Erythrocyte lysing buffer

Spleens were isolated from control BALB/c mice under aseptic conditions and prepared for cellular suspensions through a 70 μm cell strainer (BD Biosciences, San Jose, CA, USA). Single-cell suspensions were resuspended in an erythrocyte-lysing buffer (BD Biosciences) and washed. T cells were enriched by a Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s specifications. The purity (CD3ε⁺) was confirmed at > 90% by flow cytometry. Normal T cells were stimulated with 3 μg/ml anti-CD3ε plus 500 ng/ml anti-CD28 antibodies (BD Biosciences) and cultured in RPMI 1640 (Life Technologies) without l-arginine for 24 h. Media was supplemented with 4% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin.

We performed cellular phenotyping on a FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA, USA). To identify CD4 subsets, we used the following fluorochrome-labeled antibodies for surface staining, according to the manufacturer’s instructions: CD45RA-Pacific Blue and CD31-APC (Biolegend, San Diego, CA, USA), and CD4-APC-H7 and CD45RO-PE (BD Pharmingen, San Diego, CA, USA). Isotype-matched control antibodies were ordered from the same companies. After performing an automated cell count, we incubated 50 µL of blood with erythrocyte-lysing buffer (BD-Pharm Lyse, Frankin Lakes, NJ, USA) at room temperature, centrifuged the solution, and discarded the supernatant. The staining time for surface antibodies or isotypes was 30 min, followed by washing with staining buffer (1 × PBS/5% FBC, both reagents from PAA, Coelbe, Germany). Staining was performed on ice.