Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs

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1 Supplementary Fig. 1 p38 MAPK negatively regulates DC differentiation. (a) Western blot analysis of p38 isoform expression in BM cells, immature DCs (idcs) and mature DCs (mdcs). A myeloma cell line expressing all p38 isoforms was used as positive control (control). (b) Western blot analysis of p38 activity in DMSO-, SB , SB and SB treated BM cells (2-day cultures). Representative results of 3 experiments are shown. (c) Pooled data (n = 4) depicting the percentage of DC-related molecule-positive cells gated on CD11c + idcs, isb74dcs, isbdcs and isb80dcs. (d) Pooled data (n = 4) depicting the percentage of DC-related molecule-positive cells gated on CD11c + mdcs, msb74dcs, msbdcs and msb80dcs. (e) ELISA measurement of the amounts of IL-12 secreted by mdcs, msb74dcs, msbdcs and msb80dcs. (n = 3). (f) Blocking function of -CD40 mabs. idcs were treated as indicated. 48 hours later, cells were analyzed by FACS for surface expression of MHC-II and CD86. -CD40 mabs but not control IgG could inhibit the maturation of idcs by scd40l. Numbers represent mean fluorescence intensity (MFI). (g) Blocking function of - CD80 and -CD86 mabs. CFSE-labeled CD4 + CD25 Teff ( ) from WT C57BL/6 mice were cocultured with msbdcs in the presence of anti-cd3 antibody (1 g/ml) and indicated blocking mabs. Sixty hours later, CFSE intensity of Teff was measured by FACS. (n = 3). Blocking of CD86 and CD80 together could abrogate the proliferation of Teff. (h) Neutralizing function of -IL-12 mabs. Supernatant of msbdcs were treated as indicated for 2 hours and then tested by ELISA for the presence of IL-12p70. (n = 3). Representative results from one of three performed experiments are shown. Error bars in all graphs represent s.d. Statistical significance was calculated with Student s t-test. * P < 0.01

2 Supplementary Figure 2 Knockdown of p38 in DC progenitor cells induces the upregulated expression of the surface DC-related molecules. BM cells were cultured with medium containing mgm- CSF (20 ng/ml) for 24 hours, and the cells were then transfected with 100 nm non-specific or p38 specific sirna (Santa Cruz) using HiPerFect Transfection Reagent (Qiagen). After additional 6 day culture, idcs were matured by TNF- and IL-1. (a) Western blot analysis of the total and p-p38 levels in the cells 3 days after the transfection with non-specific or p38 -specific sirna. (b) FACS analysis showing the expression of DC-related molecules. Numbers are the percentages of DC-related moleculepositive cells gated on CD11c + mdcs. Data are representative of two independent experiments.

3 Supplementary Figure 3 Teff proliferation induced by p38 -specific sirna-treated mdcs (Si-p38 mdcs) was inhibited significantly by Treg when OX40L-OX40 signaling was abrogated. CD4 + CD25 Teff ( ) from wild-type C57BL/6 mice were labeled with CFSE and cocultured with mdcs, Sinon-mDCs or Si-p38 -mdcs ( ) plus anti-cd3 antibody (1 g/ml). Control medium, antibodies and/or unlabeled CD4 + CD25 + Treg ( ) were added as indicated. Sixty hours later, CFSE intensity of Teff was measured by FACS. Numbers indicate percent CFSE lo /proliferated Teff. P values were calculated with Student s t-test. Results are representative of two independent experiments.

4 Supplementary Figure 4 Effects of mdc- and Teff-expressing OX40L. (a) mdcs, msbdcs, naïve Teff, or Teff ( ) from wild-type C57BL/6 mice stimulated with mdcs or msbdcs ( ) plus anti-cd3 antibody (1 g/ml) for 24 hours were stained with antibodies against OX40, OX40L or isotype IgG. Shown are overlay histograms of mdcs, CD4 + naïve Teff or CD8 + naïve Teff. Gray-filled histograms represent isotype IgG for mdcs, OX40 and OX40L for naive Teff; thin lines indicate mdcs and Teff stimulated with mdcs; and bold lines represent msbdcs and Teff stimulated with msbdcs. Numbers are the MFI for gray-filled histograms, thin lines or bold lines from top to bottom, respectively. (b) FACS analysis of OX40L and CD86 expression by sirna-transfected mdcs and msbdcs. idcs and isbdcs (7 days culture) were transfected with 50 nm non-specific sirna or OX40L-specific sirna (Santa Cruz) by HiPerFect transfection reagent (Qiagen), and then matured by TNF- and IL-1. OX40L and CD80 expression was analyzed 2 days later. (c) CFSE-labeled CD4 + CD25 Teff ( ) from WT C57BL/6 mice were cocultured with mdcs or msbdcs, non-specific sirnatransfected mdcs (Si-non-mDCs) or msbdcs (Si-non-mSBDCs), or OX40L-specific sirnatransfected mdcs (Si-OX40L-mDCs) or msbdcs (Si-OX40L-mSBDCs) in the presence of anti-cd3 antibody (1 g/ml). Antibodies and/or unlabeled CD4 + CD25 + Treg ( ) were added as indicated. Sixty hours later, CFSE intensity of Teff was measured by FACS. Numbers indicate percent CFSE lo or proliferated Teff. P value was calculated with Student s t-test. Similar results were obtained from two independent experiments.

5 Supplementary Figure 5 msbdc hyperactivating CD8 + CTLs in the presence of Treg in vitro is dependent on OX40-OX40L ligation. CD8 + CTLs ( ) from wild-type C57BL/6 mice (a) or OX40 / C57BL/6 mice (b) were labeled with CFSE and cocultured with mdcs or msbdcs ( ) plus anti-cd3 antibody (1 g/ml). Control medium, anti-ox40l antibodies and/or unlabeled CD4 + CD25 + Treg ( ) isolated from naïve C57/BL6 mice were added as indicated. Sixty hour later, CFSE intensity of Teff was measured by FACS. Numbers indicate percent CFSE lo or proliferated CD8 + CTLs cells. P values were calculated with Student s t-test. Similar results were obtained in three independent experiments.

6 Supplementary Figure 6 Effects of p38 MAPK inhibition or silence in DCs on Treg. (a) CFSE-labeled CD4 + CD25 + Treg ( ) isolated from C57BL/6 mice were stimulated with DCs or SBDCs ( ) with anti-cd3 antibody (1 g/ml) and IL-2 (20 U/ml). Sixty hours later, CFSE intensity of Treg was measured by FACS. Numbers with s.d. indicate percent CFSE lo /proliferated Terg. (n = 3). (b) CD4 + CD25 Foxp3 Teff ( ) isolated from WT C57BL/6 mice were cocultured with mdcs, msb74dcs, msbdcs or msb80dcs ( ) in the presence of anti-cd3 antibody (1 g/ml) and IL-2 (20 U/ml) with the addition of TGF- 1 (2 ng/ml). (c) CD4 + CD25 Foxp3 Teff ( ) isolated from WT C57BL/6 mice were cocultured with mdcs, Si-non-mDCs, or Si-p38 -mdcs ( ) in the presence of anti-cd3 antibody (1 g/ml) and IL-2 (20 U/ml) with the addition of TGF- 1 (2 ng/ml). Numbers in (b-c) indicate the percentages of CD25 + Foxp3 + Treg gated on CD4 + T cells in 4-day cultures. P values were calculated with Student s t-test. (d) CD4 + CD25 Foxp3 Teff ( ) isolated from WT C57BL/6 mice were cocultured with msbdcs ( ) in the presence of anti-cd3 antibody (1 g/ml) and IL-2 (20 U/ml) with the addition of TGF- 1 (2 ng/ml). mabs were added as indicated. Numbers with s.d. indicate the percentages of CD25 + Foxp3 + Treg gated on CD4 + T cells in 4-day cultures. (n = 3). (e) CD4 + CD25 Foxp3 Teff cells ( ) were cocultured with DCs or SBDCs ( ) in the presence of TGF- 1 and IL-2 for 4 days. CD4 + T cells were purified for Western blot analysis. Shown are pstat3, Stat3, psmad3 and Smad3 levels in CD4 + T cells. Data are representative of two independent experiments.

7 Supplementary Figure 7 OX40L-blocking antibody treatment restores decreased Treg-cell population in msbdc-immunized mice. C57BL/6 mice inoculated subcutaneously with B16-OVA tumor at day 0 were immunized twice with OT-II peptide-loaded mdcs or msbdcs at days 3 and 10, with or without intraperitoneal injections of OX40L-blocking antibody (200 g every two days), starting on day 0. FACS analysis of Treg in tumor-draining lymph nodes was performed 10 days after the second immunization. Numbers represent the percentages of CD25 + Foxp3 + Treg in gated CD4 + T cells. P values were calculated with Student s t-test. Similar results were obtained from three independent experiments.

8 Supplementary Figure 8 Full unedited scans of blots shown in Figures 4. Shown are the blot scans used to generate a part of the panel of Fig. 4a. The cropped areas used in the figures are marked by boxes.

9 Supplementary Figure 9 Effects of down regulation of p38 activity on NF- B pathway. (a) BM cells were cultured with medium containing mgm-csf (20 ng/ml) for 24 hours, and the cells were then transfected with 100 nm non-specific or p38 -specific sirna (Santa Cruz) using HiPerFect Transfection Reagent (Qiagen). After additional 6-day culture, treated idcs were matured by TNF- and IL-1. Western blot analyzed the levels of cytoplasmic and nuclear p50 in untreated-, non-specific sirna treated- or p38 -specific sirna-treated mdcs. (b) Western blot analysis of I B- level in idcs, isbdcs, mdcs and msbdcs. (c) Western blot analysis of the PPARγ and pc/ebpβ protein levels in untreated-, non-specific sirna treated- or p38 -specific sirna-treated mdcs. Data are representative of two independent experiments.

10 Supplementary Figure 10 Full unedited scans of blots shown in Figures 5. Shown are the blot scans used to generate a part of the panels of Fig. 5a-b, d-e. The cropped areas used in the figures are marked by boxes.

11 Supplementary Figure 11 Antitumor activity of DC immunization. (a) C57BL/6 mice were treated with anti-cd25 antibodies (50 g/mouse) intraperitoneally. Mice (n = 3) were sacrificed at indicated days after treatment. Percentage of CD4 + CD25 + Foxp3 + Treg cells in total splenic CD4 + cells was analyzed by FACS. BT indicates before treatment. Error bars represent s.d. Representative results from one of two performed experiments are shown. (b,d,f) Comparison of antitumor activity against preestablished tumors. C57BL/6 mice (5 per group) were inoculated subcutaneously with B16-OVA tumor cells ( ) and 3 days later were immunized with unpulsed or OT-I peptide-pulsed mdcs or treated mdcs twice at a weekly interval. Arrows represent the timing of DC-therapy. Anti-CD25 antibodies (50 g/mouse) were administered intraperitoneally into some groups of mice 3 days before immunization. Error bars represent s.d. P values were calculated with Student s t-test. Representative results from one of two repeated experiments are shown. (c,e,g) IFN- ICS of splenocytes from tumorbearing mice 10 days after second immunization was performed after restimulation with the immunizing peptides overnight in vitro. Numbers represent percentages of IFN- secreting lymphocytes gated on CD8 + T cells from one representative experiment of three performed. P values were calculated with Student s t-test.

12 Supplementary Figure 12 OX40L antibody treatment abrogates the proliferation of hyperactivated OT- II T cells stimulated by msbdcs-ot-ii in tumor-draining lymph nodes. C57BL/6 mice were inoculated subcutaneously with B16 tumor cells at day 0, and were intraperitoneally injected with OX40Lblocking antibodies (200 g every two days) starting from day 9. Tumor-bearing mice were immunized with mdcs or msbdcs pulsed with 5 g/ml OT-II peptide on day 10, followed by intravenous injection of CFSE-labeled CD4 + CD25 OT-II T cells at day 11. CFSE intensity of OT-II T cells in tumor-draining lymph nodes was measured by FACS at day 15. Numbers indicate the percentage of CFSE lo /proliferated OT-II T cells. P values were calculated with Student s t-test. Similar results were obtained in two independent experiments.

13 Supplementary Figure 13 Inability of msbdcs to inhibit pre-established tumors in OX40L-blocking antibody-treated mice. C57BL/6 mice were inoculated subcutaneously with B16-OVA tumor cells at day 0. Some mice were treated with 200 g control IgG or anti-ox40l antibodies every two days starting from day 0. At days 3 and 10, mice were immunized with msbdcs-ot-ii or msbdcs-ot-i. (a,b) Tumor growth curves (5 mice/group) represent one of two independent experiments. Error bars represent s.d. Arrows represent the timing of DC-therapy. P values were calculated with Student s t-test. (c,d) IFN- ICS of lymphocytes from tumor-draining lymph nodes in tumor-bearing mice (day 20) was performed after overnight in vitro restimulation with peptides. Numbers represent the percentages of IFN- -secreting lymphocytes gated on CD4 + T cells (c) from msbdcs-ot-ii-immunized mice and CD8 + T cells (d) from msbdcs-ot-i-immunized mice. P values were calculated with Student s t-test. Similar results were obtained from two independent experiments.

14 Supplementary Figure 14 Effects of p50 signaling in msbdcs on TILs. Mice were treated as shown in Supplementary Fig 9e. (a) IFN- ICS of tumor-infiltrating CD8 + T cells isolated from mice 10 days after final immunization was performed after overnight in vitro restimulation with OT-I peptide-loaded mdcs. Numbers represent percentages of IFN- -secreting lymphocytes gated on CD8 + T cells. (b) Proliferation of TILs in the absence or presence of naïve Treg from WT C57/BL/6 mice. CFSE-labeled CD8 + Teff from TILs were cocultured with OT-I peptide-loaded mdcs, in the absence or presence of unlabeled CD4 + CD25 + Treg. Percentage of CFSE lo /proliferated TILs was analyzed by FACS 72 hours after coculture. Error bars represent s.d. (n = 3). (c) FACS analysis of the frequency of tumor-infiltrating Treg. Numbers represent the percentages of CD25 + Foxp3 + Treg in gated CD4 + T cells. (d) Reduced suppressive activity of tumor-infiltrating Treg. Splenic CD4 + CD25 Teff ( ) from OT-II mice were cocultured with OVA-loaded mdcs in the absence or presence of CD4 + CD25 + Treg ( ) from TILs. CFSE intensity of Teff was measured by FACS sixty hours later. Numbers indicate percent CFSE lo /proliferated Teff. P values were calculated with Student s t-test. Representative data from one of two repeated experiments are shown.

15 Supplementary Figure 15 Maturation status of DCs matured by different stimulus. (a) idcs and isbdcs were matured by indicated stimuli for 48 hours before analysis of express of DC-related surface molecules. All cytokines were added at 10 ng/ml. scd40l and PGE-2 were added at 0.5 g/ml. LPS, Poly I:C and CpG were used at 2 g/ml. msbsbdcs are isbdcs matured in the presence of 1.5 M of SB Shown is the percentage with s.d. of CD80-expressing CD11c + DCs, as a representative result of the maturational status of DCs. (n = 3). Similar results were obtained from two independent experiments.

16 Supplementary Figure 16 msbdcs were more resistant to cancer- and Treg cell-derived suppressive factors. CFSE-labeled splenic OT-I T cells ( ) were cocultured with DCs or SBDCs ( ) pulsed with 1 g/ml OT-I peptide. DCs or SBDCs were pretreated with B16-OVA tumor-culture supernatant (TCS), TGF- 1 (2 ng/ml), IL-10 (5 ng/ml), CD4 + CD25 + Treg cells isolated from C57BL/6 mice (at a 1:1 Treg cells to DC ratio), or control medium. Sixty hours later, CFSE intensity of OT-I T cells was examined by FACS. Shown is percentage with s.d. of CFSE lo proliferated OT-I T cells. (n = 3). Similar results were obtained from two independent experiments.