Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation
Version of Record online: 14 FEB 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 4, pages 967–978, April 2013
How to Cite
Chang, J., Thangamani, S., Kim, M. H., Ulrich, B., Morris, S. M. and Kim, C. H. (2013), Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation. Eur. J. Immunol., 43: 967–978. doi: 10.1002/eji.201242772
- Issue online: 16 APR 2013
- Version of Record online: 14 FEB 2013
- Accepted manuscript online: 15 JAN 2013 07:53AM EST
- Manuscript Accepted: 11 JAN 2013
- Manuscript Revised: 9 DEC 2012
- Manuscript Received: 27 JUN 2012
- NIH to CHK. Grant Numbers: R01AI074745, R01DK076616, R01AI080769
- SMM. Grant Number: R01GM57384
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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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