(A) GM + IL-4 DCs have a more mature phenotype. Purified DCs from BM cultures with GM or GM + IL-4 were analyzed by flow cytometry for expression of CD86, MHCII, and CD11c. (B) GM DCs produce higher levels of IL-12 than GM4 DCs following LPS stimulation. GM and GM4 DCs were stimulated with LPS (10 μg/ml) and IFN-γ (50 ng/ml), and supernatants were collected at the indicated times. The levels of IL-12p70 (left panel) and IL-12 p40 (right panel) were determined by ELISA. The results shown are representative of three independent experiments. (C) GM4 DCs induce the production of type 2 cytokines by NOD T cells. CD4⁺ T cells were purified from the spleens of NOD mice and placed in culture with GM or GM4 DC. T cells from these cultures or freshly purified T cells were stimulated in wells coated with anti-CD3 mAb. Supernatants were collected after 48 h and tested for the presence of type 1 (IFN-γ) and type 2 (IL-4, IL-5, IL-10) cytokines by ELISA. The results shown represent the mean ± sem of six to seven independent experiments. **P values < 0.01, as determined by Student t test.

The microarrays and data analysis were performed as described in Materials and Methods, and a total of 15 microarrays is included in these results. (A) SOM of the most relevant genes was constructed using Genecluster2. The original dataset of 12,488 genes was filtered to exclude transcripts with invariant expression levels across all six samples. In this manner, 1045 genes (∼8%) were obtained, which were organized in a 4 × 3 SOM. The 12 clusters are represented graphically with the number of genes in each cluster indicated at the top of each panel. In each panel, the first and second lines represent the expression pattern for GM and GM4 DCs, respectively, at 0, 6, and 20 h following LPS stimulation. The y-axis represents the normalized expression levels. (B) Six hundred sixty-three genes were identified as most differentially expressed between GM and GM4 DCs according to the algorithm defined in Materials and Methods. Plots of the expression of these genes by GM DC (x-axis) and GM4 DC (y-axis) at t = 0, t = 6, and t = 20 are shown in the indicated panels.

(A) Flow cytomettic analysis of purified GM (upper panels) and GM4 DCs (lower panels). The thin lines represent the isotype control and the thick lines, staining with the indicated antibody. The experiment shown is representative of three independent experiments. (B) Microarray results for OX40L and CD200. (C) OX40L^−/− and WT DCs induce similar expansion of Tregs at low-dose antigen. BDC2.5 CD4⁺ T cells were stimulated with NOD (■) or OX40L^−/− (▴) DCs in the presence of the indicated peptide doses. After 5 days, the cells were stained for the expression of CD3, CD4, and Foxp3, and the percentage of Foxp3⁺ cells on gated CD3⁺ CD4⁺ T cells is shown. Results represent mean ± sem of six to 10 independent experiments. (D) OX40L^−/− and WT DCs induce similar levels of Th2 differentiation. BDC2.5 CD4⁺ T cells were stimulated with NOD (■) or OX40L^−/− (▴) DCs in the presence of the indicated peptide doses. After 5 days, equal numbers of cells were restimulated on anti-CD3-coated plates. Twenty-four hours later, supernatants were collected and assayed for IL-4 levels by Luminex. Results represent mean ± sem of two to three independent experiments. AV10, .

(A) Western blot analysis of supernatants collected from GM4 and GM DCs, 24 h after stimulation with medium (Med), LPS (L), LPS + IFN-γ (L+I), or IFN-γ (I). The blots were probed with a polyclonal rabbit anti-Ym-1 antibody as described in Materials and Methods. The experiment shown is representative of three independent experiments. (B) Expression of Ym-1 mRNA, as detected by microarray, showing the same pattern as seen with the protein production.

(A) Supernatants collected from GM4 (n=8) and GM (n=5) DCs after 24 h in medium were analyzed using a 17-plex Beadlyte kit/Luminex (Upstate). (B and C) Migration of Th2 (B) and Th1 (C) cells to supernatants from GM and GM4 DCs, as detected by a transwell chemotaxis assay. Th2 and Th1 cells were derived from CD4⁺ T cells, purified from NOD spleen, which was cultured for 3 days in type 2 or type 1 skewing conditions, respectively. Positive and negative controls were CCL19 and medium (Med), respectively. *P value <0.05 as determined by ANOVA with Tukey-Kramer post test. (D) In vivo migration of Th1 cells is influenced by the presence of GM4 DCs. GM or GM4 DCs were injected (0.5×10⁶/mouse) into the flank of a NOD-SCID mouse. One day later, PFPE-labeled BDC2.5 Th1 and Th2 cells were injected i.p., and the pancreas and draining and nondraining LNs were analyzed by NMR. The cell quantification was based on NMR analysis of tissue samples (see Materials and Methods). NMR detection of Th1 or Th2 cell numbers in the pancreas in the absence or presence of GM4 or GM. Results represent means ± sd of three independent experiments. (E) Detection of Th1 but not Th2 cells in the LNs draining the site of GM4 DC injection. No T cells were detected in the draining LN (DLN) adjacent to GM DC injection. Results represent means ± sd of three independent experiments.