Multiparameter Flow Cytometry of Rhesus T Cells

The following fluorochrome-labeled monoclonal antibodies were used as described (22 (link), 33 (link)) for cell surface or intracellular staining of rhesus T cells: CD3 PerCP-Cy5.5, CD4 APC-H7, CD28 APC-H7, CD127 (IL-7Rα) PE, CD45RA PE-Cy7, CTLA4/CD152 APC, and CTLA4/CD152 VB450 (all from BD Biosciences, San Jose, CA, USA), CD8α AF700, CD25 AF700, and Foxp3 VB421 (all from Biolegend, San Diego, CA, USA), and PD1/CD279 PE (from eBioscience, San Diego, CA, USA). Following surface staining for CD3, CD4, CD8, CD25, CD28, CD127, and PD1, cells were fixed and permeabilized for 45 min at 4°C using Fixation/Permeabilization buffer (eBioscience™; ref 00-5123-43). After fixation/permeabilization, cells were stained for CTLA4 and Foxp3. No antibodies were added to the co-cultures.