Multiparametric Phenotyping of T-cell Responses

Cryo-preserved blood mononuclear cells were stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated peptide-MHC class I multimer complexes at room temperature for 20 min in Ca²⁺-free media as described [10 (link)]. Cells were then washed and stained with antibodies against CD3, CD8, CD4, HLA-G at 4°C for 20 min. For intracellular cytokine staining, cells were stimulated overnight with optimal CD8 T cell peptides in presence of brefeldin A. Cells were then stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated antibodies against CD3, CD4, CD8, HLA-G. After fixation and permeabilization for 20 min at 4°C using a commercial kit (Caltag), cells were stained intracellularly for IFN-γ, TNF-α, MIP-1β and IL-2. Cells were acquired on a Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo X (Tree star). Analysis and presentation of cell distributions were performed using Graph Pad prism (version 6) and SPICE version 5.32, downloaded from <http://exon.niaid.nih.gov/spice/>.