Cd69 pe cf594

Multi-color flowcytometric analysis was performed on cells according to standard procedures using anti-human mAbs that cross-react with rhesus macaques. The following antibodies were used at predetermined optimal concentrations: CD45 (BD clone D058-1283), CD3 APC-Cy7 (BD clone SP34-2), CD4 BV605 (BD clone L200), CD8 BV650 (BD clone SK1), CD69 PE-CF594 (BD clone FN50), CD161 PE (Biolegend clone HP- 3G10), TCR γδ PE-Cy7 (BD clone B1), TCR Vδ1 FITC (ThermoScientific clone TS8.2), TCR Vδ2 FITC (ThermoScientific clone 15D), TCR Vα7.2 BV421 (Biolegend clone 3C10), and Aqua Live/Dead amine dye-AmCyan from Invitrogen (Waltham, MA). Surface staining was carried out by standard procedures as earlier described (23 (link)). Flow cytometric acquisition was performed on the BD Fortessa instrument driven by the FACS DiVa software for at least 100,000 CD3+ T cells in PBMC or at least 10,000 CD3+ T cells for rectal biopsy lymphocytes. The data acquired were analyzed using FlowJo software (version 10.7.1; TreeStar, Ashland, OR). For evaluation of cytokine production, intracellular cytokine staining with IFN-γ BV510 (Biolegend clone 4S.B3), IL-17 PerCP-Cy5.5 (eBioscience clone eBio64DEC17), IL-22 APC (Invitrogen clone IL22JOP), and TNF-α Alexa Fluor 700 (BD clone MAb11) were utilized.

For flow cytometry, the stimulated PBMCs were stained for viability and surface markers (CD4 FITC, 1:100; CD8 BV510, 1:100; CD69 PE-CF594 1:100; CD137 BV-605 1:100; OX-40 PE-Cy7 1:100; all BD Biosciences) in FACS buffer (PBS supplemented with 2% FBS (Gibco, Invitrogen^TM, USA)) for 40 min at 4 °C. Next, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. Intracellular staining was performed by incubating the cells in perm/wash buffer supplemented with antibodies for 30 min at 4 °C (IFNγ PE, 1:50; IL-2 BV711, 1:50; TNFα BB700, 1:50; GranzymeB (GZB) PE 1:50; Perforin APC 1:100; all BD Biosciences, San Jose, CA, USA). Samples were acquired on a fluorescence-activated cell sorter (FACS) Symphony^TM instrument (BD Biosciences), using BD FACSuite software version 1.0.6 (BD Biosciences, San Jose, CA, USA), and analyzed with FlowJo software version VX (Tree Star, San Carlos, CA, USA). Functional profiles were deconvoluted by employing Boolean gating in FlowJo version XV (Tree Star, San Carlos, CA, USA) (Figure S1).