Unique immunological signatures distinguish severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia
Stefanie Kreutmair, Susanne Unger, Nicolás Gonzalo Núñez, Florian Ingelfinger, Chiara Alberti, Donatella De Feo, Sinduya Krishnarajah, Manuel Kauffmann, Ekaterina Friebel, Sepideh Babaei, Benjamin Gaborit, Mirjam Lutz, Nicole Puertas Jurado, Nisar P. Malek, Siri Goepel, Peter Rosenberger, Helene A. Häberle, Ikram Ayoub, Sally Al-Hajj, Jakob Nilsson, Manfred Classen, Roland Liblau, Guillaume Martin-Blondel, Michael Bitzer, Antoine Roquilly, Burkhard Becher* Correspondence to: firstname.lastname@example.org
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Blood samples of COVID-19 and Hospital-Aquired-Pneumonia patients and healthy controls for peripheral blood mononuclear cell (PBMC) isolation and cryopreservation were collected at indicated time points. PBMCs were kept in cell culture medium (RPMI-1640, 10% fetal calf serum (FCS; Biochrom), and 1× l-glutamine and 1× penicillin/streptomycin (both Life Technologies)) supplemented with 5U ml−1 benzonase (Sigma–Aldrich) and frozen in liquid nitrogen until experimental analysis. Then, for spectral flow analysis, cells were thawed using Cryo thaw devices (Medax). Briefly, cells were resuspended in cell culture medium supplemented with 2U ml−1 benzonase by centrifugation (300 r.c.f.; 7 min; 24 °C). Cell count was calculated using an automated cell counter (Bio-Rad). 2 million (mio) cells were directly stained for cytometry analysis (surface panel), while 1 mio cells were restimulated with 50 ng ml−1 phorbol 12-myristate 13-acetate (Sigma–Aldrich) and 500 ng ml−1 ionomycin (Sigma–Aldrich) in the presence of 1× Brefeldin A and 1x Monensin (both BD Biosciences) for 5 h at 37°C (Lymphoid panel) or in case of R848 stimulation, 2.5 mio cells using 2µg ml−1 R848 (Invivogen) in the presence of 1× Brefeldin A and 1x Monensin (both BD Biosciences) for 8 h at 37°C (Myeloid panel). Surface panel: For spectral cytometry, samples were washed in PBS and then resuspended in 100µl of Live/Dead Fixable Blue mixture (Thermo Scientific, 1:500) followed by a washing step. To avoid nonspecific binding, the samples were resuspended in 30 µl of True Stain FcX (BioLegend) and incubated for 10 min at 4°C. Anti-human flow cytometric antibodies were purchased pre-conjugated. 70 μl of the first surface-antibody mixture was added and cells were incubated for 15 min at 37°C. After another washing step, the second surface-antibody staining step (100 µl) was performed for 15 min at 4°C. Then, fixation was performed using 150 µl of 2% PFA for 15 min at 4°C. Lymphoid and myeloid panel: For intracellular spectral cytometry, after surface-antibody labeling, cells were fixed and permeabilized using Cytofix/Cytoperm reagent (BD Biosciences) for 30 min at 4°C. Intracellular labeling was then performed in 100 µl of 1x permeabilization buffer (Thermo Scientific) for 11 h (Lymphoid cytokine panel) or 10 h (Myeloid cytokine panel) at 4°C. Spectral cytometry samples were acquired on a Cytek Aurora (Cytek Biosciences). The compensation matrix was corrected using FlowJo software (Tree Star). After gating for single cells and live cells, CD45-positive cells were exported (Surface panel and Lymphoid panel) or further gated for CD3/CD19/CD56-negativity and then exported (Myeloid panel) for further R analysis. Each dataset here contains the exported FCS files.