Anti ifn γ apc xmg1.2

Cellular staining and flow cytometry analyses for T helper cells and neutrophils were conducted as previously described [9 (link)]. Briefly, following stimulation with PMA and ionomycin, NALT cells were treated with Brafeldin A and stained for surface markers with anti-CD4-FITC (GK1.5, BD Biosciences) for T helper cells and with anti- CD11b-FITC (M1/70, Biolegend) and anti- GR-1-APC (RB6-8C5, Biolegend) for neutrophils. For intracellular staining, fixed cells were permeabilized and stained with anti-IL-17A-PE (TC11-18H10, BD Biosciences) for Th17, anti-IFN-γ-APC (XMG1.2, BD Biosciences) for Th1 cells and anti-IL-4-PerCP-eFluo^®710 (11B11, eBioscience) for Th2 cells. Samples were analyzed on a FACSAriaII flow cytometer (BD Biosciences) using FlowJo software (Tree Star).

For intracellular cytokine staining (ICS), ACK lysis buffer-treated whole-blood PBMCs were incubated for 5.5 to 6 h in the presence of a peptide pool representing the antigen of interest at individual peptide concentrations of 5 μg/ml. Golgi-Plug (BD Biosciences) (1 μl/ml) and anti-CD107a PE (clone eBio/D4B) at a final dilution of 1:200 were also included. Phenotypic analysis of CD8⁺ and CD4⁺ T cells was performed by staining PBMCs using the following antibody clones: anti-CD8 PerCP-Cy5.5 (clone 53-6.7) and eFluor 650-coupled anti-CD4 (GK1.5) and anti-IFN-γ APC (XMG1.2) (BD Biosciences). Also, the surface staining of liver-resident lymphocytes was performed using the following antibodies: anti-NK1.1 FITC, anti-CD3 Alexa Fluor 700, anti-CD69 PE-Cy 7, anti-CD8 eFluor 450 (BD Biosciences). The liver-resident invariant natural killer T cells (iNKT cells) were stained with CD1d tetramer conjugated to PE. Nonspecific binding of antibodies was prevented by incubating with anti-CD16/CD32 Fcγ III/II receptor prior to staining. Flow cytometric analyses were performed using an LSRII instrument. Data were analyzed using either FACSDiva or FlowJo software. Analysis of multifunctional CD8⁺ T-cell responses was performed using Boolean analysis in FlowJo software, Pestle, and SPICE 4.0 kindly provided by M. Roederer (National Institutes of Health, Bethesda, MD).

Dendritic cells were cultured for 10 d in culturing in 6-well culture plates at a concentration of 1.5 × 10⁶ cells per well in 1.5 ml of RPMI-1640 supplemented with heat-inactivated 10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, OVA-CD4 (KISQAVHAAHAEINEAG), or OVA-CD8 (SIINFEKL) peptide, in the presence or absence of RHS2 (20 μg/ml). BMDC were cultured at 37 °C in a humidified 5% CO₂ incubator. After 36 h of incubation, 2 × 10⁶ of freshly isolated lymphocytes of culture medium was added to each well. The lymphocytes were incubated with DCs for 3 d before re-stimulation with PMA (20 ng/ml) and ionomycin (1 μM). After an additional 2 h, the cells were treated with the protein transport inhibitor brefeldin A (BD). The cells were then incubated for 4 h before staining was performed with anti-CD3 FITC (17A2), CD4 PerCP-Cy5.5 (RM4-5), anti-CD8a PerCP-Cy5.5 (53–6.7), anti-IL-2 PE (JES6-5H4), anti-TNF-α BV421 (MP6-XT22), anti-IL-4 PE (11B11) and anti-IFN-γ APC (XMG1.2) (BD Biosciences).