IL-4 enhances DC differentiation during Notch activation. (A) HPCs were plated on the fibroblasts in triplicate on 96-well plate with GM-CSF and IL-4 and cultured for 2 or 4 days. Cell proliferation was measured by [³H]-thymidine incorporation. (B) HPCs were cultured on control or Jagged-1 fibroblasts with GM-CSF and IL-4 for 7 days. Cells were labeled with APC-conjugated anti-CD11c, PE-conjugated anti-CD45, FITC-conjugated anti-IA^q, anti–B7-2, Gr-1 or anti-CD11b antibodies, and PerCP-conjugated anti-B220 antibody and analyzed on a FACSCalibur flow cytometer. Only CD45⁺ cells were evaluated. Differences between the groups were statistically significant (P < .05) for all tested cell populations. (C) DCs were generated with GM-CSF and IL-4 from HPCs as described, irradiated at 150 Gy, and cultured with lymph node cells isolated from control allogeneic BALB/c mice at different ratios. Cell proliferation was measured in triplicates as described in “Materials and methods.” Values are the average ± SE from 2 performed experiments. Differences between the groups were statistically significant (P < .05) at all LN cell/DC ratios. (D) HPCs were cultured on control or Jagged-1 fibroblasts with FL and IL-4 for 7 days. Cells were analyzed as described in Figure 6B. Differences between the groups were statistically significant (P < .05) for all tested cell populations with the exception of CD11c⁺B7-2+ cells. (E) Bone marrow–enriched HPCs were cultured with GM-CSF or GM-CSF and IL-4 on control and Jagged-1 fibroblasts. On day 5, CD45⁺ cells were isolated and nuclear protein was used for CBF-1–binding activity as described in Figure 3. Lane 1, 50-fold excess of unlabeled “cold” probe; lane 2, mutant probe; lane 3, HPCs cultured with GM-CSF on 3T3-MSCV; lane 4, cells cultured with GM-CSF on 3T3-Jagged-1 fibroblasts; lane 5, HPCs cultures with GM-CSF and IL-4 on 3T3-MSCV fibroblasts; lane 6, cells cultured with GM-CSF nd IL-4 on 3T3-Jagged-1 fibroblasts.