Blood collection and isolation of mononuclear cells
Up to 30 mL of blood were collected from each patient in vacuettes containing ethylenediamine-tetraacetic acid (EDTA). Blood was immediately processed. Isolation of peripheral blood mononuclear cells (PBMC) was performed using ficoll-hypaque according to standard procedures. For all experiments, except those related to metabolic investigation, PBMC were stored in liquid nitrogen in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO). For metabolic investigation, PBMC were used immediately after isolation. Plasma was stored at −80 °C until use. The study was reviewed and approved by each participant, including healthy donors, provided informed consent according to Helsinki Declaration, and all uses of human material have been approved by the local Ethical Committee (Comitato Etico dell’Area Vasta Emilia Nord, protocol number 199/ 2022, May 24th, 2020) and by the University Hospital Committee (Direzione Sanitaria dell’Azienda Ospedaliero Universitaria di Modena, protocol number 5974, February 24th, 2023). The patients/participants provided their written informed consent to participate in this study.
Activation induced cell marker assay (AIM) and T cell phenotype
Isolated PBMCs were thawed and rested for 6 hours. After resting, CD40-blocking antibody (0.5 mg/ml final concentration) (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the cultures 15 min before stimulation. PBMCs were cultured in 96-well plate in the presence of 15-mer peptides with 11-amino acids overlap, covering the complete sequence of Wuhan SARS-CoV-2 Spike glycoprotein (PepTivator SARS-CoV-2 Prot_S complete, Miltenyi Biotec, Bergisch Gladbach, Germany) together with 1 μg/mL of anti-CD28 (Miltenyi Biotec, Germany). PBMCs were stimulated for 18 h at 37 °C in a 5% CO2 atmosphere in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1M HEPES, 55μM β-mercaptoethanol). For each stimulated sample, an unstimulated one was prepared, as negative control. After stimulation, cells were washed with PBS and stained with PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) for 20 minutes at room temperature (RT). Next, cells were washed with FACS buffer (PBS supplemented with 2% FBS) and stained with the following fluorochrome-labeled mAbs: CXCR5-BUV661, CCR6-BUV496, CXCR3-BV785 for 30 minutes at 37 °C. Finally, cells were washed with FACS buffer and stained for 20 minutes at RT with Duraclone IM T cell panel (Beckman Coulter, Brea, CA) containing CD45-Krome Orange, CD3-APC-A750, CD4-APC, CD8-AF700, CD27-PC7, CD57-Pacific Blue, CD279 (PD1)-PC5.5, CD28-ECD, CCR7-PE, CD45RA-FITC and added with other three fluorescent mAbs i.e., CD69-BV650, CD137-BUV395 and CD95-BV605. Samples were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter). All reagents used for T cell phenotyping are listed in Supplementary Table 1. All mAbs added to DuraClone IM T cells were previously titrated on human PBMCs and used at the concentration giving the best signal-to-noise ratio. The gating strategies used to identify CD4+ and CD8+ T cells are reported in the Supplementary Figures 1,4.
Detection of SARS-CoV-2-specific B cells
Thawed PBMC were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, anti- biotics, 0.1M HEPES, 55μM β-mercaptoethanol and 0.02 mg/ml DNAse. PBMC were washed with PBS and stained using viability marker PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) for 20 min at RT in PBS. Next, cells were washed with PBS and stained for 15 min at RT with streptavidin-AF700 (decoy channel; ThermoFisher Scientific, USA) to remove false positive SARS-CoV-2-specific B cells. After washing with FACS buffer, cells were stained with biotinylated full-length SARS-CoV-2 spike protein (R&D Systems, Minneapolis) labelled with different streptavidin-fluorophore conjugates. Full-length biotinylated spike protein was mixed and incubated with streptavidin-BUV661(Becton Dickinson) or streptavidin-BV650 (BioLegend) at a 6:1 mass ratio for 15 min at RT. All samples were stained with both biotinylated streptavidin for 1h at 4°C. Then, cells were washed with FACS buffer and stained for 20 min at RT with DuraClone IM B cells (Beckman Coulter, Brea, CA) containing the following lyophilized directly conjugated mAbs: anti-IgD-FITC, CD21-PE, CD19-ECD, CD27-PC7, CD24-APC, CD38-AF750, anti-IgM-PB, CD45-KrO to which following drop-in antibodies were added: CD71-BUV395, CD20-BV785, anti-IgG-BUV496 and anti-IgA-PerCP-Vio700. Samples were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter). A minimum of 1,000,000 cells per sample were acquired. All reagents used for B cell phenotype are reported in Supplementary Table 3. All mAbs added to DuraClone IM B cells were previously titrated on human PBMCs and used at the concentration giving the best signal-to-noise ratio. The gating strategy used to identify Ag− and Ag+ B cells is reported in the Supplementary Figure 11.
Intracellular cytokine staining (ICS)
Isolated PBMCs were thawed and rested for 6 h. PBMCs were stimulated in the presence of a pool of lyophilized peptides covering the complete protein coding sequence (aa 5–1273) of spike glycoprotein ("S") of SARS-CoV-2 (PepTivator SARS-CoV-2 Prot_S Complete Miltenyi Biotec, Bergisch Gladbach, Germany) together with 1 μg/ml of anti-CD28/49d (Becton Dickinson). PBMCs were stimulated for 16 h at 37°C in a 5% CO2 atmosphere in complete culture medium (RPMI 1640 supplemented with 10% FBS and 1% each of L-glutamine, sodium pyruvate, non-essential amino acids, antibiotics, 0.1 M HEPES, 55 mM β-mercaptoethanol, and 0.02 mg/mL DNAse I). For each stimulated sample, an unstimulated one was prepared as a negative control. All samples were incubated with protein transport inhibitors brefeldin A (Golgi Plug, Becton Dickinson Bioscience, San Jose, CA, USA) and monensin (Golgi Stop, Becton Dickinson Bioscience, San Jose, CA, USA) and previously titrated concentration of CD107a-PE (BioLegend, San Diego, CA, USA). After stimulation, cells were washed with PBS and stained with LIVE/DEAD fixable Aqua (ThermoFisher Scientific, USA) for 20 min at RT. Next, cells were washed with FACS buffer and stained with surface mAbs recognizing CD3-PE.Cy5, CD4-AF700, and CD8-APC.Cy7 (BioLegend, San Diego, CA, USA). Cells were washed with FACS buffer and fixed and permeabilized with the Cytofix/Cytoperm buffer set (Becton Dickinson Bioscience, San Jose, CA, USA) for cytokine detection. Then, cells were stained with previously titrated mAbs recognizing IL-17-PE-Cy7, TNF-BV605, IFN-g-FITC, IL-2-APC, and GRZMB BV421 (all mAbs from BioLegend, San Diego, CA, USA). Samples were acquired on an Attune NxT acoustic cytometer (ThermoFisher Scientific, USA). Supplementary Table 2 reports mAb titers, clones, catalog numbers, and type of fluorochrome used in the panel.
Gating strategy used to identify and analyze the intracellular cytokine production of CD4+ and CD8+ T lymphocytes is reported in Supplementary Figure 7.
Computational analysis of flow cytometry data.
T cells analysis
Compensated Flow Cytometry Standard (FCS) 3.0 files were imported into FlowJo software version v10.7.1 and analyzed by standard gating to remove doublets, aggregates and dead cells. For ex vivo immunophenotyping of non-antigen-specific (Ag−) and antigen-specific (Ag+) T cells of both CD4+ and CD8+ we analyzed only the data of stimulated samples. For each sample, we therefore selected data from all living CD4+ or CD8+ T cells and imported them in R using flowCore package v2.4.0 f or a total of 37,397,203CD4+ T cells (of which 465,729 were SARS-CoV-2 specific) and 12,758,008 CD8+ T cells (of which 180,267 were SARS-CoV-2 specific). The further analysis was performed using CATALYST v1.17.3. All data obtained by flow cytometry were transformed in R using hyperbolic arcsine “arcsinh (x/cofactor)” applying manually defined cofactors (where x is the fluorescence measured intensity value). Clustering and dimensional reduction were performed using FlowSOM (version 2.4.0) and UMAP (version 0.2.8.0) algorithms, respectively. The Ag+ CD4+ and CD8+ T cell clusters have been analyzed using the following markers: CD45RA, CCR7, CD27, CD28, PD-1, CCR6, CXCR3, CXCR5 and CD95. The quality control (QC) of clustering for CD4+ and CD8+ T cells is reported in the respective Supplementary Figures 2,5.
B cell analysis
Compensated Flow Cytometry Standard (FCS) 3.0 files were imported into FlowJo software version v10.7.1 and analysed by standard gating to remove doublets, aggregates, dead cells, and identify CD19+ B cells. From total CD19+ B cells, to remove false positive SARS-CoV-2-specific B cells we eliminated decoy-positive B cells. For each sample, we selected the SARS-CoV-2-specific B cells as positive cells for both Spike_streptavidin-BUV661 and Spike_streptavidin-BV650 and now referred to as Ag+ B cells. The remaining double negative cells were non-SARS-CoV-2-specific B cells and mentioned to as Ag− B cells. Then, we exported for each sample separately both Ag+ and Ag− B cells and imported them in R using flowCore package v2.4.0. The unsupervised analysis was performed using CATALYST v1.17.3. All data were transformed in R using hyperbolic arcsin (arcsinh x/cofactor) applying manually defined cofactors. Clustering and dimensional reduction were performed using FlowSOM and UMAP algorithms, respectively. For each day of acquisition at CytoFLEX LX, we had a sample used as quality control (QC).
scMEP staining protocol
Thawed PBMCs were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1M HEPES, 55μM β-mercaptoethanol and 0.02 mg/ml DNAse. PBMCs were washed with Maxpar PBS and stained for 5 minutes at 37 °C with a working solution of the pre-titrated Cell-ID Cisplatin-195Pt in Maxpar PBS. For quenching the cisplatin stain, PBMCs were washed twice with Maxpar Cell Staining Buffer, using at least 5x the volume of the cell suspension. After that, PBMCs were stained with 100 μl of 1x Surface mAb Mix (see Supplementary Table 5 for all reagents used) at room temperature for 15 min. Samples were gently vortexed and incubated at room temperature for additional 15 min. Following the incubation, cells were washed twice by adding 2 mL Maxpar Cell Staining Buffer to each tube, centrifuged at 300 x g for 5 min, and supernatant was removed by aspiration, leaving a residual volume about 100 µL. For each sample, the pellet was thoroughly disrupted by pulse vortex. Cells were prepared for nuclear staining, 1 mL of Foxp3 Fixation/Permeabilization working solution was added to each sample, and they were incubated for 30 min at 4°C, protected by light. Then, 2 mL of 1X Permeabilization Buffer were added to each samples’ tube and centrifuged at 400-600 x g for 5 minutes at room temperature. PBMCs were stained with 100 μl of 1x Nuclear mAb Mix and incubate for at least 30 min at 4°C. Cells were subsequently washed twice; a first time with 2 mL of 1X Permeabilization Buffer and a second time with 2 mL of Maxpar Cell Staining Buffer. Samples were placed on ice for 10 min to chill. Then, 1 mL of 4 °C methanol was added, samples were mixed gently, and incubated on ice for further 15 min. PBMCs were washed twice with 2 mL of Maxpar Cell Staining Buffer, centrifuged at 800 x g for 5 min, and supernatants were removed by aspiration. PBMCs were then stained with 100 μl of 1x Phospho-Mix and incubated for 30 min at room temperature. Cells were washed twice by adding 2 mL Maxpar Cell Staining Buffer to each tube, centrifuged at 800 x g for 5 min, and supernatants were removed by aspiration, leaving a residual volume about 100 µL. It was essential to thoroughly disrupt the pellet by pulse vortex, before adding 1 mL of the 1.6% Formaldehyde solution to each tube. After gently mixing, PBMCs were incubated at room temperature for 10 min. When incubation was completed, cells were centrifuged at 800 x g for 5 min and supernatant was removed by aspiration. One mL of Cell-ID Intercalation solution was then added to each sample and gently vortex. Samples were incubated at 4°C overnight. After centrifuging tube at 800 x g for 5 min, each pellet was resuspended in the residual volume of iridium fix/perm solution and transferred into a labeled 1.5 mL microcentrifuge tube ready to be stored at −80°.
SCENITH assay performed on SARS-CoV-2-specific B cells
Freshly isolated PBMCs were rested overnight in RPMI 1640 supplemented with 10% FBS and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 mM β-mercaptoethanol. After resting, cells were washed with PBS and stained with biotinylated full-length SARS-CoV-2 spike protein (R&D Systems, Minneapolis, USA) labeled with different streptavidin fluorophore conjugates. Full-length biotinylated spike protein was mixed and incubated with streptavidin-BUV661 (Becton Dickinson) or streptavidin-BV650 (BioLegend) at a 6:1 mass ratio for 15 min at room temperature (RT). PBMCs were stained with both biotinylated streptavidin at 4°C for 1 h. Then, cells were washed at 1000 rpm for 7 min and resuspended in complete medium at a density of 1 x 106/0.1 ml and incubated at 37°C for 4 h. After the incubation cells were washed at 1000 rpm for 7 min and resuspended in 340 μl of complete medium and equally distributed in 4 tubes (one for each condition CO, DG, DGO, O) to proceed with SCENITH protocol. All the reagents were prepared the day of the experiment, empty tubes and 20X inhibitors CO; DG; O; P (stored at -20°C in aliquots) were equilibrated at 37°C for 30 minutes before cell treatment. 5 μl of each 20X inhibitor and 10 μl of 20X puromycin were added to the corresponding tube and incubated at 37°C for 40 minutes. In the DGO tube, DG and O were added simultaneously. After incubation, tubes with cells were filled up with ice cold MACS Buffer, centrifuged at 400 g during 5 min at 4°C and the supernatant was discarded by aspiring. Cells were resuspended in 100 μl of PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) and Fc Block (Becton Dickinson) and incubated for 15 minutes, at 4°C, in the dark. Without washing, 100 μl of 2X surface staining mix including the previously titrated mAbs CD19-PE, CD69-FITC, were added to cells and samples were incubated for 25 min at 4°C in the dark. Tubes were filled up with FACS buffer, centrifuged at 400 g, during 5 min, at 4°C and the supernatant was discarded by aspiring. Red blood cells lysis was avoided. The following intracellular staining with Invitrogen FOXP3 stain buffer was performed: cells were resuspended in 100µL of Foxp3 Fixation/Permeabilization solution, vortexed and incubated for 20 min at RT in the dark. Then 1X Permeabilization Buffer was added to cells and samples were centrifuged at 600 g for 5 minutes at RT. The supernatant was discarded by aspiration and cells were resuspended in 50 µL of intracellular block (1X Permeabilization Buffer + 20% FCS) and incubate for 10 min at RT. Without washing 50 µL of anti-puromycin-AF647 antibody solution 1/250 were added to cells and cells were incubated for 1 hour at 4°C in the dark. At the end of incubation cells were washed with 1X Permeabilization Buffer and centrifuged at 600 g for 5 min at 4°C. The supernatant was discarded, and stained cells were resuspended in 400 µL of FACS Buffer and acquired by flow cytometer CytoFLEX LX (Beckman Coulter, Hialeah, FL). A minimum of 1,500,000 cells per sample were acquired. All reagents used for the staining of cells are reported in Supplementary Table 4.
SCENITH assay performed on SARS-CoV-2-specific T cells
Freshly isolated PBMCs were incubated at a density of 1 x 106/0,1 ml in complete medium (RPMI 1640 supplemented with 10% FBS and 1% each of L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 mM ß-mercaptoethanol) with CD40-blocking antibody (0.5 μg/ml final concentration) (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 37°C before stimulation. Then, cells were stimulated by adding in the medium PepTivator SARS-CoV-2 Prot_S complete (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 15-mer peptides with 11-amino acid overlap, covering the complete sequence of Wuhan SARS-CoV-2 spike glycoprotein, together with CD28/CD49d (Becton Dickinson) and incubated for 18 h at 37°C in a 5% CO2 atmosphere. At the end of stimulation, cells were washed at 1000 rpm for 7 min and resuspended in 340 μl of complete medium and equally divided into 4 FACS tubes (one for each condition CO, DG, DGO, O) to perform the SCENITH protocol. All the reagents were prepared the day of the experiment, empty tubes and 20X inhibitors CO; DG; O; P (stored at -20°C in aliquots) were equilibrated at 37°C for 30 minutes before cell treatment. 5 μl of each 20X inhibitor and 10 μl of 20X puromycin were added to the corresponding tube and incubated at 37°C for 40 minutes. In the DGO tube, DG and O were added simultaneously. After incubation, tubes with cells were filled up with ice cold MACS Buffer, centrifuged at 400 g during 5 min at 4°C and the supernatant was aspirated. Cells were resuspended in 100 μl of PromoFluor IR-840 (Promokine, PromoCell, Heidelberg, Germany) and Fc Block (Becton Dickinson) and incubated for 15 min at 4°C, in the dark. Without washing, 100 μl of 2X surface staining mix including the previously titrated mAbs CD4-FITC, CD8-PE, CD3-PB, CD69-BV650, CD137-BUV395 were added to cells and samples were incubated for 25 min at 4°C in the dark. Tubes were filled up with FACS buffer, centrifuged at 400 g, during 5 min, at 4°C and the supernatant was discarded by aspiring. Red blood cells lysis was avoided, and the following intracellular staining with Invitrogen FOXP3 stain buffer was performed: cells were resuspended in 100µL of Foxp3 Fixation/Permeabilization solution, vortexed and incubated for 20 min at RT in the dark. Then 1X Permeabilization Buffer was added to cells and samples were centrifuged at 600 g for 5 min at RT. The supernatant was discarded by aspiring and cells were resuspended in 50 µL of intracellular block (1X Permeabilization Buffer + 20% FCS) and incubated for 10 min at RT. Without any washing step, 50 µL of anti-puromycin-AF647 antibody solution 1/250 was added to cells and followed by incubation for 1 hour at 4°C in the dark. Cells were washed with 1X Permeabilization Buffer and centrifuged at 600 g for 5 min at 4°C. The supernatant was discarded, and stained cells were resuspended in 400 µl of FACS Buffer and acquired by flow cytometer CytoFLEX LX (Beckman Coulter, Hialeah, FL). A minimum of 1,500,000 cells per sample were acquired. All reagents used for the staining of cells are reported in Supplementary Table 4.
Statistical analysis
Quantitative variables were compared using Kruskal-Wallis non-parametric test corrected for multiple comparisons by controlling the False Discovery Rate (FDR), method of Benjamini and Hochberg. Statistically significant q-values are represented. Statistical analysis of cytokines production was performed using GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, USA). Total percentage of antigen-specific (Ag+CD4+ and Ag+CD8+) T cell data have been calculated as background subtracted data. Simplified Presentation of Incredibly Complex Evaluation (SPICE) software (version 6, Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA) was used to analyze flow cytometry data on T cell polyfunctionality. Data from the total cytokine production are represented as individual values, means, and standard errors of the mean. Regarding polyfunctionality, data in pie charts are represented as median values and statistical analysis was performed using permutation test; data in graphs are represented as individual values, means, and standard errors of the mean.
Principal Component Analysis
Principal Component Analysis (PCA) was executed and visualized in R using the prcomp function (stats v3.6.2) and the pca3d package v0.1. The data used included the proportions, absolute number and scMEP matabolic state of Ag+ CD19+ B cells, CD4+, CD8+ T cells along with clinical parameters (reported in Table 1). Missing values of dataset were imputed using missMDA package v1.18. The total impact of a specific variable retained by PC1 and PC2 was computed as [(C1 * Eig1) + (C2 * Eig2)]/(Eig1 + Eig2), where C1 and C2 represent the contributions of the variable to PC1 and PC2, and Eig1 and Eig2 denote the eigenvalues of PC1 and PC2, respectively. The Euclidean distance of MS-treated groups to HD in PCA space was calculated using the phenoptr v.0.3.2 package.
PENCIL prediction analysis
PENCIL v0.7 was used to predict cell clusters associated with absence of breakthrough infection in MS patients and HD 41. As single cell input data we used our 45-parameter mass cytometry data (scMEP data) analyzed previously with R by using CATALYST v1.18.1 (see method above). We imported into Seurat v4.9.9 58 the expression matrix, containing hyperbolic arcsinh (cofactor 5) transformed data, and the metadata (also UMAP coordinates) of Ag+ T or B lymphocytes. Cells from all subjects were divided in two groups on the basis of SARS-CoV-2 breakthrough infection and were used as input to run PENCIL. For T cells prediction, to reduce the number of rejected cells due to huge differences in cell number form who experienced SARS-CoV-2 breakthrough infection (10,591 cells) and who did not (96,931 cells), a downsampled dataset was used (a total of 21,591, whose 11,000 for who experienced SARS-CoV-2 breakthrough infection; 10,591 for who did not get COVID-19). Prediction displayed 68% accuracy. Then 5,650 was used for Ag+ B cells prediction (2,825 for each group); a total of 1,047 cells were rejected. The precision of the prediction was 76%.