Anti mouse cd16 32 clone 93

To isolate mononuclear cells from NALT, NALT was first obtained from the upper jaw of the mice. NALT cells were isolated by gently rubbing the NALT sample with a needle under a stereoscopic microscope. After washing with PBS, the collected cells were treated with anti-mouse CD16/32 (clone 93; BioLegend, San Diego, California, USA) for 15 min at room temperature. After washing with PBS containing 2% newborn calf serum, the cells were stained with fluorescein isothiocyanate-conjugated hamster anti-mouse CD3ε (clone 145-2C11, BD Biosciences, San Diego, California, USA), phycoerythrin (PE)-conjugated rat anti-mouse B220 (clone RA3-6B2, BD Biosciences), PE-conjugated rat anti-mouse PD-1 (clone 29F1.A12, BioLegend), Alexa Fluor 647-conjugated rat anti-mouse GL7 (clone GL7, BioLegend), PE-Cy7-conjugated rat anti-mouse CD4 (clone RM4-5, BD Biosciences), PE-Cy7-conjugated Armenian hamster anti-mouse CD11c (clone N418, BioLegend), APC-Cy7-conjugated rat anti-mouse CD8α (clone 53-6.7, BD Biosciences), and Brilliant Violet 421-conjugated rat anti-mouse CD45 (clone 30-F11, BioLegend) for 30 min at 4 °C. After washing with PBS containing 2% newborn calf serum, cells were treated with 7-Amino-Actinomycin D (BioLegend) for 10 min at 4 °C and analyzed by means of flow cytometry (MACSQuant) (Miltenyi Biotec, Auburn, California, USA).

Following preparation of single‐cell suspensions, the cells were incubated with anti‐mouse CD16/32 clone 93 (BioLegend) for blocking of Fc‐receptors to avoid nonspecific binding. Lymphocytes were stained with fluorescent antibodies and tetramers for an hour. The following antibodies/tetramers were used for staining: FITC rat anti‐mouse CD1d clone 1B1 (BD Biosciences, Franklin Lakes, NJ), FITC rat IgG2b k isotype control (BD Biosciences), FITC anti‐mouse TCR β clone H57‐597 (BD Biosciences), PE PBS‐57 loaded tetramer and unloaded tetramer (kindly provided by the NIH Tetramer Core, Emory, GA), APC anti‐mouse CD122 clone TM‐b1 (eBioscience, San Diego, CA), APC CD3e anti‐mouse clone 145‐2C11 (BD Biosciences), PE‐Cy7 anti‐mouse CD25 clone PC61 (BioLegend), PE‐Cy7 anti‐mouse CD4 clone RM4‐5 (BioLegend), APC anti‐mouse CD8a clone 53‐6.7 (BioLegend), APC anti‐mouse CD69 clone H1.2F3 (BD Biosciences), PE‐Cy5 anti‐mouse CD5 clone 53‐7.3 (BioLegend), PerCP‐Cy5.5 anti‐mouse CD44 clone IM7 (BioLegend), PE‐Cy7 anti‐mouse CD24 clone M1/69 (BioLegend). Flow cytometric analysis was performed using a BD FACS Verse and a BD LSR II flow cytometer. The results were analyzed in FlowJo version 9.5.3 (TreeStar, Ashland, OR).

Following treatments, BMDMs were harvested from cell culture plates using 5 mmol/l EDTA in PBS for 10 min at 37 °C. Cells were washed in FACS buffer (PBS containing 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich)). Cells were incubated with 10 μg/ml anti-mouse CD16/32 (clone 93, BioLegend) to block Fc receptors, then stained with anti-F4/80-BV421 (1:400; clone BM8, BioLegend) and anti-CD11b-PE (1:400; clone M1/70, BioLegend) in FACS buffer for 20 min at 4 °C. Stained cells were washed and resuspended in FACS buffer and acquired on a BD LSRFortessa (BD Biosciences) using FACSDiva software. Analysis was carried out using FlowJo software. For analysis, live cells were identified by gating based on forward and side scatter.