Rras^−/− splenic APCs and BM-DCs show impaired T-cell proliferation capacity. (A) Bulk splenic APCs (129Sv; n = 3) or (B) splenic DCs (129Sv; n = 4) from Rras^+/+ or Rras^−/− mice in different ratios were comixed with 2 × 10⁵ T cells from WT Balb/c for 96 hours. Allogeneic T-cell proliferation was measured by adding [H³]-labeled thymidine for the last 16 hours, and the amount of incorporation was measured by a scintillation counter. Results represent triplicate measurements from a single experiment with data in panel A reproduced in 2 additional experiments. (C) A similar proliferation assay using WT T cells was conducted as in panels A and B, except that BM-DCs (129Sv) were used. Bars represent SD. (D) In vivo T-cell proliferation assays were performed. Rras^+/+ or Rras^−/− mice (129Sv) were injected intraperitoneally with PBS, 10 μg LPS, or 10 μg LPS and 1 mg OVA. Six hours after injection, splenic DCs were isolated and comixed with naive CFSE-labeled polyclonal syngeneic splenic T cells (129Sv) for 96 hours. CFSE⁺ staining was analyzed by flow cytometry to measure T-cell proliferation. All plots are gated for TCR⁺CD4⁺ cells. (E) Percentages from panel D for CFSE⁺ proliferating T cells are plotted. Results represent data from 2 independent experiments with Rras^+/+ (n = 5) and Rras^−/− (n = 6) mice. Bars represent median. ***P < .0005.