**2.12 Flow cytometry**

Cells were incubated for 20 min in the dark at room temperature with the following monoclonal-antibody panel: anti-CD3-PC7 (Cat 553,064; BD Biosciences—maximum emission (max-em): 785 nm) diluted 1:40; anti-CD4-APC-H7 (Cat 560,181; BD Biosciences—max-em: 785 nm) diluted 1:80; anti-CD8-BB515 (Cat. 564,422; BD Biosciences—max-em: 515 nm) diluted 1:160, and anti-CD19-APC (Cat MCA 1439;

Serotec—max-em: 661 nm) diluted 1:20, in PBS-BSA-HS. Cells were washed with PBS to remove unbound antibodies, fixed in 1% paraformaldehyde for 30 min in the dark at 4°C, washed again, and stored at 4°C in the dark until acquisition in flow cytometry. At least 20,000 events from each sample were acquired through CytoFlex flow cytometer (Beckman Coulter). Single-stained controls were used to set compensation parameters, while unstained cells were used to set analysis regions. After acquisition, flow cytometric analysis to evaluate the frequencies of CD8<sup>+</sup> T, CD4+ T, CD4+ /CD8+ T, and CD19<sup>+</sup> B cells was performed using CytoExpert Software (Beckman Coulter). A gate strategy was performed as follows: to exclude cell aggregates from analyses, cells were gated on Singlets region in FSC-A vs. FSC-H dot-plot; from Singlets gate an FSC-A vs. Side-Scatter-Area (SSCA), dot plot was created and the analyses region (mononuclear cells) was defined to encompass mononuclear cells and exclude dead cells from analyses; from Mono gate, CD4+ and CD8+ T lymphocytes were determined by CD3 vs. CD4 and CD3 vs. CD8 dot plots, respectively, and CD19+ B cells by CD3 vs. CD19 dot plot. CD4<sup>+</sup> /CD8+ double-positive T cells was determined by plotting CD8 vs. CD4 gated on CD3+ .
