Igg2aκ

For Foxp3 staining, whole blood samples were co-stained with anti-CD4-FITC and anti-CD25-PE (PC61.5). After fixation and permealization, cells were stained with APC-labeled anti-Foxp3 mAb (FJK-16s) (all mAbs were from e-Bioscience) as described (Chen et al., 2007 (link)). For IL-6, and IL-17 staining, 2 x 10⁶ spleen cells were stimulated with 10 ng/ml phorbol myristate acetate (PMA) (Sigma) and 10 ng/ml ionomycin (Sigma) in the presence of the Golgi inhibitor Brefeldin (Sigma) (10 μg/ml) for 4 hr. The cells were then stained with FITC-labeled anti-CD4 (GK 1.5, e-Bioscience) and PE-labeled anti-IL-6 (MP5-20F3, BD Pharmingen) or APC-labeled anti-IL-17 (eBio17B7, e-Bioscience) as described (Kappel et al., 2009 (link)). FITC-labeled anti-CD8 (53-6.7) and MHC class II (M5/114.15.2, e-Bioscience) were used for FACS analysis on T cells and BMSCs. PE-labeled IgG2a, FITC-labeled IgG1, APC-labeled IgG2a and IgG2a, κ were used as isotype controls (all from e-Bioscience). Cells were analyzed on a FACScan with Cellquest software (Beckton Dickinson). For the co-cultures with T cells and BMSCs, T cells were stimulated with Brefeldin (10 μg/ml) alone for 4 hr.

Human PBMCs were isolated by density centrifugation using Histopaque-1077 (Sigma) as described (Endres et al., 1988 (link)). For stimulation experiments, the interactions were performed in round-bottom 96-well microplates with 100 μL of cells adjusted to 5 × 10⁵ PBMCs in RPMI 1640 Dutch modification (added with 2 mM glutamine, 0.1 mM pyruvate and 0.05 mg/mL gentamycin; all reagents from Sigma) and 100 μL with 1 × 10⁵ fungal cells freshly harvested or treated. The interactions were incubated for 24 h at 37°C with 5% (v/v) CO₂. In some experiments, PBMCs were preincubated for 1 h at 37°C with either laminarin (200 μg/mL), anti-TLR2 (10 μg/mL, eBioscience, Cat. No. 16-9922) or antibodies to TLR4 (10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-293072) prior to stimulation with Candida cells. Isotype matched, irrelevant antibodies, IgG₂aκ (10 μg/mL, eBioscience, Cat. No. 14-4724-85) and IgG₁(10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-52003) were used as controls for experiments assessing TLR2 and TLR4, respectively. All reagents used for the pre-incubation experiments were negative to contamination with LPS (tested with the Limulus amebocyte lysate from Sigma), and all reactions were performed in presence of 5 μ g/mL polymyxin B (Sigma) (Schwartz et al., 1972 (link)). Plates were centrifuged for 10 min at 3000 × g at 4°C, the supernatant saved and kept at −20°C until used.