Detailed Immune Cell Phenotyping

After homogenization of spleen tissue, immune cells were purified using 35% Percoll (GE Healthcare, La Chapelle-sur-Erdre, France) and red blood cells were lyzed. 10⁶ leucocytes were incubated with anti-CD16/32 (BD Pharmingen, Rungis, France) to block non-specific binding and washed. Cells were then incubated 30 min with appropriate dilutions of anti-CD3-Pacific Blue, anti-CD8-APC-Cy7, anti-CD4-PE, anti-NK1.1-Percp-Cy5.5 and anti-CD19-APC antibodies, all purchased from BD Pharmingen. The staining of ST2 was assessed with a rat monoclonal anti-mouse ST2-FITC antibody (clone DJ8; MB Bioproducts). Cells were washed, fixed in PBS containing 2% FCS, 0.01 M sodium azide and 2% formaldehyde and analyzed on a FACS Aria II ® flow cytometer using BD FACS Diva software (BD Biosciences). Dead cells and doublet cells were excluded on the basis of forward and side scatter. The different immune cell types were identified and gated as follows: BL were CD19⁺; NK cells were NK1.1⁺/CD3⁻; T CD8⁺ lymphocytes were NK1.1⁻/CD3⁺/CD8⁺/CD4⁻; and T CD4⁺ lymphocytes were NK1.1⁻/CD3⁺/CD4⁺/CD8⁻. The gating strategy was previously described [5 (link), 12 (link)].