na¨ıve CD4+ T Cell Isolation Kit II and memory CD4+ T Cell Isolation Kit (Miltenyi Biotec), respectively. Naive CD4+ T cells (CD3+CD4+CD45RA + CD45RO2) typically had a purity of over 95%, as evidenced by flow cytometry. For some experiments, peripheral blood CD4+ T cells were isolated with the CD4+ T Cell Isolation Kit II (Miltenyi Biotec), followed by staining with APC- CD45RO + , FITC-CD45RA+, PE-Cy5-CD4+, and PE-labeled lineage mixture antibodies against CD8, CD14, CD16, CD19, CD56, CD11c, cd-TCR, CD11c, CD25, and BDCA-2, and were sorted into two fractions of CD4+CD45RO+CD45RA2 and CD4+CD45RA+CD45RO2 with cell purity over 99%, on a FACSAria (BD Bioscience). Stimulation of monocyte-derived DCs Monocyte-derived DCs were seeded at a final concentration of 106 cells per ml in RPMI culture medium in flat-bottomed 24-well plates (Falcon). Spores or hyphae of each opportunistic fungus or yeast cells of each strain of Histoplasma were counted, and added to DC cultures at a final concentration of 106 cells per ml (1:1 E:T ratio). In order to normalize for the amount of spores and hyphae that were co-cultured with DCs and minimize the effects of the increased fungal biomass of hyphae, we used early germinating hyphae (fig. 2) instead of mature, branching hyphae. In pilot experiments, increasing the amount of dormant spores to 1:10 or 1: 20 didn’t have a significant effect on the type and amount of pro-inflammatory cytokines produced by DCs. Stimulation of DCs was also induced in flat-bottomed 24-well plates by adding purified b-glucan (curdlan; 100 mg/ml), or LPS (100 ng/ml). For receptor blocking experiments DCs were pre incubated with blocking antibodies for dectin-1, or the corresponding isotype control antibody for 1 h before treatment with fungi or other stimuli. T cell stimulation Na¨ıve and memory CD4+ T cell subsets were cultured in flat- bottomed 96-well plates (Falcon) in Yssel’s medium supplemented with 2 mM L-glutamine (GIBCO-Invitrogen), 1 mM sodium pyruvate (GIBCO-Invitrogen), 10 mM HEPES (GIBCO-Invitro- gen),100 U/ml penicillin, 100 mg/ml streptomycin (GIBCO- Invitrogen), and 10% FCS (v/v; GIBCO-Invitrogen), at a density of 56104 cell per well, and stimulated with plate-bound anti-CD3 (10 mg/ml) and soluble anti-CD28 (1 mg/ml) in the presence of supernatants from unstimulated or variously stimulated monocyte- derived DCs. For some experiments with na¨ıve CD4+ T cells, anti- IFN-c and anti-IL-4 were added to the cultures at a concentration of 10 mg/ml. After 5–6 days cells were collected and washed extensively and their viability was determined by trypan blue exclusion. Cells (16106 cells/ml) were restimulated with PMA (50 ng/ml; Sigma) and Ionomycin (500 ng/ml; Sigma) for 6 h (for flow cytometry intracellular staining) or in the presence of anti- CD3 and anti-CD28 for 24 h (for ELISA). Analysis of cytokine production Supernatants of stimulated DCs or T cells were collected after overnight culture. Cytokine levels in the supernatants were determined by using ELISA kits for human IL-1b, IL-6, IL-12, IL-17, IFN-c (R&D systems), and IL-23 ELISA (eBioscience) according to the manufacturer’s instructions. Cells producing IFN- c and IL-17 were analyzed by intracellular cytokine staining after the addition of GolgiStop (10 mg/ml; BD Biosciences) during the final 4 h of restimulation. Cells were made permeable with Cytofix/Cytoperm reagents (BD Biosciences). Cells were stained with FITC-anti-IFN-c (4S.B3; BD Pharmingen) and PE-anti-IL- 17 (eBio 64DEC17; eBioscience) and washed and then were analyzed by flow cytometry (FACSCalibur; BD Biosciences). IL-23 Regulates Virulence Confocal imaging studies Live spores or hyphae of opportunistic fungi and yeast cells of Histoplasma were fixed with 4% paraformaldehyde, pelleted in propylene tubes, washed twice with PBS, blocked for 30 min in PBS plus 1% goat serum, incubated for 1 h with a mouse monoclonal antibody to linear-(1,3)-b-glucan (Biosupplies; 1 mg/ ml), washed twice in PBS plus 1% goat serum and stained by a secondary AlexaFluor 488 goat anti-mouse Ab (Molecular probes). Images of PFA-fixed fungal cells were acquired using a Leica SP2 RS laser-scanning confocal microscope with an oil-immersion objective (Leica 663/1.4 numerical aperture) using identical gain settings at room temperature. The AlexaFluor 488 goat anti- mouse Ab used as flurochrome was excited with an Argon Laser. Images were collected using LCS V2.61 software (Leica Micro- systems, GmbH) and processed with Adobe Photoshop CS2. Statistics. A standard two-tailed t-test or a t-test with Welch’s correction was applied for statistical analysis by using the GraphPad Prism software. P values of 0.05 or less were con- sidered significant. Supporting Information Figure S1 TH-17 polarizing cytokines are preferentially induced by fungal hyphae in human DCs. IL-1b (A) IL-6 (B) and TNF-a produced by human monocyte-derived DCs (1610^6 cells per ml) following overnight stimulation with purified b-glucan (curdlan, 100 mg/ml), resting (spores) or invasive (hyphae) stages of growth of each opportunistic fungus (Aspergillus and Rhizopus) at a 1:1 ratio. Data are expressed as mean 6 SEM values for DCs derived from three different donors. *, P,0.001, paired Student’s t test. Found at: doi:10.1371/journal.pone.0012955.s001 (1.08 MB TIF) Figure S2 Dectin-1 inhibitor laminarin has no effect on DC activation by TLR ligands. IL-23 production by human monocyte- derived DCs pre incubated for 1 h with increasing concentrations of the dectin-1 inhibitor laminarin (0, 0.01, 0.1, and 1 mg/ml) and subsequently stimulated with LPS (100 ng/ml). Data shown are representative of 2 independent experiments. Found at: doi:10.1371/journal.pone.0012955.s002 (3.21 MB TIF) Figure S3 TH-17 polarizing cytokines are preferentially induced by fungal hyphae in human DCs. IL-1b (A), IL-6 (B), and IL-12 (C) production by DCs stimulated with hyphae of Aspergillus (white bars) or Rhizopus (black bars), or b-glucan (scattered bars) or LPS (gray bars) with (+) or without (2) pre incubation for 1 h with an anti-dectin-1 blocking antibody (10 mg/ml). Data are expressed as mean 6 SEM values for DCs derived from three different donors. *, P,0.001; **, P,0.05 paired Student’s t test. Found at: doi:10.1371/journal.pone.0012955.s003 (4.87 MB TIF) Figure S4 Aspergillus hyphae drive the expansion of IL-17 producing memory CD4+ T cells. Flow cytometry to determine the percentage of cells producing IL-17 and IFN-c among memory CD4+ T cells primed for 5 d in plates coated with anti-CD3 and anti-CD28, in the presence of supernatants of unstimulated DCs, or supernatants of DCs stimulated with either Aspergillus spores, or hyphae (above plots), and re-stimulated for 5 h with PMA and Ionomycin. Data are expressed as mean 6 SEM values for DCs derived from five different donors. Found at: doi:10.1371/journal.pone.0012955.s004 (4.54 MB TIF) Figure S5 Polymyxin B has no effect in IL-6 production by DCs activated with b-glucan or fungal hyphae. IL-6 production by DCs stimulated with hyphae of Aspergillus (white bars) or b-glucan (black bars scattered bars) or LPS (gray bars) with or without pre incubation for 10 min with increasing concentrations of polymyx- PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12955