Il 2 pb

Fluorochrome-conjugated anti-mouse monoclonal antibodies CD3-AF700 (cat # 100216), CD4-BV785 (cat # 100453), CD8-PB (cat # 100725), CD25-BV650 (cat # 102038), GITR-PECy7 (cat # 120222), ICOS-PECy5 (cat # 107708), IFNg-PeCy7 (cat # 505826), and IL-2-PB (cat # 503820) were purchased from Biolegend. CD44-Percp-cyanine5.5 cat# 45-0441-80, CD62L-APCeFL780 (cat # 47-0621-82), Foxp3-APC (cat# 17-5773-82), Eos-eFL660 (cat # 50-5758-80), Helios-PeCy7 (cat # 25-9883-42), Aiolos-PE (cat # 12-5789-80),and bCatenin-eFL660 (cat # 50-2567-42) were purchased from Thermo Fisher Scientific. PD-1-PECy7 (cat# 25-9985-80), CXCR5-BV421 (cat # 562889), Bcl-6-PE (cat # 569522), and phospho-STAT5-PE (pY694) (cat # 612567) were procured from BD Biosciences.

After thawing and overnight incubation at 37 °C in complete X-vivo, cells were plated at the concentration of 1 × 10⁶ cells/ml in RPMI 1640 (Lonza) supplemented with glutamine 1%, penicillin/streptomycin 1%, fetal bovine serum 10%, IL-2 20 IU/ml, and in the presence of an inhibitor of metalloprotease activity (TAPI-2 200 microM^{46 (link)}). After 1-h incubation at 37 °C, GolgiStop (0.66 microl/ml, BD biosciences) and the fluorochrome-conjugated antibody CD107a-FITC (BD Biosciences) were added; in the stimulated wells only, PMA (50 ng/ml; Sigma) and Ionomycin (1 µg/ml; Sigma) were added. After 6 h at 37 °C, cells were first stained with fluorochrome-associated monoclonal antibodies specific for surface molecules, CD3-BV510, CD4-PE/Dazzle, CD8-BV785, CD45RA-PerCP/Cy5.5, CD62L-AF700 (Biolegend); next, cells underwent fixation and permeabilization for intracellular staining with monoclonal antibodies specific for the following cytokines: TFN-α-PE/Cy7, IFN-ɣ-APC/Cy7, and IL-2-PB (Biolegend). Catalogue numbers and dilutions of each fluorochrome-conjugated monoclonal antibody are reported in Supplementary Table 1. Flow-cytometry data were acquired using BD LSRFortessa cell analyzer and visualized with FlowJo software (TreeStar). Statistical analyses were performed with Prism5 software or later versions (GraphPad). Pie charts with arcs were created using SPICE software^{47 (link)}.