Igm v450

All flow cytometry used appropriate isotype controls. Gating and compensation were aided by the performing fluorescence minus one controls. Cells isolated from biopsies or blood were stained with blue-fluorescent reactive dye (Life Technologies), CD19-BV785, CD27-APC (BD Biosciences) or PE or BV421, CD10-BV605 or APC, CD38-PerCp-ef710 (eBioscience), CD24-PE/Cy7 or BV605, IgD-APC/Cy7, IgM-V450 (BD Biosciences), IgG-PE/Cy7 and IgA-FITC (Miltenyi Biotec), in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before analysis on the BD LSRFortessa (BD Biosciences). For high-throughput sequencing analysis, cells were stained with LIVE/DEAD Fixable Aqua (Life Technologies) or DAPI, CD19-BV785, CD27-FITC or APC, IgD-APC/Cy7, CD38-PerCp-ef710 (eBiosciences), CD10-BV605 in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before sorted on the BD FACSAria (BD Biosciences). Four subsets from PBMC and three subsets from paired biopsy mononuclear cells from Donors 1 to 4. The numbers of cells used to generate sequences for each sample from Donors 1 to 4 are shown in Supplementary Fig. 14d.

S2P spike trimer-specific memory B cells were isolated and sequenced using the protocol previously described by Liu et al.²¹ (link)^,27 (link) In brief, peripheral blood mononuclear cells (PBMCs) from patient 12 were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Invitrogen) at room temperature for 20 min, washed with RPMI-1640 complete medium, and incubated with 10 μg/mL B.1.351 S2P spike trimer at 4 °C for 45 min. Cells were then washed and incubated with a cocktail of flow cytometry and hashtag antibodies, containing CD3 PE-CF594 (BD Biosciences), CD19 PE-Cy7 (Biolegend), CD20 APC-Cy7 (Biolegend), IgM V450 (BD Biosciences), CD27 PerCP-Cy5.5 (BD Biosciences), anti-His PE (Biolegend), and human Hashtag 3 (Biolegend) at 4 °C for 1 h. Cells were then washed again, resuspended in RPMI-1640 complete medium, and sorted for S2P spike trimer-specific memory B cells (CD3−CD19+CD27+S trimer+ live single lymphocytes). These sorted cells were mixed with PBMCs from the same donor, labeled with Hashtag 1, and loaded to a 10X Chromium chip for the 5′ Single Cell Immune Profiling Assay (10X Genomics) at the Columbia University Human Immune Monitoring Core (HIMC; RRID:SCR_016740). Library prep and quality control were performed according to the manufacturer’s instructions and then sequenced on a NextSeq 500 (Illumina).