Reconstitution and function of DCs in WT and RelB^−/− bone marrow chimeras. Splenocytes from WT or RelB^−/− BMCs were examined by flow cytometry for engraftment and cell subset enumeration by staining with (A) CD45.1-FITC, CD45.2-PE, and 7AAD and (B) CD19-FITC, CD3-PE, and 7AAD or I-A/I-E–FITC, PDCA-1–PE, 7AAD, and CD11c-allophycocyanin. Results represent mean ± SEM from individual animals. (C) The density gradient–enriched APC fraction was stained with 120G8-FITC, CD11c-allophycocyanin, and 7AAD; CD11c^hi 120G8^neg DCs were sorted and restained with CD8-FITC and CD4-PE. Numbers in quadrants indicate the frequency of each DC subset. Results are representative of 5 such analyses. (D) Costimulatory molecule expression by freshly sorted or cultured (without anti-CD40 mAb) sort-purified CD11c^hi DCs from naive (fresh and cultured) or irradiated (irradiated) WT (gray fill) or RelB^−/− (thick line) BMCs. Isotype control is shown as a fine line. (E) Cytokine levels in tissue culture supernatants from CD11c^hi DCs from WT or RelB^−/− BMCs cultured with anti-CD40. The dotted line in each histogram represents the cytokine concentration in supernatants of RelB^−/− BMC DCs cultured without anti-CD40 which were equivalent to levels from unstimulated WT BMC DCs. Results represent mean ± SEM from individual animals. (F) Sort-purified (H-2^b) WT and RelB^−/− BMC CD11c^hi DCs were used to stimulate naive (H-2^d) CD4⁺ T cells in allogeneic MLCs. Proliferation and cytokine content of day 4 culture supernatants were measured as described in “Materials and methods.”