Cd45ra pe cy7

For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4⁺ T cells were identified as CD3⁺CD8⁻ T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).

PBMCs (1 × 10⁶) were stained with CD4 PerCP, CD25 Alexa488, and FoxP3 PE for the identification of Treg populations (BD FoxP3 staining protocol, according to the manufacturer’s instructions). CD4 PerCP, CD25 Alexa488, CD27 APC, and CD45RA PE-Cy7 (eBioscience) were used to assess surface phenotype. In some experiments, CD39 APC, PD1 PE-Cy7, and glucocorticoid-induced tumor necrosis factor receptor (GITR) APC (Biolegend) were analyzed on Treg populations. Intracellular cytokine staining was performed to determine IFN-γ and IL-10 production at the single-cell level as described previously using anti-human IFN-γ-PE-Cy7 and anti-human IL-10 APC (both from Biolegend). For the evaluation of TGF-β secretion, the surface expression of the latency-associated peptide (LAP) protein (anti-human LAP-PE-Cy7, Biolegend) was assessed. For all flow cytometry experiments, sample acquisition and analysis were carried out on a FACSCanto flow cytometer using the BD FACSDiva software (BD Biosciences). Negative control samples were incubated with irrelevant, isotype-matched mAbs in parallel with experimental samples.