Cd56 5.1h11

For biomarker analysis, 15 mL of blood was collected at baseline and at the time of each disease evaluation. Peripheral blood mononuclear cells (PBMCs) were obtained by standard density gradient centrifugation. To evaluate the prognostic implications of immune cells, multicolour flow cytometry analyses using the CytoFLEX flow cytometry platform (Beckman Coulter, Brea, CA) were performed to determine the proportions of different immune cell populations in the PBMCs. Panels for multicolour flow cytometry included CD3 (UCHT1; BioLegend, San Diego, CA, USA), CD4 (OKT4; BioLegend), CD8 (SK1; BioLegend), CD14 (63D3; BioLegend), CD11c (3.9; BioLegend), CD56 (5.1H11; BioLegend), γδ TCR (B1; BioLegend), HLA-DR (L243; BioLegend), CD69 (FN50; BioLegend), FoxP3 (236A-E7; eBioscience, San Diego), PD-1 (EH12.2H7; BioLegend), LAG-3 (11C3C65; BioLegend), CTLA-4 (L3D10; BioLegend), and TIGIT (A15153G; BioLegend).

At each WIHS visit, PBMCs were isolated from venous blood by standardized methods, frozen, and stored in liquid nitrogen in a specimen repository. Cryopreserved PBMCs stored in liquid nitrogen were warmed in 37°C water bath, then removed and immediately diluted with 1 mL of warm cRPMI and transferred and diluted again in an additional 8mL of warm cRPMI. Samples were then washed with PBS and stained with viability reagent (Ghost Dye^TM Red 710, Tonbo Bioscience) according to manufacturer’s recommendation. Samples were stained with antibody cocktail containing CD14 (M5E2, Biolegend), CD16 (3G8, Biolegend), and dump channel: CD3 (OKT3, Tonbo Bioscience), CD19 (HIB19, Tonbo Bioscience), CD56 (5.1H11, Biolegend), CD66b (G10F5, Biolegend), gated based on living cells and excluding dump channel-positive cells. Intermediate monocytes were defined as CD14⁺CD16⁺ and non-classical monocytes were defined as CD14^dimCD16⁺. Monocytes were sorted directly into TRIzol® LS Reagent (Life Technologies) and frozen at -80°C. FCS files were exported from FACS Diva (BD Bioscience) and processed using FlowJo v10.2 (FlowJo, LLC). The quantification of intermediate and non-classical monocytes are estimated from the FACS data from all PBMC samples.