Anti cd3 plus anti cd28 mabs

Cells from five patients and five controls were stimulated for 6 days in resting conditions, or after stimulation with anti-CD3 plus anti-CD28 mAbs (1 μg/mL each, Miltenyi Biotech, Bergisch Gladbach, Germany) and with 20 ng/mL IL-2. The fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester was used at a concentration of 1 μg/mL (TechnoFisher) according to standard procedures. Flow cytometric analyses for the identification of cycling cells belonging to different T cell populations were performed by gating TN, TCM, TEM, TE among CD4⁺ and CD8⁺ T cells^{16 (link)}.

PBMC were stimulated for 6 days in resting conditions (as a control) or after stimulation with anti-CD3 plus anti-CD28 mAbs (1 μg/mL each, Miltenyi Biotech, Bergisch Gladbach, Germany) and with 20 ng/mL IL-2. The fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester was used at a concentration of 1 μg/mL (ThermoFisher Scientific) according to standard procedures^{61 (link)}. Flow cytometric analysis for the identification of cycling cells belonging to different lymphocyte populations was performed on CD19⁺ B cells and on electronically gated population of T_N, T_CM, T_EM, T_EMRA CD4⁺ or CD8⁺ T cells. Supplementary Table 2 reports the complete list of mAbs used.

Cells were stimulated for six days in resting conditions, or after stimulation with anti-CD3 plus anti-CD28 mAbs (1 μg/mL each, Miltenyi Biotech, Bergisch Gladbach, Germany) and with 20 ng/mL IL-2. The fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester was used at a concentration of 1 μg/mL (ThermoFisher) according to standard procedures^{52 (link)}. Flow cytometric analyses for the identification of cycling cells belonging to different T-cell populations were performed by gating CD4⁺, CD8⁺ T cells, and CD19+ B cells. mAbs used are listed in Sup Data 6.