7 aad viability staining solution

PBMCs were incubated with Fc receptor blocking solution (BioLegend) and then stained with mixtures of the following monoclonal antibodies against human surface antigens: anti-CD3-Brilliant Violet 421 (clone UCHT1), anti-CD4-APC/Fire 750 (clone RPA-T4), anti-CD45RA-Brilliant Violet 605 (clone HI100), anti-CD279 (PD1)-PE (clone EH12.2H7), and anti-CD185 (CXCR5)-PE/Dazzle594 (clone J252D4) (all from Biolegend) for T cell subset analysis; anti-CD19-PE/Dazzle594 (clone HIB19), anti-CD20- Alexa Fluor 700 (clone 2H7), anti-CD27-APC/Fire 750 (clone M-T271), anti-IgD-Brilliant Violet 421 (clone IA6-2) (all from Biolegend), and anti-CD38-FITC (clone T16) (Beckman Coulter, Brea, CA, USA) for B cell subset analysis. Cells were washed and stained with 7-AAD Viability Staining Solution (BD Biosciences). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc.).

For the proliferation assay with 1.5 µm carboxyfluorescein succinimidyl ester (CFSE) (#150347‐59‐4; Dojindo, Rockville, MD, USA), 293/null cells were resuspended in growth medium at a density of 10⁶·mL⁻¹ and cultured in six‐well plates with 0, 10 or 20 µm fatostatin. CFSE‐treated cells that were cultivated in the absence of DMSO or fatostatin were used as negative controls. Cells were labeled with CFSE in accordance with the manufacturer’s instructions (#13‐0850‐U50; TONBO Biosciences, San Diego, CA, USA). The 293/null cells were incubated at 37 °C in 5% CO₂ and harvested 44 h after fatostatin treatment. Cells were washed once with 1 × phosphate‐buffered saline and 1 × binding buffer (#00‐0055; Invitrogen, Waltham, MA, USA) each, at 620 g for 3 min. The pellet was resuspended in 200 µL of 1 × binding buffer, and incubated on ice for 15 min after the addition of 5 µL of 7‐AAD Viability Staining Solution (#559925; BD, Franklin Lakes, NJ, USA). Cell divisions were measured using a LSRFortessa cytometer (BD) and data were analyzed using flowjo, version 10.4 (BD).

Recombinant biotinylated SARS-CoV-2 spike RBD proteins of the wild type and the Omicron variant (Acrobiosystems) were tetramerized with streptavidin APC and streptavidin BV605 (both from BioLegend) as previously described^{17 (link),52 (link),57 (link)}. Two to four million cryopreserved PBMCs were first incubated with Fc receptor blocking solution and then stained with Fluorescent-labeled Spike RBD and mixtures of the following monoclonal antibodies against human surface antigens: anti-CD3- PerCP/Cyanine5.5 (clone UCHT1), anti-CD14-PerCP/Cyanine5.5 (clone M5E2), anti-CD20- Alexa Fluor 700 (clone 2H7), anti-CD27-APC/Fire 750 (clone M-T271), anti-IgM-PE/Cyanine7 (clone MHM-88), and anti-IgG Fc-Brilliant Violet 421 (clone M1310G05) (all from BioLegend); anti-CD8- PerCP/Cyanine5.5 (clone SK1), anti-CD19- V500 (clone HIB19), and anti-IgD-PE-CF594 (clone IA6-2) (all from BD Biosciences); and anti-CD38-FITC (clone T16) (Beckman Coulter). Cells were washed and stained with 7-AAD Viability Staining Solution (BD Biosciences). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc.). Data at each time point were obtained by subtracting the frequency before the vaccine.

Enriched B cells from 17 samples were resuspended in 100 μl of 1× PBS/1% BSA labeling buffer containing the following anti-human antibodies: anti-CD3-BV510 (diluted 1/20) (BD Pharmingen), anti-CD19-BV421 (diluted 1/50) (BD Pharmingen), BKPyV-gI-VLP-Alexa Fluor 555 (1.34 μg/ml), BKPyV-gI-VLP-Alexa Fluor 647 (0.54 μg/ml), and MPyV-VLP-Alexa Fluor 488 (1.34 μg/ml). Patient-specific and timepoint-specific TotalSeq-C oligonucleotide-labeled antibodies (BioLegend) were added into the mix as shown in Fig S4, then incubated at 4°C for 30 min. Cells were washed three times in 13 ml of 1× PBS/1% BSA, and centrifuged at 300g for 5 min at 4°C. After the final wash, cells were resuspended in 1 ml of 1× PBS/0.04% BSA. 10 μl 7-AAD viability staining solution (BD Pharmingen) was added and incubated for 5 min in the dark. The 17 samples were pooled before sorting through a BD Aria FACS sorter (Becton-Dickinson). 1 × 10⁵ CD19⁺ B cells were sorted first, followed by BKPyV-specific B cells. The sorted cells were immediately used in the single-cell RNAseq procedure.

TC treated cell culture dishes (Cat. F611203) and noTC treated polystyrene culture dishes (Cat. F611003) were purchased from Sangon Biotech (Shanghai). SYBR Green PCR Master Mix (2×) (Cat. 4913914001) was from Roche. RNAiso Plus (Cat. 9109) and PrimeScript™ RT reagent kit (Cat. RR047A) with gDNA eraser were from TaKaRa. LPS (Cat. L2630) and accutase solution containing 0.5 mM EDTA (Cat. A6964) were obtained from Sigma-Aldrich. 0.5 M EDTA was obtained from Solarbio (Cat. 420404). 0.25% Trypsin containing 0.02% EDTA was from Zhejiang Senrui Biological Technology (Cat.CR-25200). Recombinant mouse M-CSF (Cat. 415-ML) and GM-CSF (Cat. 416-ML) were purchased from R&D Systems. Debris Removal Solution (Cat. 130-110-203) and antibodies against Ly6C (APC) (Cat. 130-111-779) were from Miltenyi Biotec. 7-AAD Viability Staining Solution (Cat. 51-68981E) and antibodies against F4/80 (PE) (Cat. 565410) were from BD Bioscience. Anti-MHCII (Brilliant Violet 421) (Cat. 107632) antibodies were from Biolegend. Antibodies against CD11b (PE-CY7) (Cat. 25-0112-82) and CD16/CD32 (Cat. 14-0161-86) and Rat IgG1 kappa isotype control (Cat. 14-4301-85) were from Invitrogen.