Cd27 bv421

PBMCs were isolated from leukapheresis samples by Ficoll density centrifugation. CD4⁺ cells were isolated from PBMCs using the EasySep™ Human CD4⁺ T-cell Isolation Kit (Stemcell Technologies). Resting CD4⁺ cells were then isolated by negative depletion of cells expressing CD69, CD25, or HLA-DR (CD69 MicroBead Kit II, CD25 MicroBeads, Anti-HLA-DR MicroBeads; Miltenyi Biotec).
Cells were stained with CD27-BV421, CD3-BV510, CCR7-PE, CD45RO-APC, CD4-PECy7 (Biolegend) and sorted on a MoFlo Legacy (Beckman Coulter). Viability was determined using a propidium iodide stain. Cells were sorted from gated live singlet CD3⁺CD4⁺ lymphocytes based on the following expression patterns: (TN)CD45RO⁻/CCR7⁺/CD27⁺, (TCM)CD45RO⁺/CCR7⁺/CD27⁺, (TTM)CD45RO⁺/CCR7⁻/CD27⁺, (TEM)CD45RO⁺/CCR7⁻/CD27⁻.

Frozen PBMCs obtained from the patient and two healthy donors were thawed when cells (10⁶) were either unstimulated or stimulated with PMA (20ng/mL; Sigma) and ionomycin (1μM; Sigma) in the presence of brefeldin A (10μg/mL; Sigma) at 37°C overnight. For detection of intracellular cytokines, cells were stained with Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen) as well as for extracellular CD3 (eVolve605), CD8 APC-eFluor780 (eBioscience), CD45RO PE-CF594 (BD Biosciences), and CD27 BV421 (BioLegend) for 30 min. Cells were fixed (paraformaldehyde 4%), permeabilized, and stained for intracellular antigens with CD4 PE-Cy7 (BD Biosciences), IL-17A PE, IFN-γ FITC, and TNF-α APC (eBioscience). Data were collected using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Treestar) by gating on lymphocytes (forward & side scatter) and on viable CD3+CD4+CD8-CD45RO+ CD27− cells.