Pe vio615

Samples were acquired, processed, and analyzed on the same day to minimize inter-experiment variability. All samples were assigned a disease severity based on the worst symptoms present at the time of blood draw according to National Institutes of Health COVID-19 Treatment Guidelines Panel, available at https://www.covid19treatmentguidelines.nih.gov/. Discarded blood samples were obtained from patients from August 2020-April 2021 at the Louis Stokes Cleveland Medical Center, Cleveland, Ohio, USA. Peripheral blood samples from patients were directly stained with the following antibodies CD159a (FITC, Miltenyi), CD158e (APC, Miltenyi), CD314 (PE-VIO 615, Miltenyi), CD335 (PE, Beckman Coulter), CD3 (Krome-Orange, Beckman Coulter), CD56 (APC-Alexa Flour 700, Beckman Coulter), CD94 (Pacific blue, Beckman Coulter), CD19 (APC-Alexa Flour 750, Beckman Coulter), CD158b1/b2 (PC7, Beckman Coulter) and CD158a,h (PC5.5, Beckman Coulter). Samples were run on a Beckman Coulter Navios EX Flow Cytometer. Flow data analysis was performed using FlowJo 10.

Mononuclear cells were isolated using Histopaque 1083 (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, using 1.5 mL of peripheral blood diluted 1:1 in PBS. Cells were resuspended in 500 µL of RPMI-1640 for maintenance or cytometry staining.
CD68 antibody anti-mouse coupled to PE-Vio615 (Miltenyi, Bergisch Gladbach, Germany; 130-112-674), CD4 antibody anti-mouse coupled to FITC (Miltenyi, 130-120-750), CD8a antibody anti-mouse coupled to PerCP-Vio700 (Miltenyi, 130-120-756), and CD335 antibody anti-mouse coupled to PE (Miltenyi, 130-112-201) were used for flow cytometry. The BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD, Franklin Lakes, NJ, USA) was used to fix and permeabilize the cells. Washing steps and resuspension for analysis were performed in FACS buffer (PBS/EDTA/FBS).
Flow cytometry was performed according to the manufacturer’s instructions, following Miltenyi’s cell surface flow cytometry staining protocol for CD4, CD8a, and CD335, as well as their intracellular flow cytometry staining protocol for CD68.

Human primary CD4⁺ T cells (0.5 × 10⁶ cells/well) were activated with anti-CD3 (plate-coated, 5 μg/mL, eBioscience) and anti-CD28 (1 μg/mL, eBioscience) on 96-well plates. CD4⁺ T cells were differentiated for 2 – 8 days at 37°C in 100 μL RPMI 1640 medium (Sigma) supplemented with 10% A+ serum and 100 μL supernatants from TLR2/TLR8 stimulated monocytes stimulated with TLR ligands. CD4⁺ T cell effector cytokine production was analyzed 48 h post activation and on day 8 after 6 h short-term re-stimulation with Cell Stimulation Cocktail (eBioscience). Protein Transport Inhibitor Cocktail (eBioscience) was added for the last 4 h before harvest of the cells. Cell were stained with Fixable Viability Dye eFluor 780 (eBioscience) and surface-stained with fluorescent antibodies to CD3 (BV 785, BioLegend), CD4 (Alexa Fluor 700, eBioscience) before fixation and permeabilization (FOXP3 buffer set, BD Biosciences). Staining for intracellular cytokine production was performed with fluorescent antibodies to IFN-γ (FITC, Miltenyi Biotec), IL-17 (BV 510, BioLegend), IL-2 (PE/Cy7, eBioscience), IL-10 (APC, eBioscience) IL-4 (PE-Vio615, Miltenyi Biotec) and TNF-α (BV421, BioLegend) and Multicolour flow cytometry was performed on a BD LSRII flow cytometer and analyzed with FlowJo software (FlowJo, LLC).