Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A. Ratio of plasma antibody binding of CoV-2 wildtype Spike RBD to viral variant RBD for <1 Month, 1 Month, and 3 Month. p-values were calculated using unpaired Wilcoxon rank-sum test with Bonferroni multiple testing correction. B. Overview of the experimental design for sorting and single cell sequencing of antigen-specific B cells. A panel of up to 25 viral antigens was used to sort specific B cells from vaccinees, CoV-2 patients, and organ donors. Figure created using Biorender. C. A representative CoV-2 Spike-staining for a vaccinee and a patient sample. D. Change in Spike-specific B cell frequencies in vaccinees and patients was analyzed using a mixed-effect model. Multiple comparisons were performed using Tukey’s test (vaccinated), or Dunnett’s test versus the 3-month timepoint (infected).

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Infection, Clinical Proteomics, Binding Assay, Variant Assay, Single Cell, Sequencing, Staining

Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A. UMAP clustering of all included cells based on total gene expression (not including VDJ and antigen-sequencing data) results in 22 distinct cell clusters. B. Antigen-isotypes from the VDJ sequencing library are displayed on the UMAP from ( A ). C. B cell subsets assigned to clusters in ( A ). B cell subsets identified are naïve B cells, non-switched memory B cells (nsMBC), class-switched memory B cells (csMBC), activated memory B cells (actMBC), atypical memory B cells (atMBC), antibody-secreting B cells (ASC), and proliferating ASC (pASC). D. Antigen-specificities are displayed on the UMAP from ( A ). E. Antigen-specificities displayed as percentage (%) of each cluster. Legend same as in ( D ). F. Canonical B cell marker genes are displayed that distinguish the different B cell subsets shown in ( C ). G. Volcano plot shows differential gene expression between actMBC and total CD27+ MBC. H. Volcano plot shows differential gene expression between atMBC and total CD27+ MBC. I. Volcano plot shows differential gene expression between cluster 13 and csMBC J. Circos plot depicts clonal relationships between cells of different B cell subsets based on identical V H -, and J H -gene, same CDHR3 length, as well as an 85 % amino acid sequence similarity of the CDR-H3 region. K. UMAPs showing CoV-2-specific and total B cells before month 1, month 1, and month 3. RBD, Receptor-binding domain; MBC, memory B cell. See also Figure S1 , S2 .

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Gene Expression, Sequencing, Marker, Binding Assay

Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A . UMAP clustering of all included cells based on total gene expression (not including VDJ and antigen-sequencing data) results in 19 clusters. B. Antigen-isotypes from the VDJ sequencing library are displayed on the UMAP from ( A ). C. B cell subsets assigned to clusters in ( A ). B cell subsets identified are naïve B cells, non-switched memory B cells (nsMBC), class-switched memory B cells (csMBC), atypical memory B cells (atMBC), antibody-secreting B cells (ASC), germinal center B cells (GC), and Low viability cells which are excluded from further analysis. D. Antigen-specificities are displayed on the UMAP from ( A ). E. Selected marker genes distinguish the different B cell subsets shown in ( C ). F - G . Up- and down-regulated genes from peripheral actMBC ( F ), and csMBC cluster 13 ( G ) are shown on splenic B cell data. H. Antigen-specificities displayed as percentage (%) of each cluster. The antigen legend is the same as in ( D ). I. Circos plot depicts clonal relationships between cells of different B cell subsets based on same V H -, and J H -gene, same CDR-H3 length, as well as a 90% nucleotide sequence identity of the CDR-H3 region. J. Antigen-specificity of B cells stratified by concentration of N-specific antibodies; control, negative; N-seropositive, >3000 AU/ml; N-seronegative high, 1500–3000 AU/ml; N-seronegative low, <1500 AU/ml. K . Convergent BCR sequences (same VH-, and JH-gene, same CDR-H3 length, 90 % nucleotide similarity per person) of antigen-specific B cell that were identified from different B cell subsets are found in bone marrow of organ donors that were vaccinated, infected, or actively infected. See also Figure S3 .

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Gene Expression, Sequencing, Marker, Concentration Assay, Control, Infection

Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A . Antigen-specific B cells were sorted from three organ donors (donor 3, 5, and 15) using Antigen panel 2 with 25 viral proteins, including full Wuhan-Hu-1 Spike, Spike RBD, S1 NTD, S2, and Nucleocapsid, as well as 20 viral variant Spike RBD and OC43 Spike RBD. Heatmap shows binding signal from 210 expanded B cell clones from these donors that were detected both with the large antigen panel 2 and the smaller antigen panels 1a/b (100 % amino acid sequence similarity of CDR-H3) using a smaller B cell panel (italic *), as well as in the large antigen panel of 25 antigens. B. MAbs were produced based on sequences derived from sorted CoV-2-specific B cells. MAbs were tested using electrochemiluminescence assays (MSD © V-PLEX © Coronavirus panel 2) and ELISA to assess binding specificity. Antigen-binding from sorted B cells (blue) and sequence derived antibodies (orange) is shown for 100 mAbs. C. 59 mAbs specific for Spike RBD were tested with MSD V-PLEX CoV-2 Panel 11, 22, and 28 kits to determine binding to RBD viral variants. See also Figure S4 .

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Binding Assay, Variant Assay, Clone Assay, Sequencing, Produced, Derivative Assay, Electrochemiluminescence, Enzyme-linked Immunosorbent Assay

Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A-B. Heatmap showing normalized antigen binding of CoV-2 specific B cells in infected and vaccinated participants, based on antigen DNA-tag counts ( A ) and deceased organ donors ( B ). Rows grouped by Euclidean distance and average hierarchical clustering. C-D. Unsupervised clustering of binarized binding scores for BCRs that bound the Wuhan-Hu-1 RBD. Blood data shown on the left panel ( C ) and deceased organ donor data shown on the right ( D ). RBD variants group into two clusters (less broad and broad) by Euclidean distance and complete linkage hierarchical clustering. PID, patient identifier; See also Figure S5 , S6 .

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Single Cell, Variant Assay, Binding Assay, Infection

Journal: Immunity

Article Title: Multiplexed antigen panel analysis identifies B cell phenotype and receptor genetic contributions to antibody breadth

doi: 10.1016/j.immuni.2026.03.006

Figure Lengend Snippet: A. Bar plot depicting the frequency of IGHV gene use considering the B cells in stepwise categories of increasing antigenic binding breadth. Blood samples depicted in the top panel and organ donors depicted on the bottom. Only IGHV genes with a frequency of 2 or more depicted (n Blood =247, n Organ =1096). Bars colored by participant identifier, for blood see Figure S6 , for organ donors see Figure S5 . B. Intersection plots of binarized binding scores for BCRs from cluster 13 and 15 that bound RBD. Vertical bars: antigen–specific IGH sequences per variant combination colored according to cluster origin (top) and time (bottom). Left bars: total number of BCRs able to bind RBD variant. C. Cartoon of weighted accumulative binding score. BCRs able to bind less evasive variants (Alpha [N501Y]; K417N; and L452R) receive a score of 0.5 per variant. BCRs able to bind evasive variants (E484K alone; Gamma [E484K, N501Y, K417T]; and Beta [E484K, N501Y, and K417N]) receive a score of 1 per variant. D. Boxplots comparing weighted variant breadth of cluster 13 and 15 RBD antigen-specific cells. E-F. Boxplots comparing weighted variant breadth ( E ) and SHM ( F ) of cluster 13 and 15 RBD-specific cells, split by time of sampling. Statistical significance was determined using a two-tailed Wilcoxon rank-sum test. G. Key variables selected by applying DIABLO as most informative in predictive models discriminating BCRs that are broad and less broad binders. Bars indicate loading coefficient weights of selected features (ranked from most to least informative in cluster prediction, according to binding group). H. Circos plot depicting the strongest correlated variables determined by (G). The variables selected by applying DIABLO to cell type, IGHV and IGKLV gene use and isotype are depicted using a circos plot. The variables within the Circos plot are highlighted and linked to other variables using red or blue lines, depending on whether the correlation is positive or negative. Only correlations above a threshold of 0.5 are depicted. The lines around the Circos plot are drawn by connecting the average expression value of a given variable for a certain phenotypic group.

Article Snippet: CoV-2 Spike S2 Protein, His Tag , Acrobiosystems , Cat# S2N-C52H5.

Techniques: Binding Assay, Variant Assay, Sampling, Two Tailed Test, Expressing