Il 10

The cells were thawed and washed with PBS first and PBS/1% BSA later and then stained with surface antibodies for 30–60 minutes. Surface antibodies used were CD3 - Amcyan, CD4 - APC-H7 and CD8 - PE-Cy7 (all from BD Biosciences). The cells were washed and permeabilized with BD Perm/Wash buffer (BD Biosciences) and stained with intracellular cytokines for an additional 30 min before washing and acquisition. Cytokine antibodies used were IL-10 (BD Pharmingen), IL-19, IL-24 and IL-26 (R&D Systems). Flow cytometry was performed on a FACS Canto II flow cytometer with FACSDiva software v.6 (Becton Dickinson). The lymphocyte gating was set by forward and side scatter and 100,000 lymphocyte events were acquired. FMO gating was used for intracellular cytokine detection. Data were collected and analyzed using Flow Jo software. All data are depicted as frequency of CD4⁺ or CD8⁺ T cells expressing cytokine(s). Values following media stimulation are depicted as baseline frequency while frequencies following stimulation with antigens or anti-CD3 are depicted as net frequencies (with baseline values subtracted).

To assess the effector activity of CMV-CTL by intracellular cytokine staining, expanded cells were co-cultured for 6 h with CMV-loaded or control feeders labelled with CFSE at a 2.5:1 ratio, in the same way as for surface activation marker expression analysis. The incubation was done in the presence of 1 μg/ml anti-CD28 (BD Bioscience) and 1 μg/ml of brefeldin A (Sigma-Aldrich). Following the incubation, 1 × 10⁶ cells were stained with either PE-conjugated anti-human IFN-γ, IL-2, IL-10, TNF-α, or Granzyme B (BD Biosciences) and with CD8-PerCP and CD4-APC-Cy7 monoclonal antibodies, then fixed and permeabilized (Intrastain; DakoCytomation, Ely, UK), according to the manufacturer’s instructions.

For ex vivo stimulation experiments splenocytes were obtained from mice at day 5 of i.v. infection with C. albicans and stimulated ex vivo with LPS (10 ng/mL) heat-killed Candida yeast (1 × 10⁷/mL) or heat-killed C. albicans hyphae (1 × 10⁶/mL). Splenocytes were obtained by gently squeezing spleens in a sterile 100 mm filter. After centrifugation and washing, splenocytes were resuspended in complete RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM 2-mercaptoethanol, and counted using a hematocytometer. Splenocyte concentration was adjusted to 5 × 10⁶/mL. 200 μL of the cell suspension were cultured in round-bottom 96-well plates (Corning, Durham, NC) and stimulated with RPMI or 1 × 10⁶ heat-killed C. albicans yeast or hyphae/mL. The measurement of cytokine concentrations was performed in supernatants collected after 48 h of incubation at 37°C in 5% CO_2. Cytokine production from mouse cells was determined in supernatants using commercial ELISA kits for IL-1β, TNFα, IL-6, IFNγ and IL-10, all from BD Pharmingen (San Diego, CA).

Cells suspensions were prepared from lymph nodes and pancreatic islets. Single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies: anti-mouse CD3, CD4, CD8, ST2, and CXCR3 (BD Biosciences), CD11c and CD11b antibodies (BioLegend, San Diego, CA) or with isotype-matched control and analyzed on a FACSCalibur (BD) using CELLQUEST software (BD). The intracellular staining was performed with lymph node cells incubated for 6 h in the presence of Phorbol 12-myristate13-acetate (50 ng/ml) (Sigma, USA), Ionomycin (Sigma, USA) (500 ng/ml), and GolgyStop (BD Pharmingen) at 37°C, 5% CO2, stained with anti-CD4 monoclonal antibodies or appropriate isotype controls, fixed and permeabilized with a Cytofix/Cytoperm solution. Intracellular staining was performed using monoclonal antibodies: IFN-γ, IL-17, IL-10, IL-5, IL-13, IL-2, and Foxp3 (BD Biosciences) or appropriate negative controls. Cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).