shown. (B,C) Ag presentation measured in vitro. BM DCs were prepared from WT and C3^−/− mice and loaded with OVA protein at the indicated concentrations. The Ag-loaded DCs (10⁴) were cocultured with naive OT2 CD4 T cells (10⁵) (B) or OT1 CD8 T cells (10⁵) (C). Specific T-cell responses were measured by IFN-γ and IL-2 production and thymidine uptake. All data are shown as mean plus or minus the standard error of the mean (SEM) (n = 4 for ELISA, or n = 8 for thymidine uptake). Data were analyzed by 2-way ANOVA. P values are for comparisons between WT DCs and C3^−/− DCs. A representative of 4 independent experiments is shown. (D) OVA-specific T-cell responses measured ex vivo. OVA-loaded WT or C3^−/− DCs were administered to WT syngeneic recipient mice by intraperitoneal injection (4 mice in each group). After 10 and 60 days of injection, splenocytes from these mice were restimulated ex vivo with OVA. Each dot represents a single animal and is shown as mean of 4 replicate wells of the ex vivo culture. All data were analyzed by 2-way ANOVA. A representative of 2 independent experiments is shown. (E) OVA-specific T-cell proliferation in vivo. OVA-loaded WT or C3^−/− DCs and CFSE-labeled OT2 CD4 T cells were coinjected into syngeneic WT recipient mice (3 mice per group). After 3 days, CFSE⁺ cells from spleen and lymph nodes were gated to analyze cell division. Control mice received CFSE-labeled OT2 CD4 T cells only. A representative of 2 independent experiments is shown.