diffDCs preferentially chemoattract Th1 via autocrine IP-10 in vitro. imDCs and diffDCs (1 × 10⁶ cells/mL) cultured in medium alone or stimulated with 500 ng/mL LPS for 24 hours. Then IP-10–neutralizing antibody was incubated with the conditioned supernatant of diffDCs for 30 minutes. The supernatant was 10-fold diluted and used to chemoattract the Th1 or Th2 cells stained with CD4-FITC in 3.0-μm pore size transwell insert at 37°C. After 2 hours, Th1 (A) or Th2 (B) cells that had migrated into wells were harvested and counted by FACSCalibur flow cytometer and analyzed by CellQuest software. The results are expressed as migration index (number of cells that migrated in response to IP-10/number of cells that migrated in response to medium alone). *P < .05. (C) diffDCs inhibit Th1 cell proliferation. Purified CD4⁺ Th1 cells were cocultured with diffDCs as indicated in the presence of OVA323-339 peptide at a 1:10 (DC/T) ratio in round-bottom 96-well plates for 5 days. Then cells were collected and double stained with anti–CD4-FITC and 7-AAD and counted by FACS. Data are shown as mean ± SD of 3 independent experiments. (D) diffDCs sustain the intracellular expression of IL-2 and IFN-γ in Th1 cells. Purified CD4⁺ Th1 cells were cocultured with maDCs or diffDCs (LPS treated or not) in the presence of OVA peptides and brefeldin A for 24 hours. Intracellular expression of IL-2 and IFN-γ in Th1 cells was detected by flow cytometry (cytokine and CD4 double staining).