The procedures used in this experiment have been published previously [25 (link),26 (link)]. Immune cells were evaluated in each experiment and treated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), stained with fluorescence-labeled antibodies as indicated, and incubated for 20 min at 4 °C in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin. The following mouse antibodies were purchased from BioLegend (San Diego, CA, USA): anti-CD3 (labeled with FITC, PE, and APC-Cy7), anti-CD4 (PE), anti-CD8 (FITC and APC-Cy7), anti-CD11b (APC-Cy7), anti-CD11c (BV421), anti-CD40 (APC), anti-CD44 (PE), anti-CD45 (PerCP-Cy5.5), anti-CD45.1 (Pacific Blue), anti-CD45.2 (Pacific Blue), anti-CD62L (PerCP), anti-CD69 (PE), anti-CD80 (PerCP-Cy5.5), anti-CD86 (PE), anti-CD103 (PE-Cy7), anti-CD127 (BV510), and anti-NK1.1 (PerCP-Cy5.5.). We also used 7-AAD (BioLegend) to stain dead cells for the evaluation of cell viability. The stained cells were subjected to FACS Verse for data acquisition (Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
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