*2.5. Flow Cytometry Analysis*

Lymphocyte immunophenotyping performed from peripheral blood and spleen cell suspension were done for all experimental groups by flow cytometry, using a BD FAC-SCanto II cytometer (BD Biosciences, San Jose, CA, USA). We have quantified T lymphocytes (CD3ε + ), with T helper (CD4+CD8−) and T suppressor/cytotoxic (CD8a+CD4−) subsets, B lymphocytes (CD3ε <sup>−</sup>CD19<sup>+</sup> ), NK cells (CD3ε <sup>−</sup>NK1.1<sup>+</sup> ), and the expression levels of several maturation markers (CD49b, CD27, CD11b, CD43, KLRG1) and activation (CD69, CD28, CD11c, NKp46) markers on NK cells were assessed.

Both types of samples (peripheral blood and spleen cell suspension) were incubated with TruStain fcX (anti-mouse CD16/32, isotype Rat IgG2a, λ) Antibody (BioLegend, San Diego, CA, USA) for 7 min on ice and stained in the dark for 20 min at room temperature with the following monoclonal antibodies conjugated with fluorochromes: 0.5 µL Alexa Fluor 647 anti-mouse CD3ε (clone 145-2C11, isotype Armenian Hamster IgG); 0.5 µL Alexa Fluor 488 anti-mouse CD8a (clone 53–6.7, isotype Rat IgG2a, κ); 1.25 µL PE-Cy7 anti-mouse CD4 (clone GK1.5, isotype Rat IgG2b, κ); 1,25 µL PerCP-Cy5.5 anti-mouse CD19 (clone 6D5, isotype Rat IgG2a, κ); 1,25 µL PE anti-mouse NK1.1 (clone PK136, isotype Mouse IgG2a, κ); 0.5 µL FITC anti-mouse CD3ε (clone 145-2C11, isotype Armenian Hamster IgG); 2.5 µL Brilliant Violet 510 anti-mouse NK1.1 (clone PK136, isotype Mouse IgG2a, κ); 0.6 µL PerCP/Cy5.5 anti-mouse/rat/human CD27 (clone LG.3A10, isotype Armenian Hamster IgG); 0.6 µL APC/Cy7 anti-mouse CD43 (clone RA3-6B2, isotype Rat IgG2a, κ); 1.25 µL PE anti-mouse CD28 (clone 37.51, isotype Syrian Hamster IgG); 2.5 µL PE/Cy7 anti-mouse CD69 (clone H1.2F3, isotype Armenian Hamster IgG); 2.5 µL PE/Cy7 anti-mouse CD335 (NKp46) (clone 29A1.4, isotype Rat IgG2a, κ); 2.5 µL PerCP/Cy5.5 anti-mouse CD11c (clone N418, isotype Armenian Hamster IgG) (all from BioLegend, San Diego, CA, USA); 2.5 µL eFluor 450 anti-mouse CD49b (clone DX5, isotype Rat IgM, κ); 0.3 µL APC anti-mouse CD11b (clone M1/70, isotype Rat IgG2b, κ); 0.6 µL PE anti-mouse KLRG1 (clone 2F1, isotype Syrian Hamster IgG) (all from eBioscience Inc, San Diego, CA, USA). Red blood cells lysis was performed with BD FACS Lysing Solution (BD Biosciences, San Jose, CA, USA) for 10 min in the dark at room temperature, followed by centrifugation for 5 min at 350× *g* and two washing steps with Cell Staining Buffer. Flow cytometry analysis was preceded by daily check-up of cytometer performances (BD Cytometer Setup and Tracking Beads Kit, BD Biosciences, San Jose, CA, USA) and compensation of spectral

overlaps (UltraComp eBeads, Invitrogen by Thermo Fischer Scientific, San Diego, CA, USA). Unlabeled cells were used as negative control. Data were acquired and analyzed using BD FACSDiva v 6.1 software (BD Biosciences, San Jose, CA, USA).

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