Arc amine reactive compensation beads

Validation experiments using flow cytometry (FACS) was performed on total PBMCs from n=40 individuals in our cohort (25 LC, 15 non-LC). Sample preparation and cell revival were performed as described above for CyTOF preparation, and analyzed at baseline, or following SARS-CoV-2 peptide stimulation. For staining, cells were first treated with Zombie UV^™ or Zombie NIR^™ (BioLegend) as viability indicators, and then blocked with Human TruStain FcX^™ (BioLegend). FACS antibodies that were used in this study are shown in Table S7. For intracellular staining, cells were fixed with 2% PFA and incubated with Foxp3 fix/perm buffer (eBioscience) per manufacturer’s instructions, following completion of surface antibody staining. The BD Horizon^™ Brilliant stain buffer (BD Biosciences) was used according to manufacturer’s instructions when staining with antibodies conjugated to brilliant dyes. All cells were fixed in 2% PFA prior to analysis on a Fortessa X-20 (BD Biosciences). UltraComp eBeads^™ Compensation Beads (Invitrogen) served as a single fluorescence dye control, and ArC^™ Amine Reactive Compensation Beads (Invitrogen) served as a loading control for live/dead cell staining. FCS files were exported into FlowJo (BD, version 10.9.0) for further analyses. FACS data were arcsinh-scaled prior to analyses.

A 14-color, 17-parameter flow cytometry panel was used to enumerate different cell populations as described [26 (link)]. Briefly, fresh whole blood samples were stained for 30 min at room temperature with a panel of antibodies to lymphocyte surface markers (Supplementary Table S2) and 1:500 live/dead fixable near-infrared (near-IR) dye (Thermo Fisher Scientific, UK), followed by fixation and red blood cell lysis using red blood cell lysis/fixation solution (BioLegend, UK). After washing, CountBright™ absolute counting beads (Invitrogen, UK) were added to the sample and data was acquired using an LSR-Fortessa cell analyzer (BD Bioscience). Instrument calibration using cytometry setting and tracking (CST) beads, and compensation using AbC total compensation beads (Thermo Fisher Scientific, UK) and ArC Amine reactive compensation beads (Invitrogen, UK), were performed for each experiment.

Whole limbs were dissected at the joints, and the tissue was digested in collagenase (as described earlier). Isolated cells were counted using a nucleocounter NC-250 (ChemoMetec, Allerod, Denmark). A sample (5 μl) of the cell suspension was diluted in PBS (1:4), and a solution of acridine orange and 4′,6-diamidino-2-phenylindole (Solution 18; ChemoMetec) was added to provide counts of total and dead cells, respectively. For flow cytometric analysis, the remaining cells were subjected to live/dead staining (LIVE/DEAD Fixable Blue Dead Cell Stain Kit; Thermo Fisher Scientific) for 20 min. ArC Amine Reactive Compensation Beads (Thermo Fisher Scientific) were used to obtain compensation controls for live/dead staining. After washing off the live/dead stain, an Fc block (CD16/CD32) was applied for 15 min, and then cells were stained with a cocktail of antibodies (Additional file 1: Tables S2 and S3). Cells were analysed on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) after compensation set-up with OneComp eBeads (Thermo Fisher Scientific). Gating strategies are outlined in Additional file 1: Figures S4 and S5. Data were analysed using FlowJo software (FlowJo, Ashland, OR, USA). In some experiments FLS (CD45⁻CD90.2⁺) were sorted from these samples (BD Influx cell sorter; BD Biosciences) into lysis buffer, and RNA was prepared using RNeasy Plus Micro kits (Qiagen).

Compensation controls were prepared by using unstained and single stained cells, eBioscience Ultracomp eBeads (San Diego, CA), and/or ArC Amine Reactive Compensation Beads (ThermoFisher Scientific). Cells were stained following the protocol outlined above for either surface staining or Live/Dead staining. Beads were stained following manufacturer protocol.

Following overnight stimulation, cells were washed with PBS and stained with LIVE/DEAD Fixable Aqua dead cell stain (Thermo Fisher) for 30 min in the dark at 4°C to label dead cells. Nonspecific binding to the FcR was blocked by incubation with saturating doses of rat anti-mouse CD16/CD32 (BD Biosciences) for 15 min in the dark at 4°C. Afterwards, cells were labeled with antibodies specific for surface markers, including CD3 (clone 145-2C11, PE-eFluor 610), CD4 (clone GK1.5, eFluor 450), CD45 (clone 30-F11, APC-Cy7), and CD8 (clone 53.6.7, APC). Cells were fixed with IC Fixation Buffer (eBioscience), permeabilized with Permeabilization Buffer (eBioscience), and labeled with antibodies for intracellular markers, including IFN-γ (clone XMG1.2, FITC), TNF-α (clone MP6-XT22, PE), and IL-2 (clone JES6-5H4, PE-Cy7). All antibodies were obtained from eBioscience (CD3, CD4, IFN-γ, TNF-α) or Biolegend (CD45, CD8, IL-2) and were used at the manufacturers’ recommended dilutions. Fluorescence was acquired on a MoFlo Astrios cell sorter (Beckman Coulter) using the UltraComp eBeads (Thermo Fisher) and ArC Amine Reactive Compensation Beads (Thermo Fisher) to provide compensation controls. Additional controls included fluorescence minus one (FMO) controls and unstained cells. Results were analyzed using FlowJo software (Tree Star).

Tumoral cells were purified on a MOFLO Astrios EQ cell sorter (Beckman Coulter, Villepinte, France). Briefly, cells were sorted using a 70 µm nozzle, sheath fluid under 59 psi pressure, and droplet frequency around 93,000 Hz. Cells were sorted using the purify mode, with a 1.2 droplet mask.
Cells were stained with the following markers: LIVE/DEAD^® Fixable Near-IR Dead Cell Stain Kit (L10119, ThermoFisher), Cell Prolif Dye eFluor450 (65-0842-85; ThermoFisher), anti-CD45-PECy7 (MHCD4512; ThermoFisher), and anti-EMA-PE (355604; BioLegend).
To determine the spectral overlap and spillover matrix, monostaining was performed on compensation beads (AbC™ anti-mouse beads and ArC™ Amine Reactive Compensation Beads; ThermoFisher).
Regarding a gating strategy for cell sorting, cells were first selected on an FSC SSC plot.
Then, cells were selected as negatively stained for live dead, positive for proliferation dye, CD45-negative, and finally EMA-positive. To determine EMA-positive cells, EMA staining was observed on CD45+ cells (negative for EMA) and compensation beads.
Only bright EMA+ cells were sorted by the instrument (the gating strategy is available in Figure S1).

Compensation controls were prepared by using unstained and single stained cells, eBioscience Ultracomp eBeads (San Diego, CA), and/or ArC Amine Reactive Compensation Beads (ThermoFisher Scientific). Cells were stained following the protocol outlined above for either surface staining or Live/Dead staining. Beads were stained following manufacturer protocol.