Cytobank software

After acquisition, samples were cleaned up according to the latest standard of data pre-gating (Fluidigm) using the Cytobank™ software (www.cytobank.org, Beckman-Coulter, Brea, CA, USA)²⁸ . To avoid leukocyte contamination, we gated the acquired events for platelet-specific markers: only CD41 (GPIIb)- and CD61-(GPIIIa) positive events were selected for further analysis and defined as platelets (Supplemental Fig. I). CD3 marker was included in the panel as an additional negative control (Supplemental Fig. II).

Upon stimulation with the SARS-CoV-2 PepTivator, PBMCs were stained using fluorophore-conjugated antibodies. A master mix containing all the antibodies was prepared directly prior to staining. At first, the cells were stained using live/dead marker Viobility 405/452 dye (Miltenyi Biotech) for 10 min at RT in the dark. Following fixation and permeabilization, the cells were stained using fluorochrome-conjugated antibodies (Table S2) for 10 min at RT in the dark. All samples were washed thoroughly with PBS containing 0.5% bovine serum albumin (AMERESCO, USA) and 2 mM EDTA. Data were acquired using the Cytoflex 5L cytometer (Beckman Coulter) followed by analysis using Cytobank software (Beckman Coulter, Brea, CA, USA). Live CD14^–, CD20^–, CD3⁺, CD4, and CD8 T-cells specific to SARS-CoV-2 S protein were defined based on the double expression of two activation markers exhibiting the highest expression levels of CD40L⁺ TNFα⁺ CD4 T-cells and CD40L⁺ IFNγ⁺ CD8 T-cells. The percentage of S-specific peptide CD4 for each patient was calculated as the percentage of CD40L⁺ TNFα⁺ CD4 in the S peptide-stimulated sample minus the percentage of CD40L⁺ TNFα⁺ CD4 in the DMSO control sample. The same calculation was performed for S-specific peptide CD8 and for each marker (CD40L, TNFα, and IFNγ) separately.

Tumors were dissociated using the Miltenyi mouse tumor dissociation kit, and CD45+ cells were enriched using Percoll differential centrifugation. Live cells were stained using Cell-ID cisplatin, and Fc receptors were blocked with TruStain FcX (1:100) prior to extracellular staining using the University of North Carolina mass cytometry core antibody panel. Cells were fixed and permeabilized for intracellular staining of FoxP3 using the eBioscience FoxP3 (ThermoFisher Scientific, Waltham, MA, US) staining buffer set. Finally, cells were intercalated with iridium overnight. Live CD45+ single cells were gated using Cytobank software (version 9.0, Beckman Coulter, Brea, CA, USA) and then analyzed in R as previously described [33 (link)]. Briefly, following hyperbolic inverse sine transformation, a self-organizing map was built, and consensus clustering was performed. Over-clustered clusters were collapsed manually and visualized by tSNE plots. The differential abundance of each cell type and the differential expression of each marker with each cell type was determined by a generalized linear mixed logistic model.

Allo-stimulator B cells were obtained from previous study where transplant donor primary B cells were expanded using CD40L-transfected fibroblasts and IL-4 according to previously published protocol^{18 (link),42 (link)}. Wildtype and HLA-E+ K562 cell lines were provided by Deepta Bhattacharya. All stimulator cells were profiled for activating and inhibitory ligands by staining with a viability dye and surface antibodies (Supplementary Table 8) in FACS buffer (DPBS, 2% heat-inactivated FBS, 2 mM EDTA) for 30 minutes on ice; cells were stained in triplicate with isotype controls. After washing, cells were fixed with 2% paraformaldehyde (PFA) (EMS, cat #15710) for 10 minutes at room temperature, resuspended in FACS buffer, and stored at 4°C until acquisition on LSRFortessa (BD Biosciences). Results were analyzed on Cytobank software (Beckman Coulter) and R software version 4.0.3.