Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: The HLA64-RC panel was labeled with fluorochrome-conjugated Abs or with unconjugated primary Abs followed by fluorochrome-conjugated secondary Abs. Following flow cytometry acquisition, data were analyzed using FlowJo ™ software. Live cell events were demultiplexed into 64 populations based on the six FP channels as in . Stacked histograms of the demultiplexed populations show fluorescence intensities corresponding to Ab labeling. Histograms are grouped by HLA-A (dark red), HLA-B (green), HLA-DQ (blue), and HLA-DR (purple) alleles, displayed upward in the order indicated on the far-right. Blue labels positioned in the upper-right (top row) or lower-right (bottom row) corners denote the reported specificities of the corresponding mAbs. hIgG1K and hIgG1L, human IgG1κ and IgG1λ isotype control Abs. BB7.2-hIgG1K, BB7.2 in human IgG1κ format. Anti-hIgG-PE and anti-mIgG-PE, PE-conjugated anti-human or anti-mouse IgG secondary Abs. SA-PE, PE-conjugated streptavidin.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Labeling, Flow Cytometry, Software, Fluorescence, Control

Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: Using the HLA64-RC panel, ten serum samples (#01–#10) from sensitized transplant candidates were tested for HLA-binding activities at 1/3 serial dilutions from 1:2 to 1:486, followed by secondary labeling using PE-conjugated anti-human IgG at 2 µg/ml. Raw binding data are shown in Supplementary Document 1. The dilutions yielding the highest signal-to-background ratio for individual samples were selected and MFI values (Supplementary Data 3) were compared with those obtained from clinical SAB assays (Supplementary Data 4) using the same samples. a) Heatmaps comparing log 10 (MFI) values for shared alleles between the HLA64-RC and SAB assays. The selected dilutions for the HLA64-RC assay were indicated in parentheses. For the SAB assay, serum samples were tested neat. Alleles with MFI values below the binding threshold (see Methods) are shown in grey. Reactivities detected by the HLA64-RC assay but absent in the SAB assay are highlighted with blue boxes. b) Dot plots comparing log 10 (MFI) values for shared alleles between the HLA64-RC and SAB assays, as in ( a ). Data from multiple samples were pooled and plotted separately for HLA-A, -B, -DQ, and -DR loci, with color and shape indicating individual alleles within each locus. For consistency, only serum samples tested at 1:2 dilution in the HLA64-RC assay in ( a ) were included. Within each locus, Lin’s CCC with 95% bootstrap confidence interval (CI), Spearman correlation, and SMA regression with 95% bootstrap CI were calculated using log 10 (MFI) values from the two assays. Solid diagonal lines represent SMA regression fits; dotted lines indicate the 95% CI.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: Profiling Allogeneic HLA-specific B-cell Responses Utilizing a 64-plex Single-HLA Reporter Cell Panel

doi: 10.64898/2026.01.19.700453

Figure Lengend Snippet: HLA monomer-binding single B-cell culture supernatants were screened using the HLA64-RC assay. R952-X06 and R952-X08 to -X12 cultures were derived from phenotyping antibody Panel 1 mediated sorting, and the rest from Panel 2 sorting. Secondary labeling was performed using PE-conjugated anti-human IgG (2 μg/ml). Flow cytometry data were analyzed and visualized using the HLA64 R package, and MFI values were exported (Supplementary Data 5). Demultiplexed HLA alleles corresponding to individual reporter cell populations are indicated on the right. Data are shown for HLA-specific cultures, and only HLA-A and -B loci are plotted. Human IgG1κ (hIgG1K), W6/32 in human IgG1κ format (W6/32-hIgG1K), and a no-B-cell culture supernatant were included as labeling controls. Populations exceeding the binding threshold (see Methods) relative to the no-Bcell control were automatically highlighted in red.

Article Snippet: Following three additional washes, a cocktail of HRP-conjugated anti-human Igκ (Southern Biotech, 2061-05) and anti-human Igλ (Southern Biotech, 2071-05) Abs was added.

Techniques: Binding Assay, Cell Culture, Derivative Assay, Labeling, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Flow cytometry gating strategies to identify B-cell populations in LN cells derived from SHIV-infected RMs. (B) Alluvial plots showing percentages of IgG + GC, IgG + Bmem and IgM + Bmem populations among CD20 + LN cells during the infection time course in the three RMs in each infection group. (C-E) Alluvial plots showing percentages of SOSIP-binding (C), gp140-binding (D), and autologous neutralizing (E) IgG + single B-cell cultures from different B-cell populations over the infection course. See also Figures S1 and S2, and Tables S1 and S2.

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Flow Cytometry, Derivative Assay, Infection, Binding Assay

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Kinetics of IgG production in culture supernatants from RM IgM + and IgG + single B cells cultured with the MS40L feeder cell line. Each dot represents an individual IgG + culture well. Dashed lines indicate detection limits, and error bars (in red) represent the median values. (B-C) Comparison of the original MS40L and engineered MEC-147 feeder cell lines in supporting single B-cell cultures from different B-cell populations. Panel (B) shows the culture of B cells isolated from the lymph nodes of an RM after DTaP immunization, while panel (C) shows the culture of B cells isolated from PBMCs of a healthy human donor. The top panels display the percentage of IgG + culture wells among all single-cell sorted wells. Error bars indicate the mean + SD across three 96-well culture plates. The bottom panels show OD450 values from IgG ELISA assays, with each dot representing an individual IgG + culture well. Dashed lines indicate detection limits, and error bars (in red) represent the median values. (D) Single nucleated cells were isolated from LN biopsies of infected RMs at various time points following SHIV infection. B cells of different phenotypes, including mature follicular (MF), IgM + Bmem, IgG + GC, and IgG + Bmem cells, were identified from isolated LN cells by flow cytometry and sorted into single B-cell cultures with MEC-147 feeder cells. Supernatants were harvested after 18 days of culture, and IgG-producing cultures were identified using standard ELISA assays. IgG-containing culture supernatants were tested for neutralization activity against autologous SHIV pseudo-viruses using standard TZM-bl assays. Each dot represents an individual single B-cell culture supernatant containing clonal IgG. Error bars in red indicate median values, while blue dashed lines indicate cutoff thresholds for defining neutralizing versus non-neutralizing cultures. Inserted red numbers represent the percentage of neutralizing cultures among IgG + cultures for the corresponding B-cell populations (see Table S2 for details).

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Cell Culture, Comparison, Isolation, Enzyme-linked Immunosorbent Assay, Infection, Flow Cytometry, Neutralization, Activity Assay

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: The same IgG-containing single B-cell culture supernatants prepared as described in were tested for binding activity against autologous SOSIP and gp140 antigens using standard Luminex assays, conducted in parallel and in a blinded manner to the autologous neutralization assays shown in . RMs were infected with either the BG505 (A) or CH505 (B) SHIV strains. B-cell populations are color-coded as indicated in the keys. Dashed lines indicate cutoff thresholds for defining antigen-binding versus non-binding cultures. Inserted numbers represent the percentage of antigen-binding cultures among IgG + cultures for the corresponding B-cell populations. Additional antigens or capture antibodies, including CH505.gp120, MN.gp41, anti-RhIgG, and non-relevant controls (streptavidin, BSA, and ovalbumin), were included in the Luminex bead panel (see Table S2 for details).

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Cell Culture, Binding Assay, Activity Assay, Luminex, Neutralization, Infection

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Related to Table S3. Proportions of identified autologous SOSIP- or gp140-binding B cells for which BCR sequences were determined. The blue horizontal line separates two categories: cultures with binding MFI > 300 (upper) and MFI ≤ 300 (lower). Stacked bars represent proportions normalized to the total counts of Env-binding B cells with MFI > 300, with pink indicating sequenced and grey indicating not sequenced. Numbers within bars denote the counts of Env-binding B cells in each category. (B) Related to . VH mutation frequency and neutralization differences between Bmem and IgG + GC B cells for lineages containing both IgG + GC and Bmem members at the same timepoints in individual RMs. Each dot represents a Bmem–IgG + GC B cell pair. Only RM–Bmem population groups with more than two data points are shown. (C) Related to . Relationship between rAb binding MFI values to heterologous gp140 protein and the binding MFI values of corresponding single B-cell culture supernatants to CH505.gp120 and MN.gp41 proteins in the initial screening. Functional types of source B-cell clones are color-coded, and dashed lines indicate binding cutoffs. (D) Related to Methods. UMAP plots showing the same coordinates and clusters as in , with colors indicating individual assay batches. (E-F) Related to . Normalized density plots display the distribution of distances calculated from the pairwise IGV gene distance matrix at two categorical levels: IGV gene (E) and family (F). Distances within the same category (“Within”) are shown in black, while distances between different categories (“Between”) are shown in grey. Dashed vertical lines in blue represent cutoff values, determined either at the intersection of within and between distance distributions or at the point of minimum difference when no intersection is observed. (G) Related to . Distribution of MADM of IGV allele usage frequencies across RMs, calculated from percentages smoothed within IGV families. The distribution for uCDR3 represents the combined MADM values from 1000 multinomial down-sampled simulations (see Methods). Empirical p values were calculated between the median MADM of xBCR and 1000 median values of randomly-sampled and size-matched uCDR3 subsets (see Methods). ***, empirical p < 0.001. Alleles with zero frequencies across all three RMs were excluded to prevent distortion by an excess of MADM = 0 values. (H) Related to . Cosine similarity scores of IGV allele usage between RMs. For the uCDR3 source, dots indicate medians from 1000 multinomial down-sampling simulations (see Methods), with error bars showing 25th – 75th percentiles. Lines connect the same RM pair comparisons from uCDR3 or xBCR sources, with solid lines for original percentages and dashed lines for percentages smoothed within IGV families. Line colors indicate the significance of empirical p values: grey (***) for p < 0.001, blue (**) for p < 0.01, green (*) for p < 0.05, and orange (ns) for not significant.

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Binding Assay, Mutagenesis, Neutralization, Cell Culture, Functional Assay, Clone Assay, Sampling

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Expansion/contraction of Env-reactive neutralizing and non-neutralizing IgG + GC B-cell clones in each infection group over time. ND, not detected. (B-C) Distributions of timepoint occurrence (B) and clone size (C) were compared between neutralizing (Neut) and non-neutralizing (NonN) clones. Two-sided Wilcoxon rank-sum tests at the clone level (pooled across RMs within each SHIV group) were used. (D) Clone counts of neutralizing and non-neutralizing IgG + GC B-cell clones in individual RMs. (E) IGHV mutation frequency changes in Env-reactive clones spanning multiple timepoints. All nine clones (named “RM_Clone-ID”) that persisted across at least two timepoints and contained more than two clonal members at each timepoint are shown. Adjacent timepoints were compared using two-sided Wilcoxon rank-sum tests. Statistical annotations: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. See also Table S3 and Document S1.

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Clone Assay, Infection, Mutagenesis

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Proportions of neutralizing and non-neutralizing Env-reactive B cells from different populations. Inserted numbers indicate cell counts. ND, not detected. (B) Clonal relationship between Env-reactive IgG + GC and Bmem cells. Top sectors show B-cell populations (color-coded); lower sectors represent individual neutralizing or non-neutralizing clones, including lineages and singletons. Color gradients (red for neutralizing and blue for non-neutralizing) were used solely to visually distinguish adjacent individual clones. (C) Venn diagrams showing the distribution of B-cell clones across single or multiple populations. Blank regions indicate zero counts. (D-E) For lineages containing both IgG + GC and Bmem members at the same timepoints: (D) VH mutation frequencies and neutralization activities for all seven RM_Clone-ID_Timepoint groups identified with >3 clonal members; (E) VH mutation frequency and neutralization differences between Bmem and IgG + GC B cells for all 19 RM_Clone-ID_Timepoint groups identified. Each dot represents a Bmem–IgG+ GC B cell pair. See also Table S3.

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Clone Assay, Mutagenesis, Neutralization

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Binding activities of reference bnAbs and nnAbs (human IgG1 format) for BG505 and CH505 SOSIP and gp140 antigens were tested in standard Luminex assays using serial dilutions. Vertical dashed lines indicate the concentration point (206 ng/ml) used for calculating MFI ratios in . (B) Binding activities of rAbs (rhesus IgG1 format) derived from representative B-cell clones of different functional types were similarly tested. Results for selected rAbs from BG505-infected (top row) and CH505-infected RMs (bottom row) are shown (named as “RM_Clone-ID (Type)”). Full data for all 146 rAbs are provided in Document S3. Antigens are color-coded as in panel (A). Dashed grey curves represent MFI values detected with Luminex beads conjugated to an anti-RhIgG capture antibody. Vertical dashed lines indicate the concentration points where anti-RhIgG MFI values align closely with those detected using culture supernatants during initial screening. (C) Comparison of MFI values for autologous SOSIP and gp140 antigens detected using culture supernatants (MFI (sup)) during initial screening and rAbs (MFI (rAb)) at selected concentration points (indicated by vertical dashed lines in panel (B) and Document S3). Linear regression models were fitted, and R 2 values indicating the proportion of variance explained are shown in parentheses in the legends for corresponding antigens.

Article Snippet: For human IgG ELISA assays, the coating antibodies used were goat anti-human Igκ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2061-01) and goat anti-human Igλ (2 μg/ml, polyclonal, Southern Biotech, Cat# 2071-01), the standard antibodies used were human IgG1 Kappa (polyclonal, Southern Biotech, Cat# 0151K-01) and human IgG1 Lambda (polyclonal, Southern Biotech, Cat# 0151L-01), and the detection antibody used was goat anti-human IgG-HRP (1/5000 dilution, polyclonal, Southern Biotech, Cat# 2040-05).

Techniques: Binding Assay, Luminex, Concentration Assay, Derivative Assay, Clone Assay, Functional Assay, Infection, Comparison