Stages of cell differentiation of murine BM DCs generated in GM-CSF + IL-4.

(A-B) Phenotype of BM DCs analyzed by flow cytometry. Cells were labeled with Cy-Chrome anti-CD11c, PE anti-CD86, and one of the following FITC-labeled mAbs: anti–MHC-I, anti–MHC-II, anti-CD40, anti-CD80, anti-CD11b, anti-CD54, or anti–OX-40L. Flow profiles illustrate the expression of specific markers on CD86⁻ DC (gray profiles) and on CD86⁺ DC (open profiles, thick line). Isotype controls are represented by open profiles in dashed lines. (C) FITC-dextran and FITC-albumin uptake by CD11c⁺immunobead-sorted CD86⁻ and CD86⁺ DC. Only CD86⁻ DCs internalized FITC-dextran and FITC-albumin at 37°C, a phenomenon down-regulated at 0°C. Results are representative of 3 independent experiments. (D) Allostimulatory activity of γ-irradiated, FACS-sorted CD86⁻ (▾), or CD86⁺ B10 DC (▿), assessed using C3H splenic T cells as responders. B10 (H2^b) DC (d 7) were set up at graded concentrations with 2 × 10⁵ responder naive C3H (H2^k) T cells, and the cultures were maintained for 72 hours. [³H]TdR was added 18 hours before harvesting. The MLR stimulatory activity of freshly isolated allogeneic (B10 [○]) or syngeneic (C3H [●]) bulk spleen cells is also shown. Results are expressed as mean cpm ± 1 SD and are representative of at least 3 separate experiments.