Cd3 bv510

Spleens were removed from sacrificed mice according to Kalli et al.^{45 (link)}. Frequency of memory T lymphocytes was assessed ex vivo on splenocytes stained by the following conjugated mAbs: CD3-BV510 (BD PharMingen, Haryana, India), CD4-APC-eFluor 780, CD8-PE-Cy7, CD44- PE (Thermo Scientific,Waltham, MA, USA). To assess ex vivo the percentages of non-lymphoid APCs (monocytes and DCs), splenocytes were labelled with the following conjugated mAbs: CD80- FITC (Biolegend, San Diego, CA, USA), HLA-DR-PE, CD86-APC (Thermo Scientific,Waltham, MA, USA). The samples were analyzed by a FACS Canto II flow-cytometer (BD Bioscience, Frankin Lake, NJ, USA) using FACS DIVA software. In both analyses, splenocytes were stained with Live/dead fixable violet dead cell stain (Thermo Scientific,Waltham, MA, USA) to exclude dead cells. Statistical analyses were performed by the Mann-Whitney unpaired T test for nonparametric measures using the GraphPad Prism 4.0 Software (GraphPad Software, Inc, La Jolla, CA).

NK cell production of the cytokines IFN-γ, TNF-α and GM-CSF was determined by intracellular staining under two stimulatory conditions as described previously [9 (link), 44 (link)]. After isolation, PBMCs were incubated in the presence of either K562 cells (E:T of 25:1) or phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) (Sigma-Aldrich, St Louis, USA) plus ionomycin (I, 0.5 μg/ml) (Sigma-Aldrich, St Louis, USA) for 1 h at 37 °C with 5 % CO₂. Brefeldin A (BD Biosciences, San Diego, USA) was added to prevent cytokine secretion during stimulation and the cells were incubated for an additional 5 h [9 (link), 44 (link)]. PBMCs incubated in RPMI 1640 media alone served as the unstimulated control sample. Following 6 h incubation, the PBMCs were washed and incubated with monoclonal antibodies (mAbs) for CD56-PE-Cy7, CD16-BV711 and CD3-BV510 (BD Biosciences, San Diego, USA) for 25 min. The PBMCs were subsequently washed, incubated in BD fixation/permeabilisation solution (BD Biosciences, San Diego, USA) for 20 min, washed in BD perm/wash buffer (BD Biosciences, San Diego, USA) and then incubated for 30 min with mAbs against IFN-γ- allophycocyanin (APC), TNF-α- peridinin chlorophyll protein-cyanine (PerCP-Cy)-5.5 (BD Biosciences, San Diego, USA) and GM-CSF-PE (Biolegend, San Diego, USA) for flow cytometric detection of intracellular cytokines.

PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF7a, ORF8, RBD) or 1:200 dilution (spike). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19⁺/antigen-PE⁺ were sorted as probe positive. The PE⁺ gate was drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.

PBMC were plated and stimulated with CpG-ODN 2006 (Aurogene Srl) alone or in the presence of 100 μl of CM-hAMSC as described above. After 5 days of culture, cells were collected and protein expression of the transcription factors BCL6, PAX-5, IRF-4, and BLIMP-1 was analyzed by flow cytometry. After exclusion of dead cells by eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific) staining, cells were stained for 20 min at 4°C with CD19 BB700 (SJ25C1, 1:200), CD3 BV510 (UCHT1, 1:100), CD14 BV510 (MΦP9, 1:200), CD27 PE-Cy7 (M-T271, 1:100), all from BD Biosciences. After washing in stain buffer, the cells were fixed and permeabilized with Transcription Factor Buffer Set (BD Biosciences) for 40 min at 4°C, according to the manufacturer's instructions. Then, cells were stained for 40 min at 4°C with specific antibody against BCL6 BV421 (K112-91, 1:30), PAX-5 PE (1H9, 1:100), IRF-4 PE (Q9–343, 1:100), BLIMP-1 Alexa Fluor® 647 (6D3, 1:100). Finally, the cells were washed with Transcription Factor Buffer Set (BD Biosciences), acquired on a FACSAria III (BD Biosciences), and analyzed using FCS express v5.0 (DeNovo Software, Los Angeles, CA, USA).

Cryopreserved samples were thawed, washed, and resuspended in PBS. Surface staining was performed according to standard protocols as published before [18 (link)]. Immunophenotyping was performed using fluorochrome-conjugated anti-human monoclonal antibodies (mAb) as follows: CD3-BV450 (UCHT1), CD3-BV510 (UCHT1), CD4-Alexa Fluor 700 (RPA-T4), CD8-APC-Cy7 (SK1), CD27-BV421 (M-T271), CD45RO-APC (UCHL1), CD197 (CCR-7)-PE-Cy7 (3D12), and CD69-FITC (L78) (BD Biosciences); CD158b-PE-Cy7 (DX27) and TCR Vγ9-FITC (B3) (BioLegend); TCR Vδ1-FITC (TS8.2) (Thermo Scientific); and TCR pan γδ-PE (REA591) and TIM3-APC (F38-2E2) (Miltenyi Biotec). FACS CANTO (BD Biosciences, San Jose, CA, USA) was used to acquire samples, and FlowJo V10 (TreeStar) was used to analyze the results. The gating strategy is shown in Figure 1(a).
Manual gating was used to characterize individual samples, and subsequently, data were downsampled and merged (concatenated) for further visualization using dimensionality reduction algorithm plugin, t-Distributed Stochastic Neighbor Embedding (tSNE).