T bet

The cells were harvested and stained for cell surface antigens CD4. After washing, cells were fixed and permeabilized, using Cytofix/Cytoperm kit (BD Biosciences), and then stained for T-bet, GATA-3, and Foxp3 fluorescein-conjugated antibodies from eBiosciences. The cells were analyzed by means of a flow cytometer.

Spleen cells were washed and maintained in ice-cold RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 2-mercaptoethanol (50 μM), L-glutamine (2 mM), sodium pyruvate (1 mM) and 3% heat-inactivated fetal bovine serum (FBS). All supplements were purchased from Life Technologies (USA). Cells (1.0 × 10⁶/well) were surface labeled with fluorochrome-conjugated monoclonal antibodies (mAbs) to CD4 (H129.19 or GK1.5), CD11a ((M17/4), CD25 (PC61), CD44 (IM7), CD127 (A7R34) (BD Bioscience, USA) and CD45.1 (104), CD45.2 (A20) (eBioscience, USA). For intracellular labeling, splenocytes (1.0 × 10⁶/well) were fixed with Cytofix/Cytoperm buffer (BD Bioscience) and permeabilized with PermWash (BD Bioscience). Cells were labeled with fluorochrome-conjugated mAbs to Blimp-1 (6D3), Bcl6 (K112-91), Ki67 (B56), IFNγ (XMG-1.2) (BD Bioscience) and T-bet (eBio4B10), FoxP3 (FJK-16s), Phospho-Akt1 (SDRNR), Phospho-mTOR (MRRBY) (eBioscience). In some experiments, cells were also stained with TMRE (100 nM, Invitrogen, USA), DCFDA (10 μM, Invitrogen), 2-NBDG (10 μM, Invitrogen) and MTG (50 nM, Invitrogen), for 15 min at 37°C, to assess MMP, ROS, glucose uptake and mitochondrial mass, respectively. Samples were acquired using FACS Fortessa or FACSCanto BD flow cytometers and the data analyzed with the FlowJo program.

Cells were washed and stained for 30 min in ice-cold PBS containing 10% bovine serum albumin. All the antibodies were purchased from Becton Dickinson except CD71 (Biolegend; 334108), granzyme B (Invitrogen; MHGB04), Ki-67 (EBioscience; 11-5699-42), T-Bet (EBioscience; 50-5825-80), and ICOS (EBioscience; 46-9948-42). For intracellular protein staining, cells were permeabilized with Cytofix/Cytoperm buffer (Becton Dickinson), followed by staining for 60 min with antibodies that were diluted in Perm/Wash buffer (catalog no. 554723; Becton Dickinson). The fixable viability dye eFluor 780 (EBioscience; 65-0865-18) was used for excluding dead cells during analysis. Flow cytometry data acquisition was performed either on a FACSCanto II or an LSR-II (Becton Dickinson). Flow cytometry data were analyzed using FlowJo software (TreeStar Inc.). Phenotypes and functions were analyzed among gated CD8 T cells, defined as cells that expressed both CD3 and CD8. Absolute cell numbers per milliliter volume of blood were calculated using the BD Trucount Tubes bead system (BD; 340334) according to the manufacturer's protocol.