Human b cell isolation kit

Human peripheral blood mononuclear cells were donated by the Chungbuk Red Cross Blood Center (Cheongju, Korea). Lymphocytes were isolated from these cells by density gradient centrifugation (Ficoll-Paque) 34 (link). hB cells were isolated from lymphocytes using a human B cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). mB cells were purified from spleen cells of MRL.Fas^lpr mice by a negative depletion method using mouse B cell isolation kit (Miltenyi Biotec) 9 (link). Purity of hB and mB cells was typically >90%. B cells (1 × 10⁵ cells/well) and MSCs (0.01-0.1 × 10⁵ cells/well) were added in 200 µL to the wells of 96-well plates. CpG-oligodeoxynucleotide (ODN; 5 μg/mL) was used to activate hB cells, and lipopolysaccharide (LPS; 1 μg/mL) was used to activate mB cells. To measure B cell proliferation, cells were pulsed with [³H]-thymidine (113 Ci/nmol; NEN, Boston, MA, USA) at a concentration of 1 μCi/well for the last 18 h and were harvested on day 3 using an automated cell harvester (Inotech, Dottikon, Switzerland). The amount of [³H]-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac, Turku, Finland) 3 (link).

mRNA or sham (no mRNA) electroporated NK cells and target cells were co-cultured at an effector-to-target (E:T) ratio of 1:1 for 1 hour at 37 °C. NK cells without target cells were used as negative control detecting spontaneous background activity. Following incubation, cells were stained with fluorescently conjugated antibodies for CD107a expression and markers to identify NK cells. ADCC was measured in co-cultures containing either 0.1 or 10 μg/mL of rituximab (Roche). Autologous healthy B-cells were isolated on the same day of the experiment using the human B-cell isolation kit (Miltenyi). Autologous patient lymphoma cells from fine-needle aspirations were also thawed on the same day as the assay but were not subject to further cell isolation due to limitations in cell number.

The human‐related study was approved by the Ethics Committee of Hangzhou Centre for Disease Control and Prevention (Approval No. 2020‐7). All participants proved written informed consent before participation in this study and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. All experiments with samples from COVID‐19 convalescent patients were performed in a BioSafety Level 2⁺ level laboratory and with approval from the Ethics Committee of Hangzhou Centre for Disease Control and Prevention (Approval No. 2020‐7). Thirty COVID‐19 convalescent patients were recruited and plasma RBD‐binding titer was determined by ELISA. Blood samples from three convalescent patients with high plasma RBD‐binding titer were drawn. Total B cells were isolated from COVID‐19 convalescent patients’ peripheral blood mononuclear cells using a human B cell isolation kit according to the manufacturer’s instructions (Miltenyi). B cells from three convalescent patients were pooled (total 1.2 million B cells) and incubated with biotin‐labeled SRAR‐CoV‐2 S‐RBD protein, and S‐RBD specific B cells were enriched using anti‐biotin magnetic beads (Miltenyi). The isolated B cells, counted 5,000, were suspended in 1 ml TRIzol (Invitrogen), flash‐frozen in liquid nitrogen, and stored in −80°C before RNA extraction.