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Figure S1. Effects of RA and Ro41-5253 on survival of BM-DCs. BM-DCs were prepared by culturing mouse bone marrow cells with GM-CSF for 9-10 days. RA (10 nM) or Ro41-5253 (Ro41; 100 nM) was added as indicated during culture. Dead cells were detected with staining first with anti-CD11C-PerCP and then with propidium iodide and annexin V (BioLegend) before flow cytometric analysis. The experiment was repeated 3 times and one set of representative data are shown.

Figure S2. Cell surface phenotype and T cell priming activities of RA and Ro41 BM-DCs. BM-DCs were prepared by culturing mouse bone marrow cells with GM-CSF for 9-10 days. RA (10 nM) or Ro41-5253 (100 nM) was added as indicated during this culture. BM-DCs were examined at the immature state or after activated with LPS for 24 h. (A) Indicated antigens were examined with antibodies to CD40 (3/23), CD80 (16-10A1), CCR7 (4B12), and MHC I-A/I-E (M5/114.15.2). An isotype control antibody was used to establish the background fluorescence level for pooled DCs. (B) DCs were co-cultured with naïve CFSE-labeled OT-II CD4+ T cells at 1:10 ratio for 3 days in the presence of OVA323-339 and CFSE dilution as the result of T cell proliferation was examined. This experiment was repeated 3 times, and the results were pooled and shown in the graph.

Figure S3. Genes that are regulated by RA in DCs in a heat map format. Microarray analysis on the RA and Ro41 BM-DCs was performed using Mouse 430 2.0 chips (Affymetrix, Inc.). The study was performed also on LPS-activated BM-DCs. Fold ratios of RA-regulated over Ro41-regulated genes are shown. Two sets of data, one with BALB/C and the other with C57BL/6 mice, are shown.

Figure S4. Flow cytometry detection of Arg1 expression by BM-DCs generated with GM-CSF and FLT3L. BM-DCs were prepared by culturing mouse bone marrow cells with GM-CSF or FLT3L for 9-10 days. RA or Ro41 was added as indicated during this culture. The expression level of intracellular Arg1 based on mean fluorescence intensity was determined with flow cytometry. Pooled data from 3 different experiments are shown. * Significant differences. P values based on Student t test from the left to right and top to bottom are 0.013, 0.01, 0.046, and 0.044.

Figure S5. Effects of arginase inhibitors on survival of BM-DCs. BM-DCs were prepared by culturing mouse bone marrow cells with GM-CSF for 9-10 days as described in Fig. S1 and the DCs were further cultured with BEC (10 μM) or Nor-NOHA (10 μM) for 3 days. Dead cells were detected with staining with propidium iodide and annexin V. The experiment was repeated 3 times and one set of representative data are shown.

Figure S6. Impact of arginase on Th17 cells. Arginase inhibitors (A) or Arg1-deficient BM-DCs (B) were used. Control, RA, and Ro-DCs were generated from the marrow cells of 6-8 week-old WT (A) or 12 to 13 day-old WT and Arg1-deficient pups (B). These DCs were activated for 24 h with LPS before co-culture with T cells. CD4+ T cells (1 × 105/well) and DCs (1 × 104/well) were co-cultured in the presence of SEB for 6 days in U-bottomed 96-well plates. TGF-β1 (5 ng/ml), mIL-6 (20 ng/ml), mIL-21 (10 ng/ml), mIL-23 (10 ng/ml), mIL-1β (10 ng/ml), mTNF-α (10 ng/ml), anti-mIL-4 (11B11, 10 μg/ml), anti-mIFN-γ (XMG2.4, 10 μg/ml), and LPS (1 μg/ml) were added. Induction of Th17 cells was examined after culture. Error bars indicate SEM of 5 data sets. P values based on Student t test from left to right are 0.0001, 0.018, 0.0005, and 0.0001.

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