Immunomagnetic separation technique

Thawed PBMC were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol and 0.02 mg/ml DNAse.
CD2⁺ cells were isolated through immunomagnetic separation technique (Miltenyi Biotec, Bergish Gladbach, Germany). Purified CD2⁺ cells were washed with PBS and stained 20’ at room temperature using LIVE DEAD Red (ThermoFisher) viability marker. Then, cells were washed and stained for 20’ at room temperature with the following mAbs: anti-CD4 FITC, -CCR7 PE and -CD45RA PE-Cy7 (Biolegend, San Diego, CA, USA).
CM CD4⁺ T cells were identified and sorted with a purity more than 98% by using a Biorad eS3 Sorter (Bio-Rad, Hercules, California, USA), operating inside a specifically designed Benchtop Biocontainment Enclosure (bioBUBBLE, Fort Collins, Colorado, USA). The gating strategy and purity are shown in Supplementary Fig. 9.

Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation, using standardized protocols and Lymphoprep reagent from Stemcell (Cambridge, MA, United States). Plasma was then stored at −80°C until use (10 (link)). A minimum of 1.5 million CD14+ monocytes were sorted starting from 20 million PBMC through immunomagnetic separation technique (Miltenyi Biotec, Bergish Gladbach, Germany). Purity of monocyte population was always >95%.