Ionomycin

Surface staining was performed on PLN cells incubated with 10% rabbit serum to block non-specific binding for 20 min and then incubated with anti-CD3, CD4, CD8, CD11c, B220, MHC-II, CD86, PD-L1, CD25, CD69, and CCR4 conjugated to the following fluorochromes: PE, PE-Cy7, FITC, APC, APC-Cy7 or PerCP. For the Foxp3 staining, the cells were fixed and permeabilized using the BD Pharmingen mouse Foxp3 buffer set (BD Bioscience, San Diego, CA, USA) and then incubated with anti-Foxp3 antibody conjugated to PE for 30 min. For the intracellular cytokine staining, PLN cells were incubated with PMA (500 μg/ml), ionomycin (50 μg/ml), and GolgiStop (BD Biosciences, San Diego, CA, USA) for 6 h at 37 °C. Extracellular staining was performed, followed by fixation and permeabilization using the Cytofix/Cytoperm kit (BD Bioscience, San Diego, CA, USA). Anti-IFN-γ and IL-17 antibodies conjugated to PE or APC were added to the cell suspension and incubated for 30 min. The cells were acquired on a flow cytometer (FACS CantoII; BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

Whole blood (1 mL) was mixed with 1 mL of either non-activating medium [10% fetal calf serum (FCS)-supplemented RPMI 1640 with 4 μL/mL GolgiStop (BD Biosciences, Franklin Lake, NJ)] or activating medium [non-activating medium with 50 ng/mL phorbol myristate acetate (PMA) and 5 μg/mL ionomycin (BD Biosciences, Franklin Lake, NJ)], and incubated at 37°C in an atmosphere with 5% CO₂ for 5 hours. After washing with PBS, the cells were collected by centrifugation and adjusted to 5 × 10⁵ white blood cells per test, and stained with PECy5-labeled anti-human CD4 monoclonal antibody (BD Biosciences, Franklin Lake, NJ). Cell fixation and permeabilization were performed with FACSTM Perm 2 (BD Biosciences), according to the manufacturer's instructions. Intracellular cytokines were stained with FITC-labeled anti-human IFN-γ and PE-labeled anti-human IL-4 monoclonal antibodies (BD Biosciences). IFN-γ- and IL-4-producing CD4+ T cells were analyzed on a FACS Calibur (BD Biosciences). Nonspecific staining with an isotype-matched control monoclonal antibody was <1%.

The mouse spleens were digested with 1 mg/mL collagenase I (Gibco) in Hank’s balanced salt solution (HBSS; Life Technologies) and stained. The bone marrow (BM) was prepared from femur and BD Pharm Lyse (BD Biosciences) was added to lyse red blood cells. TruStain FcX antibody (BioLegend, San Diego, CA) was applied to block non-specific binding for 10 min at 4 °C in FACS buffer (Ca²⁺/Mg²⁺-free PBS with 1% human bovine serum albumin, 4% FBS, and 0.5 M EDTA) before staining (30 min) with appropriate antibodies. For intracellular staining, SVCs isolated from epididymal white adipose tissue (eWAT) were stimulated for 5 h at 37 °C with PMA, ionomycin, and GolgiStop (BD). Stimulated cells were washed with PBS, fixed, and permeabilized by Cytofix/Cytoperm kit (BD) as per the manufacturer’s protocol. Abs were purchased from BioLegend or R&D Systems: For mouse, CD45R/B220 (30-F11), F4/80 (BM8), Ly-6C (HK1.4), Ly-6G (1A8), CD3 (17A2), CCR7 (4B12), CD8a (53-6.7), CD11b (FAB1124S), CD4 (FAB554S), DARC (FAB6695A), CD206 (C068C2), IL-22Ra1 (FAB42941P), IL-22 (Poly5164), and IL-10 (JES5-16E3); for human, CD4 (RPA-T4), CD8 (SK1), CD14 (63D3), CD11b (ICRF44), CD16 (3G8), DARC (Clone #358307), IL-22Ra1 (Clone #305405), CD86 (IT2.2), and CD206 (15-2). Isotype control forward- and side-scatter parameters were used to remove the cell aggregates and debris.

To ascertain cytokines in CD4 T-cells, co-cultures were treated with phorbol-12-myristat-13-acetat (PMA) (2 μg/ml)/Ionomycin (1 μM, both Sigma-Aldrich, Munich, Germany) for 4 h on day 6 of co-culture. Intracellular staining was performed as indicated above. For measurement of secreted cytokines on day 6 of co-culture, DN T-cells and CD4 T-cells were separated using anti-CD4+ magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity was confirmed with flow cytometry (>95%). CD4 T-cells were stimulated with PMA/Ionomycin, supernatants were collected after 6 h and analyzed simultaneously for IL-2, IL-4, IL-6, IL-10, IL-17A, TNF, and IFN-γ secretion using the human Th1/Th2/Th17 cytokine cytometric bead array kit (BD Biosciences, Heidelberg, Germany).

The following monoclonal antibodies were used for flow cytometric analysis of immune cells: anti-mouse CD4 (BD Pharmingen, Cat. 553088), anti-mouse IFN-γ (BD Pharmingen, Cat. 560660), anti-mouse IL-4 (BD Pharmingen, Cat. 560699), IL-17a (BD Pharmingen, Cat. 561020), CD8 (Biolegend, Cat. 100744), B220 (BD Pharmingen, Cat. 563894), CD11c (BD Pharmingen, Cat. 746392), CXCR5 (Biolegend, Cat. 560617), CD138 (BD Pharmingen, Cat. 568626), IgD (BD Pharmingen, Cat. 553510), CD19 (Biolegend, Cat. 115530), CD45 (BD Pharmingen, Cat. 560510), CD11b (BD Pharmingen, Cat. 564454), and Gr-1 (Biolegend, Cat. 108410). For cytokine analysis, cells were stimulated in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, HyClone), PMA, ionomycin, and GolgiPlug (BD Biosciences, Cat. 550583) at 37°C and 5% CO₂ for 6 h. For intracellular staining, cells were fixed and permeabilized with a Human Foxp3 Buffer Set (BD Biosciences, Cat. 562574) or Cytofix/Cytoperm (BD Biosciences, 554722) according to the manufacturer’s instructions.