Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Representative flow cytometry plots (left) and quantification (right) of spleen cells from 8- to 10‐week‐old wild-type, 10E8-NGS-04 H and 10E8-UCA H mice and gating strategy for the quantification of splenic B220 + TCRβ − B cells and TCRβ + B220 − T cells. Data are pooled from two independent experiments ( n = 3–4 mice per independent group). b , Representative flow cytometry plots (left) and quantification (right) of spleen cells from 8- to 10‐week‐old wild-type, 10E8-NGS-04 H and 10E8-UCA H mice and gating strategy for the quantification of CD21 hi CD24 lo follicular B cells, transitional CD21 lo CD24 hi T0/T1 B cells, CD21 hi CD24 hi CD23 + T2 B cells and CD21 hi CD24 hi CD23 − marginal zone B cells. Data are pooled from two independent experiments ( n = 3–4 mice per independent group). Error bars indicate mean ± s.d. from mice in pooled groups. c , Representative flow cytometry (left) and quantification (right) of B220 + CD4 − CD8 − F4/80 − Gr-1 − naive B cells from peripheral blood binding biotinylated 10E8-GT9 in wild-type or 10E8-NGS-04 H mice. Data are pooled from two independent experiments. Error bars indicate mean ± s.d. from mice in each group ( n = 3–6). d , Representative flow cytometry (left) and quantification (right) of naive B cells binding to biotinylated 10E8-GT9 in wild-type or 10E8-UCA H mice. Error bars indicate mean ± s.d. from mice in each group ( n = 3). Data from a single experiment are presented and are representative of three independent experiments. e , Top, 10x Genomics scBCR-seq data from 10E8-GT10.2-specific naive B cells from 10E8-NGS-04 H mice ( n = 1,026 pairs amplified) showing human 10E8-NGS-04 IGH-V gene frequency (left) and mouse IGK-V genes (right). Cells from three mice were sequenced, and representative data from one mouse are presented. Bottom, 10x Genomics scBCR-seq data from 10E8-GT10.2-specific naive B cells from 10E8-UCA H mice ( n = 1,418 pairs amplified) showing the frequency of the human 10E8-UCA IGH-V gene (left) and mouse IGK-V genes (right). Cells from three mice were sequenced, and representative data from one mouse are presented; HC, heavy chain; LC, light chain.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Binding Assay, Amplification

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Representative flow cytometry of bone marrow progenitors isolated from 8–10‐week‐old wild-type, 10E8-NGS-04 H and 10E8-UCA H mice separated by Hardy classification into: early (B220 + CD43 + ) and late (B220 + CD43 − ) B cells (top panel); early (A–C) B cell subfractions (middle panel); and the late (D–F) subfractions in the bone marrow (bottom panel). Data is representative of two independent experiments (n = 3–4 mice/independent group). b , Quantification of flow cytometry of bone marrow progenitors, as in a. Error bars indicate mean ± SD from mice in each group. Data are pooled from two independent experiments (n = 3–4 per treatment) for wild-type, 10E8-NGS-04 H and 10E8-UCA H mice; MPER-HuGL18 H data presented for comparative purposes (n = 4 mice from one experiment). c , Representative flow cytometry of CD4 + /CD8 + T cells and T2/MZB (marginal zone B cells) cells isolated from spleen samples of 8–10‐week‐old wild-type, 10E8-NGS-04 H and 10E8-UCA H mice, as in Fig. . d , Quantification of T and B cell subfractions, as in c. Error bars indicate mean ± SD from mice in each group. Data are pooled from two independent experiments (n = 3–4 per treatment) for wild-type, 10E8-NGS-04 H and 10E8-UCA H mice, with some values repeated from Fig. 1a, b for comparison; MPER-HuGL18 H data also presented for comparative purposes (n = 4 mice from one experiment). e , Representative flow cytometry histograms showing expression of B220, IgM and IgD in follicular B cells in spleen (green, WT; red, 10E8-NGS-04; yellow, 10E8-UCA and blue, MPER-HuGL18). Data representative of two independent experiments (n = 3–4 mice per treatment) for wild-type, 10E8-NGS-04 H and 10E8-UCA H mice; MPER-HuGL18 H data presented for comparative purposes (n = 4 mice from one experiment).

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Isolation, Comparison, Expressing

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Representative flow cytometry of immature (B220 + CD93 + ) and mature (B220 + CD93 − ) B cells isolated from spleen samples of 8–10‐week‐old wild-type, 10E8-NGS-04 H and 10E8-UCA H . b , Representative flow cytometry of T1, T2 and T3 subfractions from immature B cells isolated from spleen samples of wild-type, 10E8-NGS-04 H and 10E8-UCA H . c , Quantification of transitional cells subset T3 as in b, with MPER-HuGL18 H shown for comparison. Error bars indicate mean ± SD from mice in each group (n = 4). Data representative of two independent experiments (n = 3 mice per treatment) for wild-type, 10E8-NGS-04 H and 10E8-UCA H mice with one presented; MPER-HuGL18 H data from one experiment presented for comparative purposes (n = 4 mice from one experiment). d , Gating strategy for single-cell sorting of naïve B cells from blood of 10E8-NGS-04 H and 10E8-UCA H mice. e, f , 10x Genomics single-cell BCR sequences from B220 + IgM + naïve B cells sorted from 10E8-NGS-04 H (e) and 10E8-UCA H (f) mice. Human NGS-04 IGHV gene frequency in teal (94%), human 10E8UCA IGHV gene frequency in green (65%), and grey murine HC. Pies on the right shows the respective murine IGKV genes in various colors; IGKV families used in key. n=pairs amplified, NGS-04 (n = 1046) and 10E8-UCA (n = 1260). g , Left, crystal structure of the T298 scaffold (PDB 3T43) that served as a starting point for the iterative vaccine design of 10E8-GT9 and 10E8-GT10 immunogens. Purple: MPER; Cyan: scaffold backbone. Right, schematic illustration of the multimerization of the 10E8-GT immunogens into self-assembling 12mer nanoparticles.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Isolation, Comparison, FACS, Amplification

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Representative equilibrium binding curves of 10E8-GT9.2 interacting with human 10E8-UCA H /UCA L and 10E8-NGS-04 H /10E8-NGS-04 L as measured by the monomer SPR assay. b , Quantification of precursor frequencies corresponding to transfer of 5,000, 50,000, 100,000, 200,000 and 500,000 CD45.2 + 10E8-UCA H B cells from donor mice to the host wild-type CD45.1 + mice. (n = 4). Error bars indicate mean ± SD from mice in each group. Experiment performed once. c , Flow cytometry of CD45.2 + cells in splenic GCs of mice transferred with varying numbers of CD45.2 + 10E8-UCA H precursors and immunized with 50 μg of 10E8-GT9 12mer and the Ribi adjuvant system 7 DPI. d , Representative gating strategy used for the GC response in adoptive transfer experiments.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Binding Assay, SPR Assay, Flow Cytometry, Adjuvant, Adoptive Transfer Assay

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Affinities of human 10E8-UCA H /UCA L or 10E8-UCA H /mouse L Fabs to 10E8-GT9.2 12mer in a nanoparticle SPR assay. Symbols represent individual antibodies, and the line denotes the median 10E8-UCA H /mouse L value. b , Representative flow cytometry plots of CD95 hi CD38 lo B220 + GC B cells and their distribution into CD45.2 + and CD45.1 + B cells at day 7 p.i. in CD45.1 wild-type mice transferred with 50,000 CD45.2 + 10E8-UCA H B cells (to reach a frequency of 4:10 5 CD45.2 + 10E8-UCA H B cells) 1 day before immunization with 50 µg of 10E8-GT9 12mer or 10E8-GT9-KO and Ribi adjuvant. c , Representative flow cytometry plots showing the percentage of CD45.2 + 10E8-GT9 ++ (top) CD45.1 + 10E8-GT9 ++ (bottom) GC B cells at day 7 p.i. with 50 µg of 10E8-GT9.2 12mer in recipients as in b . d , Ribbon diagrams showing the modifications in immunogens 10E8-GT9.4 and 10E8-GT9.6 derived through the reduction of off-target effects (10E8-GT9.4) plus the addition of the PADRE T cell help epitope (10E8-GT9.6). Mutation T95A (red) was removed, as candidates containing this mutation failed to express as 12mers. e , f , Representative flow cytometry plots of CD45.2 + GC B cells and CD45.1 + GC B cells ( e ) and quantification of GC B cells and CD45.2 + GC B cells ( f ) at day 7 p.i. in CD45.1 wild-type mice transferred with CD45.2 + 10E8-UCA H B cells (frequency of 6:10 5 CD45.2 + 10E8-UCA H B cells) and immunized 1 day later with 50 µg of 10E8-GT9.2 12mer, 10E8-GT9.3 12mer, 10E8-GT9.4 12mer, 10E8-GT9.5 12mer, 10E8-GT9.6 12mer or 10E8-GT9-KO 12mer with Ribi adjuvant. Error bars indicate mean ± s.d. from mice in each group ( n = 6). Data from one representative experiment are presented, and two independent experiments were performed.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: SPR Assay, Flow Cytometry, Adjuvant, Derivative Assay, Mutagenesis

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Sequences of new immunogens. Mutations relative to 10E8-GT9.2 12mer are red; nanoparticles are purple; the PADRE epitope is in brown; N-linked glycosylation sites are blue; flexible linkers are green. Sequences are wrapped over multiple lines. b , Table and schematic describing the 10E8-GT9 nanoparticle design elements including GT version, added glycan sites, presence of PADRE T-help epitope, and off-target reduction. c , Representative FACS gating strategy for 10E8-GT9 libraries designed to reduce off-target responses. A selection strategy was employed to maintain high binding to 10E8-UCA (y-axis) while reducing binding of mouse competitors (x-axis). d , Graph showing affinities of human 10E8-UCA H / UCA L Fab and Fabs of 10E8-UCA H /mouse L to 10E8-GT9.2 12mer, 10E8-GT9.3 12mer, 10E8-GT9.4 12mer, 10E8-GT9.5 12mer and 10E8-GT9.6 12mer, using the nanoparticle SPR assay. Symbols represent individual antibodies; lines represent median 10E8-UCA H / mouse L values.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Glycoproteomics, Selection, Binding Assay, SPR Assay

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Affinities of 10E8-UCA H /mouse L Fabs or human 10E8-UCA H /UCA L for 10E8-GT9.2, 10E8-GT9.6 and 10E8-GT10.2 12mers in nanoparticle SPR assays. Symbols represent individual antibodies, and lines represent 10E8-UCA H /mouse L median values. b , c , Representative flow cytometry plots of CD45.2 + and CD45.1 + GC B cells ( b ) and percentage of GC B cells and CD45.2 + GC B cells ( c ) at day 7 p.i. in the spleens of CD45.1 wild-type mice transferred with CD45.2 10E8-UCA H B cells (frequency of 1:10 4 CD45.2 + 10E8-UCA H B cells) and immunized 1 day later with 50 µg of 10E8-GT9.6 12mer, 10E8-GT10.2 12mer and 10E8-GT9-KO 12mer with Ribi adjuvant. Error bars indicate mean ± s.d. from mice in each group ( n = 4–6). Significance was calculated with a two-tailed, paired t- test. Data shown are from one of two independent experiments; NS, not significant. d , Representative flow cytometry plots (left) and quantifications (right) of 10E8-UCA H CD45.2 + B cells in GC B cells at day 7 p.i. in the spleens of CD45.1 wild-type mice transferred with CD45.2 10E8-UCA H B cells (frequency of 1:10 4 CD45.2 + 10E8-UCA H B cells) immunized with 10E8-GT10.2 12mer (40 µg, 10 µg and 2.5 µg) or 10 µg of 10E8-GT9-KO 12mer with Ribi adjuvant ( n = 4; top) or with 10E8-GT10.2 12mer (20 µg, 5 µg and 1.25 µg) or 5 µg of 10E8-GT9-KO 12mer with alhydrogel adjuvant (bottom). Error bars indicate mean ± s.d. from mice in each group ( n = 3). Alhydrogel data are representative of three independent experiments, and Ribi data are representative of two independent experiments, with one quantified. e , Representative immunohistochemistry image of the spleen (top) and percentage (bottom) of 10E8-UCA H CD45.2 + B cells in GCs in recipients at day 7 p.i., as in d . Scale bar, 50 µm. The image is representative of images obtained from four mice.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Adjuvant, Two Tailed Test, Immunohistochemistry

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Graph showing mean fluorescence intensity (MFI, % of initial) of 10E8-GT10.2 dissociation from 10E8-UCA H (green) and 10E8-NGS-04 H BCRs (teal). Error bars indicate SD (n = 4). Experiment performed once. b , Schematic showing wild-type mice adoptively transferred with 10E8-NGS-04 H precursors and immunized with either 50 µg of 10E8-GT9.6 12mer, 10E8-GT10.2 12mer or 10E8-GT9-KO 12mer with ribi adjuvant (control), as in Fig. . c , Schematic showing wild-type mice adoptively transferred with 10E8-NGS-04 H precursors and immunized with either 5 μg 10E8-GT10.2 12mer and alhydrogel or 5 μg 10E8-GT9-KO 12mer control and alhydrogel. d , Representative flow cytometric plots of CD45.2 + 10E8-NGS-04 H B cells recruitment to GCs 7 and 14 DPI in spleens of mice immunized as in c. e , Quantification of GC size and CD45.2 in GC in spleen of mice immunized in c,d. Data are pooled from two independent experiments (n = 5–6 per treatment). Error bars indicate mean ± SD from mice in each group.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Fluorescence, Adjuvant, Control

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Representative flow cytometry plots of CD45.2 + 10E8-UCA H recruitment to GCs 7 DPI in spleens of mice immunized with 5 μg of 10E8-GT10.2 12mer or with 25 μg, 6.2 μg or 1.5 μg of GT10.2 60mer and Ribi adjuvant. b , Quantification of a: (Left) GC size; (right) CD45.2 + 10E8-UCA H cells in GCs. Error bars indicate mean ± SD from mice in each group (n = 3). Two independent experiments were performed; data from one representative shown. c , ELISA of Ab response to immunizations as in a. Green dots represent IgG level specific to 10E8-GT10.2 probe and gray dots represent IgG level to 10E8-GT10-KO probe. Each symbol represents a different mouse. Bars indicate geometric mean and geometric SD from mice in pooled groups. n = 2–3 mice in each group. d , Quantification of GC size and epitope specific (GT10 ++ KO − ) CD45.2 + 10E8-UCA H in GC from mice immunized with different doses of 10E8-GT10.2 12mer with Ribi adjuvant, as shown in Fig. . Error bars indicate mean ± SD from mice in each group (n = 4). Two independent experiments performed, one shown. e , Quantification of GC size and epitope specific (GT10 ++ KO − ) CD45.2 + 10E8-UCA H in GC from mice immunized with different doses of 10E8-GT10.2 12mer with alhydrogel, as shown in Fig. . Error bars indicate mean ± SD from mice in each group (n = 3). Three independent experiments were performed; data from one representative shown. f , Quantification of lymph node response in mice. GC size, CD45.2 + 10E8-UCA H B cells in GCs and epitope specific CD45.2 + in GCs from 7 DPI with 10E8-GT10.2 12mer and alhydrogel, from mice immunized as in Fig. . Error bars indicate mean ± SD from mice in each group (n = 3).

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Adjuvant, Enzyme-linked Immunosorbent Assay

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Schematic showing mice adoptively transferred to reach ~1:10 4 10E8-UCA H precursors and immunized with 5 µg either different variants of 10E8-GT9 12mer and GT10.2 12mer with alhydrogel or 10E8-GT9-KO 12mer with alhydrogel (control); experiment performed once. b , Quantification of GC size, CD45.2 + 10E8-UCA H B cells in GC and epitope specific CD45.2 + 10E8-UCA H in GC in spleen of mice immunized as in a. Error bars indicate mean ± SD from mice in each group (n = 5). c , Quantification of GC size, CD45.2 in GC and epitope specific CD45.2 in GC in lymph nodes of mice immunized in a. Error bars indicate mean ± SD from mice in each group (n = 5).

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Control

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Schematic of adoptive transfer of CD45.2 + 10E8-UCA H B cells into CD45.1 + wild-type mice followed by immunization with 10E8-GT10.2 12mer with alhydrogel and analysis until 21 DPI. b , Quantification of percent of B cells in GC of spleen from mice immunized with 10E8-GT10.2 12mer at 7, 14, and 21 DPI, as in a. Green lines show the mean percentage of 10E8-UCA H GC B cells in mice immunized with 10E8-GT10.2 12mer and gray lines show mean percentage of cells immunized with 10E8-GT9-KO 12mer (n = 3). Data from one representative of three independent experiments shown. c , Quantification of epitope specific CD45.2 + 10E8-UCA H in mice immunized with 10E8-GT10.2 12mer at different time points, as in a. Error bars indicate mean ± SD from mice in each group (n = 3). Data from one representative of three independent experiments shown. d , ELISA of Ab response of 10E8-UCA H to immunizations with 10E8-GT10.2 12mer, as in a. Green dots represent IgG titer specific to 10E8-GT10.2 probe and gray dots represent IgG level to KO probe. Data pooled from three independent experiments. Each symbol represents a different mouse. Bars indicate geometric mean and geometric SD from mice in all three groups. n = 4-5 mice in each group. e , Graph showing the MFI of mAb-GT10.2-CD45.2 10E8-UCA H -14DPI-PE with varying amounts of competing antibodies (µg) on x-axis (yellow: CD45.2 Ab; red: CD45.1 Ab). Dotted line represents the ‘no competition’ control. f , Representative flow cytometric plots of CD45.2 + 10E8-UCA H B cells recruitment to GCs 7 and 14 DPI in spleens of mice transferred to reach frequencies of ~1:10 4 5:10 4 and 25:10 4 CD45.2 + 10E8-UCA H precursors and immunized with 5 μg of 10E8-GT10.2 12mer with alhydrogel or 10E8-GT9-KO 12mer with alhydrogel (control). g , Quantification of CD45.2 + in GC in spleen of mice immunized in f. Data are pooled from two independent experiments. Error bars indicate mean ± SD from mice in each group (n = 3 mice/independent group).

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Adoptive Transfer Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Flow cytometry plots of CD45.2 + GC B and CD45.1 + GC B cells at days 7, 14 and 21 p.i. in the spleens of wild-type CD45.1 mice adoptively transferred (intravenously) with 200,000 CD45.2 10E8-UCA H B cells to establish a frequency of 1:10 4 10E8-UCA H B precursors and immunized 1 day later with 5 µg of 10E8-GT10.2 12mer ( n = 3 mice) or 5 µg of 10E8-GT9-KO 12mer ( n = 3) with alhydrogel. Representative data from one of three independent experiments are shown. b , Percentage of CD45.2 + GC B cells in splenic GCs (top) and percentage of CD45.1 + GT10 ++ KO − GC B cells in splenic GCs (bottom) at days 7, 14 and 21 p.i. as in a ( n = 3). Each circle represents one mouse. Top, representative data from one of three independent experiments; lines mark the means. Bottom, data are from pooled groups from three independent experiments; lines mark the respective means. c , Amino acid SHM of 10E8-UCA H CD45.2 + GT10 ++ KO − B cells isolated from spleen GCs at days 7 and 14 p.i. with 10E8-GT10.2 12mer, endogenous CD45.1 + GT10 ++ KO − B cells isolated from spleen GCs at day 14 p.i. with 10E8-GT10.2 60mer and pretransfer (day −1, naive) 10E8-UCA H GT10 ++ KO − and wild-type GT10 ++ KO − B cells as a control. Circles represent individual sequences, and lines indicate median values. d , Affinities of monomeric mAbs expressed from 10E8-UCA H CD45.2 + GT10 ++ KO − B cells (GT10.2-10E8-UCA H ), endogenous CD45.1 + GT10 ++ KO − B cells (GT10.2-WT) and pretransfer (day −1) 10E8-UCA H GT10 ++ KO − B cells and wild-type GT10 ++ KO − B cells (naive), as in c , determined by monomer SPR assay. Circles represent individual antibodies, and lines represent median values; LOD, limit of detection.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Flow Cytometry, Isolation, Control, SPR Assay

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Orientation of mature 10E8 Fab in relation to the MPER peptide in the published Protein Data Bank (PDB) 4G6F crystal structure (heavy chain, white; light chain, dark gray; MPER peptide, purple; top left) and surface rendering and positioning of the critical YxFW residues of HCDR3 (bottom left), representative GT10.2-10E8-UCA H mAb in complex with a glycan-knockout (KO) version of 10E8-GT10.2 (heavy chain, yellow; light chain, dark gray; targeted MPER graft with GT mutations, purple; top center; structure aligned and oriented to the MPER peptide as in the top left) and designed binding pocket (green) and engagement of the GT10.2-10E8-UCA H mAb HCDR3 (yellow) compared to mature 10E8 HCDR3 (white; bottom center) and representative GT10.2-WT mAb in complex with 10E8-GT10.2 (heavy chain, red; light chain, dark gray; targeted MPER graft with GT mutations, purple; top right) and binding pocket (green; bottom right). b , Overlay of the GT10.2-10E8-UCA H and GT10.2-WT mAbs described in a , showing the representative competitor response in relation to on-target response (top) and overlay of the GT10.2-WT mAb with mature 10E8 mAb from a (bottom). Arrows indicate clashes of the antibodies. c , Representative flow cytometry histograms showing the mean fluorescence intensity of fluorescently labeled GT10.2-10E8-UCA H -PE mAb to 10E8-GT10.2-coated streptavidin beads in the presence of GT10.2-10E8-UCA H mAb or GT10.2-WT mAb or in the absence of other mAbs. Data are derived from one of two experiments. d , e , Representative flow cytometry plots ( d ) and quantifications ( e ) of GC B cells and CD45.2 + B cells in GC B cells in the spleen at day 7 p.i. in CD45.1 wild-type mice adoptively transferred with CD45.2 10E8-UCA H B cells to reach a frequency of 1:10 4 CD45.2 10E8-UCA H B cells 1 day before i.p. injection with or without 10 µg of GT10.2-WT mAb, followed by immunization with 5 µg of 10E8-GT10.2 12mer with alhydrogel 12 h later. Error bars indicate mean ± s.d. from mice in each group ( n = 5). Data were analyzed with a two-tailed Mann–Whitney test and are derived from one experiment.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Glycoproteomics, Knock-Out, Binding Assay, Flow Cytometry, Fluorescence, Labeling, Derivative Assay, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Location of mutations introduced in 10E8-GT10.2 to generate 10E8-GT10.3 (MPER, purple; scaffold backbone, cyan; mutations, orange). b , Affinities of human 10E8-UCA H /UCA L Fab and Fabs of 10E8-UCA H /mouse L antibodies (sorted based on B220 + IgM + expression on B cells) to 10E8-GT10.2 12mer and 10E8-GT10.3 12mer in nanoparticle SPR assays. Symbols represent individual antibodies, and lines represent 10E8-UCA H /mouse L medians. c , d , Representative flow cytometry plots of CD45.2 + and CD45.1 + B cells ( c ) and percentages of CD45.2 + B cells ( d ) in spleen GCs at days 7, 14 and 21 p.i. in CD45.1 wild-type mice transferred with CD45.2 10E8-UCA H B cells to reach 1:10 4 10E8-UCA H B cells and immunized 1 day later with 5 µg of 10E8-GT10.2 12mer, 10E8-GT10.3 12mer or 10E8-GT9-KO 12mer with alhydrogel. Data in d are pooled from two independent experiments ( n = 5–8). Error bars indicate mean ± s.d. from mice in pooled groups. Significance was calculated with a Student’s t -test (unpaired, two tailed). e , Affinities of mAbs expressed from epitope-specific 10E8-UCA H CD45.2 + GT10 ++ KO − GC B cells (GT10.3-10E8-UCA H mAbs) and endogenous CD45.1 + GT10 ++ KO − GC B cells (GT10.3-WT mAbs) at days 7, 14 and 21 p.i. with 10E8-GT10.3 12mer as in c and the affinity for corresponding iGL sequences (naive), as measured by monomer SPR assay. Circles represent individual antibodies, and lines indicate median values. f , Representative immunohistochemistry image of a spleen section at day 21 p.i. from a mouse immunized with 10E8-GT10.3 12mer and alhydrogel, as in c (left), and percentage of total GCs containing CD45.2 + B cells (right). White boxes show GCs with CD45.2 + B cells. Scale bar, 200 μm (left). g , Phylogenetic tree of IGHV sequences from 10E8-UCA H B cells, colorized on the basis of affinities at day 21 p.i., as in e .

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, SPR Assay, Immunohistochemistry

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Schematic of CD45.1 + mice adoptively transferred to establish a frequency of 1:10 4 CD45.2 + 10E8-UCA H B cells and then immunized with either 5 μg of 10E8-GT10.3 12mer, 10E8-GT10.2 12mer, or a control KO immunogen with alhydrogel, as in Fig. . b , Quantification of percentage of GC B cells post immunization with 10E8-GT10.3 12mer, 10E8-GT10.2 12mer, or 10E8-GT9-KO 12mer. Data are pooled from two independent experiments (n = 5–8) with lines marking the respective means. c , Quantification of epitope specific (GT10 ++ KO − ) CD45.2 + 10E8-UCA H B cells in GC in mice immunized with 10E8-GT10.2 12mer or 10E8-GT10.3 12mer at different time points, as in a. Error bars indicate mean ± SD from mice in each group (n = 5–8). Data are pooled from two independent experiments. d , Quantification of epitope specific CD45.1 + cells in GC in mice immunized as in a. Data are pooled from two independent experiments. *p =0.0137(unpaired t test, two tailed); ns, not significant. N = 5–8 mice in each group; lines mark means. e , Mutations in IGHV of GT10-specific 10E8-UCA H B cells 7, 14, and 21 DPI. (Left) Nucleotide (nt); (right) amino acid (aa). The black dashed line indicates the median number of mutations. f , Diversification of IGHV sequences from 10E8-UCA H B cells isolated at 7, 14, and 21 DPI with 10E8-GT10.3 12mer as in e. g , Graph comparing the number of mutations in heavy chain of 10E8-UCA cells ( x- axis) against their affinity ( y -axis) (shown in Fig. ) 21 DPI with 10E8-GT10.3 12mer. h , HCs (top) LCs (bottom) sequenced from GT10-specific 10E8-UCA H B cells 7, 14, and 21 DPI by 10E8-GT10.3 and alhydrogel, as in a. Numbers inside pie shows the number of cells sequenced. i , Graph showing the affinities of the 10E8-UCA H 21 DPI as per in Fig. with the associated mouse light chains.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Control, Two Tailed Test, Isolation

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Affinities of human MPER-HuGL18 H /MPER-HuGL18 L Fabs and MPER-HuGL18 H /mouse L Fabs to 10E8-GT10.3 12mer in the nanoparticle SPR assay. Symbols represent individual antibodies, and lines represent median MPER-HuGL18 H /mouse L values. b , c , Representative flow cytometry plots showing CD45.2 + and CD45.1 + B cells ( b ) and quantification of the percentage of GC B cells and CD45.2 + B cells ( c ) in spleen GCs at days 7, 14 and 21 p.i. in CD45.1 wild-type mice transferred with CD45.2 MPER-HuGL18 H B cells (1:10 4 MPER-HuGL18 H B cells) 1 day before immunization with 5 µg of 10E8-GT10.3 12mer or 5 µg of 10E8-GT9-KO 12mer with alhydrogel. Data were pooled from two to three independent experiments ( n = 4–10 per treatment), and lines show respective means. d , Number of nucleotide (nt; left) and amino acid (aa; right) mutations in IGHV of epitope-specific MPER-HuGL18 H CD45.2 + GT10 ++ KO − GC B cells at days 14 and 21 p.i., as in b . The black line indicates the median number of mutations. e , Phylograms showing diversification of IGHV sequences from MPER-HuGL18 CD45.2 + GT10 ++ KO − GC B cells at days 14 and 21 p.i., as in d . f , Pie plots showing MPER-HuGL18 heavy chains (top) and associated mouse light chains (bottom) sequenced from epitope-specific MPER-HuGL18 H CD45.2 + GT10 ++ KO − GC B cells at days 14 and 21 p.i. with 10E8-GT10.3 12mer and alhydrogel as in b and pretransfer (day −1, naive) MPER-HuGL18 GT10 ++ KO − B cells as a control. g , Affinities of mAbs expressed from MPER-HuGL18 H CD45.2 + GT10 ++ KO − GC B cells (GT10.3-MPER-HuGL18 H ) and endogenous CD45.1 + GT10 ++ KO − GC B cells (GT10.3-WT) at days 14 and 21 p.i. with 10E8-GT10.3 12mer as in b and affinities of iGL and pretransfer (day −1) MPER-HuGL18 H , with iGL wild-type mAbs for monomeric 10E8-GT10.2 (naive) as a control, as measured by monomer SPR assay. Circles represent individual antibodies, and lines mark median values.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: SPR Assay, Flow Cytometry, Control

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Schematic of CD45.1 + mice adoptively transferred to establish a frequency of 1:10 4 CD45.2 + MPER-HuGL18 H B cells and then immunized with either 5 μg of 10E8-GT10.3 12mer immunogen and alhydrogel, or 10E8-GT9-KO 12mer (control), as per Fig. . b , Quantification of epitope specific CD45.2 + MPER-HuGL18 H B cells in mice immunized with 10E8-GT10.3 12mer as in a. Error bars indicate mean ± SD from mice in each group (n = 6–8). Data pooled from two independent experiments. c , Immunohistochemistry of spleen at 21 DPI. Red: GL7 (GC marker); White: CD45.2; Green: IgD; Blue: TCRβ. Scale, 250 um. d , Quantification showing CD45.2 recruitment in GCs from 10E8-UCA H or MPER-HuGL18 H transferred mice immunized with 10E8-GT10.2 12mer and 10E8-GT10.3 12mer (merged data from Figs. , , and ref. ). Data are pooled from 2–3 independent experiments (n = 4–6). Error bars indicate mean ± SD. (*p = 0.0131, **p = 0.0085) (unpaired t test, two-tailed). e , Representative flow cytometry showing gating strategy used for the identification of memory B cells (MBC) 36 DPI with 10E8-GT10.3 12mer with alhydrogel (n = 8) or 10E8-GT9-KO 12mer with alhydrogel (n = 5). f , Quantification of memory B cells (MBC) 36 DPI with 10E8-GT10.3 12mer, as in e. Experiment performed once. Error bars indicate mean ± SD. (p = 0.024) (unpaired t-test, two-tailed). g , Graph of the affinities of MPER-HuGL18 H mAbs 21 DPI with 10E8-GT10.3 12mer (panel with affinities) (y-axis) sorted by light chain ( x -axis) LOD: limit of detection. h , Graph showing the affinities ( y- axis) of the subset using IGKV1-117 light chains from the MPER-HuGL18 H 21 DPI with 10E8-GT10.3 12mer (selection from Fig. ) vs. number of heavy chain mutations (x-axis). LOD: limit of detection. i , Tree showing phylogenetic relationship between MPER-HuGL18 H B cell IGHV sequences, colorized on the basis of 21 DPI affinities shown in (Fig. ).

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Control, Immunohistochemistry, Marker, Two Tailed Test, Flow Cytometry, Selection

Journal: Nature Immunology

Article Title: Affinity gaps among B cells in germinal centers drive the selection of MPER precursors

doi: 10.1038/s41590-024-01844-7

Figure Lengend Snippet: a , Quantifications of the percentages of GC B cells (left) and epitope-specific CD45.2 + GT10 ++ KO − GC B cells (right) in spleens at day 7 p.i. in CD45.1 wild-type mice adoptively transferred with 10E8-NGS-04 H , 10E8-UCA H and MPER-HuGL18 H CD45.2 B cells to reach a frequency of 1:10 4 for each precursor lineage 1 day before immunization with 5 µg of 10E8-GT10.2 12mer or 10E8-GT9-KO 12mer with alhydrogel. Data are pooled from two independent experiments ( n = 5–7 per treatment). Error bars indicate mean ± s.d. from mice in the pooled group. Significance was calculated by using a two-tailed unpaired t -test. b , Pie plots and bar graphs showing the frequencies of MPER-HuGL18, 10E8-UCA and 10E8-NGS-04 heavy chains sequenced from CD45.2 + GT10 ++ KO − GC B cells at days 7 and 21 p.i. as in a ( n = 12 mice). Significance was calculated by using an unpaired t -test with Welch’s correction. c , Uniform manifold approximation and projection embedding of 10E8-UCA H and MPER-HuGL18 H CD45.2 + GT10 ++ KO − GC B cells from the dark zone (DZ) and light zone (LZ) of spleen GCs at day 7 p.i., as in b (left), and stacked bar plot showing cluster composition of 10E8-UCA H and MPER-HuGL18 H light zone and dark zone B cells. Bars represent the fraction of cells in dark zone/light zone clusters in each cell line.

Article Snippet: Our study highlights the need for bnAb-class-specific approaches to GT immunogen design and offers a route toward inducing MPER-class bnAbs through the generation of new 10E8 precursor models without autoreactivity, the characterization of lineage-specific acquisition of affinity-enhancing mutations and the identification of the dynamic gap between competing BCR affinities over time as a determinant of GC survival.

Techniques: Two Tailed Test