Pe cyanine7 anti human cd4

Stimulated PBMCs were recovered from the culture plates and resuspended in 100 μL PBS. Cell viability was assessed by staining with Viobility™ Fixable Dyes (Miltenyi Biotec, Germany). Cells were washed, fixed, permeabilized, and then stained with an antibody cocktail containing Pacific Blue™ anti-human CD3 (Biolegend, clone: HIT3a), PE/Cyanine7 anti-human CD4 (Biolegend, clone:A161A1) and PerCP/Cyanine5.5 anti-human CD8 (Biolegend, clone: SK1) for T cell identification; APC anti-human CD69 (Biolegend, clone: FN50) and PE anti-human IFN-γ (Biolegend, clone:4S.B3) for the activation analysis; and FITC anti-human CD14 (Biolegend, clone:HCD14) and FITC anti-human CD20 (Biolegend, clone:2H7) for the exclusion of non-specific signals and B cells. Fifty thousand events were analyzed by a BD LSR-II flow cytometer. (BD Biosciences, San Jose, CA) The gates applied for the quantification of the stimulated T cells are illustrated in Figure S1.

All antibodies used were commercially available and validated for specificity by the manufacturer. Allophycocyanin (APC) anti-human IL-2 (MQ1-17H12, 1:20) and BD Horizon™ BV786 anti-human IFN-γ (4S.B3, 1:20) were purchased from BD Biosciences. Alexa Fluor 700 anti-human CD28 (CD28.2, 1:20), APC anti-human CD161 (HP-3G10, 1:10), Brilliant Ultraviolet (BUV) 395 anti-human CD3 (SK7, 1:20), Brilliant Violet 605 anti-human TCR Vα7.2 (3C10, 1:10), Brilliant Violet 650 anti-human CX3CR1 (2A9-1, 1:20), Brilliant Violet 711 anti-human IL-17A (BL168 1:20), Brilliant Violet 711 anti-human perforin (dG9, 1:20), PE/Dazzle 594 anti-human/mouse granzyme B (QA16A02, 1:100), PE/Dazzle 594 anti-human TNF-α (MAb11, 1:100), PE/Cyanine7 anti-human CD4 (SK3, 1:20), and PerCP/Cyanine5.5 anti-human CD8 (SK1, 1:20) were purchased from BioLegend. APC-eFluor™ 780 anti-human CD27 (O323, 1:20) was purchased from eBioscience. Phycoerythrin (PE) anti-human TCR Vδ2 (123R3, 1:100), fluorescein isothiocyanate (FITC) anti-human TCRγ/δ (REA591, 1:10), and VioBlue anti-human TCR Vδ1 (REA173, 1:10) were purchased from Miltenyi Biotec. Dead cells were excluded using the viability dye Zombie Aqua™ Fixable Viability Kit (BioLegend) according to the manufacturer’s instructions.

To validate the scRNA-seq findings, the following antibodies were used: anti-human CD45 (AmCyan, 339203, Biosciences), Anti-Human CD3 (Pacific Blue, 300330, Biolegend), anti-human CD4 (PE/Cyanine7, 300512, Biolegend), anti-human CD8a (APC/Cyanine7, 300926, Biolegend), Anti-Human CD274 (APC, 329707, Biolegend), Anti-Human CXCL13 (APC, WA3170553, Invitrogen), anti-human/mouse Granzyme B (FITC, 515403, Biolegend), anti-human Granzyme K (FITC, 370507, Biolegend), Anti-Ki-67 (PE, 556027, Biosciences), anti-human CD68 (PE, 333808, Biolegend), κ isotype Ctrl (PE, 400112, Biolegend), κ isotype Ctrl (FC) (APC, 400122, Biolegend), κ isotype Ctrl (FITC, 981802, Biolegend). Stained cells were detected using a BD FACSCanto^TM II and analyzed using FlowJo software.