Anti ifnγ alexa fluor 647

At day 0 or day 7 post stimulation performed as above, 4x10⁵ PBMC or CTLs were washed and stimulated or not in a 96-well plate pre-coated with anti-mouse IgG (4 μg/ml; Southern Biotech) followed by anti-CD3 antibodies (5 μg/ml; UCHT1, Biolegend) and soluble anti-CD28 antibodies (2 μg/ml; Beckman Coulter) or (Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and Ionomycin (1 μg/ml). Two hours later, brefeldin A (10 μg/ml, Sigma-Aldrich) was added. At 6 hours of stimulation, cells were fixed with 4% PFA for 15 min, at room temperature, permeabilized in PBS 1X BSA 0,5%, saponine à 0,05% for 15 min at room temperature and subsequently stained with anti-IL-2-PerCP-Cy5.5 (1/50; clone MQ1-17H12; BD Biosciences), anti-TNF-FITC (1/100; clone cA2; Miltenyi biotech), anti-IFNγ-Alexa fluor 647 (1/50; clone 4S.B3; BD Biosciences) and only for day 7 anti-Granzyme B-PE (1/50; clone GB12; Molecular Probes), in the presence of 0.05% saponin. FACS analyses were performed as above. Similar experiments were performed at day 7, after removing IL2.

MALC obtained at day 10 of growth, were co-cultured with NK cells at ratio E:T 0.5:1 or 1:1 and treated or not with mAbs at 10 μl/ml during 4 h in presence of brefeldin A at 10 μl/mL (except of CD69 detection). Cells were then washed, fixed with 2% PFA during 4 min, washed and permeabilized with 1% saponin during 8 min. Cells were stained with anti-CD56-PE (Miltenyi Biotec), anti-CD3-PE/Cy7 (Beckman Coulter, Roissy, France), anti-CD69-PE-Cy5 (BD Biosciences), anti-perforin (PFN)-FITC (BD Biosciences), anti-IFN-γ-Alexa fluor 647 (BD Biosciences), anti-granzyme B-Alexa647 (BD Biosciences) antibodies during 30 min at 4°C in the dark. Cells were analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

ICS was processed according to our previous method [87 (link)]. Briefly, 1×10⁶ freshly isolated mouse splenocytes were stimulated with HSV-1 peptides (Genscript, Nanjing, China, listed in S3 Table at a final concentration of 2 mg/mL for 2 h at 37°C. The splenocytes were then incubated with brefeldin A (BD Pharmingen) for 16 h at 37°C. After incubation, the cells were collected and stained with anti-CD3-FITC, anti-CD4-BB700, and anti-CD8-PE-Cy7 monoclonal antibodies (BD Biosciences) for 1 h. Then the resuspended cells were permeabilized in FACS Perm/wash buffer for 20 min before staining with anti-IFN-γ-Alexa Fluor 647, anti-TNF-α-PE, and anti-IL-2-BV605 (BD Pharmingen). Samples were tested using the flow cytometer (CytoFLEX S, Beckman, America) instrument with CytExpert software (version 2.4).