Cd43 1b11

Cell surface and intracellular stainings were performed using antibodies as follows: CD4 (RM 4–5; BioLegend), CD8 (53–6.7; eBioscience), CD43 (1B11; BioLegend), CD62L (MEL-14; BioLegend), CD11b (M1/70; BioLegend), KLRG1 (2F1/KLRG1; BioLegend), Ki67 (16A8; BioLegend), FasL (MFL3; BD Biosciences), TRAIL (N2B2; eBioscience), Foxp3 (FJK-16s; eBioscience), GzmB (GB12; ThermoFisher Scientific), GzmA (GzA-368.5; eBioscience) and Gzm K (Orb102688; Biorbyt). Dead cells were excluded by fixable viability dye (eBioscience) staining. For FasL staining, lymphocytes were isolated and restimulated with anti-CD3 (145-2C11, eBioscience) and anti-CD28 (37.51, eBioscience) antibodies for 5 hours at 37 °C. BD Cytofix/Cytoperm Fixation/Permeabilization kit was used for intracellular staining following the manufacturer’s instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences). Analyses were done using FlowJo 5.0 software (Tree Star Inc., Ashland, OR).

Procedures for staining cells with antibodies for flow cytometry have been described in detail in earlier studies.15 Anti‐CD16/32 (FcBlock: 5 µg/mL) (Biolegend, San Diego, CA, USA) was used to block non‐specific antibody binding through Fc receptors. Fluorochrome‐ or biotin‐conjugated antibodies used were specific for CD11b, (M1/70), CD11c (N418), Ly6C (Al‐21), Ly6G (1A8), CD8 (53‐6.7), CD43 (1B11) and Siglec‐F (E50‐2440) (Biolegend). Propidium iodide staining (PI; 1 µg/mL) prior to flow cytometry was used to identify and gate live (PI⁻) cells. Flow cytometric analysis of labelled subsets was performed on a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). FACSDiva software (Becton Dickinson) was used to acquire data. FloJo software (Tree Star, Ashland, OR, USA) was used for data analysis.

Cell surface staining was performed for 15 min in the dark using PBS. The exclusion of dead cells was achieved using Zombie UV dye (BioLegend). Cells were stimulated with Ionomycin (500 ng/ml), PMA (25 ng/ml), Monesin (1×, BioLegend) and Brefeldin A (2 μg/ml) diluted in IMDM buffer and incubated for 3 h at 37 °C to detect cytokines and FasL expression. For intracellular stainings BD Cytofix/Cytoperm Fixation/Permeabilization kit was used. Surface and intracellular stainings were performed using following antibodies: CD3 (17A2, eBioscience), CD43 (1B11, BioLegend), CD49b (Dx5, eBioscience), CD69 (H1.2F3, eBioscience) CD86 (GL1, BioLegend), CD107a (ID4B, BioLegend), FasL (MFL3, BD Pharmingen), IFNγ (XMG1.2, eBioscience), IL-10 (JES5-16E3, eBioscience) IL-13 (eBio13A, eBioscience), NK1.1 (PK136, eBioscience), and TNFα (MP6-XT22, BioLegend).