Percp conjugated cd3

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by density gradient centrifugation on Ficoll-Paque PLUS (Cytiva). The isolated PBMCs were then analyzed with an 8-color flow cytometer (FACSCanto™II, BD Biosciences, CA, USA) after labeling with the following anti-human fluorochrome-conjugated antibodies: FITC-conjugated CD38 (clone HIT2); APC or FITC-conjugated PD-1 (clone EH12.2H7); FITC-conjugated CD45RA (clone HI100); PE-conjugated CD24 (clone ML5); PE-conjugated ICOS (clone C398.4A); PE-conjugated CCR6 (clone G034E3); PerCP-conjugated CD3 (clone UCHT1); PE/cyanine7-conjugated CD27 (clone O323); PE/cyanine7-conjugated CD4 (clone OKT4); APC-conjugated CD19 (clone HIB19); APC-conjugated CD45RO (clone UCHL1); APC-conjugated CXCR3 (clone G025H7); and Brilliant Violet 421-conjugated CXCR5 (clone J252D4) (all antibodies were from BioLegend, CA, USA). Cell viability was assessed using a live/dead assay (Fixable Viability Dye eFluor 780; eBioscience). All data were collected and analyzed using FlowJo software. The gating strategies of the isotype-matched IgG control for specific cell populations are shown in the supplementary figure (Figure S1).

Fluorescent‐conjugated anti‐human antibodies including fluorescein isothiocyanate (FITC)‐conjugated CD16 (clone: B73.1), phycoerythrin (PE)‐conjugated CD56 (5.1H11), Peridinin‐chlorophyll‐protein complex (PerCP)‐conjugated NKp44 (clone: P44‐8), Allophycocyanin (APC)‐conjugated CD3 (clone: OKT3), FITC‐conjugated CD3 (clone: OKT3), PerCP‐conjugated CD3 (clone: HIT3a), PerCp‐Cy5 conjugated CXCR3 (clone: G025H7), PerCp‐Cy5‐conjugated NKG2D (clone: 1D11), APC‐conjugated CD11b (clone: ICRF44), PerCP‐Cy5.5–conjugated CD27 (clone: M‐T271), PerCP‐Cy5.5‐conjugated perforin (clone: B‐D48), Alexa Fluor® 647‐conjugated granzyme B (clone: GB11), PerCP‐Cy5.5‐conjugated CD96 (clone: NK92.39) and their cognate isotype controls (all from Biolegend) as well as PerCP‐Cy5.5‐conjugated NKG2C (clone: 134591, R&D system) antibodies were used for immunophenotyping. Lysing solution (BD Biosciences) was used for removing the red blood cells. For washing, fixing, and cell permeabilizing, 1X phosphate buffer saline (PBS), 1% paraformaldehyde solution (Sigma), and 1X perm/wash solution (BD Biosciences) were used, respectively.