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A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.

Frank GM, Angeletti D, Ince WL, Gibbs JS, Khurana S, Wheatley AK, Max EE, McDermott AB, Golding H, Stevens J, Bennink JR, Yewdell JW - MBio (2015)

Bottom Line: A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens.Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity.In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus

Recombinant HA can specifically identify HA-specific GC B cells. Briefly, we treated cells with receptor-destroying enzyme for 60 min at 37°C to remove sialic acid from the cell surface to minimize nonspecific binding. We stained cells first with surface MAb, then with rHA, and finally with anti-6×His MAb to detect rHA binding. (A) Representative flow plots demonstrate that H17 L7, a PR8 HA-specific hybridoma, retains high levels of surface Ig expression. (B) Representative flow plots indicate that H17 L7 binds strongly to A/Puerto Rico/8/1934 molecular clone rHA (rHAPR8) but not to A/Vietnam/1203/2004 rHA (rHAViet 04), a serologically distinct HA. (C) Plot of percent 6×His-positive cells versus rHAPR8 concentration illustrating that H17 L7 reacts to rHAPR8 in a dose-dependent fashion. At 14 days after PR8 i.n. infection, MLNs were excised and dispersed into single-cell suspensions, and GC B cells were stained using the rHA approach. (D) representative plots depict gating strategy to observe GC B cells based on B220+ GL-7+ CD38− surface expression. Only GC B cells reacted specifically to rHAPR8. (E) B6 mice were i.n. infected with an H3N1 reassortant (J1), and MLN-resident GC B cells were stained with rHAPR8. Representative flow plots of MLN resident GC B cell 6×His staining show that J1-responding GC B cells did not react to rHAPR8 at staining concentrations up to 66 nM, providing the maximum staining concentration of rHAPR8 that can be used to identify H1 HA-specific B cells with confidence. Data represent 3 independent experiments.
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fig1: Recombinant HA can specifically identify HA-specific GC B cells. Briefly, we treated cells with receptor-destroying enzyme for 60 min at 37°C to remove sialic acid from the cell surface to minimize nonspecific binding. We stained cells first with surface MAb, then with rHA, and finally with anti-6×His MAb to detect rHA binding. (A) Representative flow plots demonstrate that H17 L7, a PR8 HA-specific hybridoma, retains high levels of surface Ig expression. (B) Representative flow plots indicate that H17 L7 binds strongly to A/Puerto Rico/8/1934 molecular clone rHA (rHAPR8) but not to A/Vietnam/1203/2004 rHA (rHAViet 04), a serologically distinct HA. (C) Plot of percent 6×His-positive cells versus rHAPR8 concentration illustrating that H17 L7 reacts to rHAPR8 in a dose-dependent fashion. At 14 days after PR8 i.n. infection, MLNs were excised and dispersed into single-cell suspensions, and GC B cells were stained using the rHA approach. (D) representative plots depict gating strategy to observe GC B cells based on B220+ GL-7+ CD38− surface expression. Only GC B cells reacted specifically to rHAPR8. (E) B6 mice were i.n. infected with an H3N1 reassortant (J1), and MLN-resident GC B cells were stained with rHAPR8. Representative flow plots of MLN resident GC B cell 6×His staining show that J1-responding GC B cells did not react to rHAPR8 at staining concentrations up to 66 nM, providing the maximum staining concentration of rHAPR8 that can be used to identify H1 HA-specific B cells with confidence. Data represent 3 independent experiments.

Mentions: To identify HA-specific B cells, we used recombinant HA from A/Puerto Rico/8/34 (PR8) secreted by insect cells with its carboxy-terminal domain modified to promote trimerization and enable detection with an anti-His tag monoclonal antibody (MAb). This HA preparation (rHAPR8) is remarkably native, as shown by biochemical analysis in conjunction with a panel of MAbs to assess conformation (20). To establish the properties of rHAPR8 as a B cell probe, we screened hybridoma cell lines to identify a line that expresses cell surface Ig, which is unusual (21). H17-L7, an HA Cb site-specific hybridoma, exhibits surface IgG expression (Fig. 1A) and binds rHAPR8 in a dose-dependent fashion but does not bind to the serologically distant H5 rHAVietnam 04, demonstrating the specificity of staining with rHAPR8 (Fig. 1B and C).


A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.

Frank GM, Angeletti D, Ince WL, Gibbs JS, Khurana S, Wheatley AK, Max EE, McDermott AB, Golding H, Stevens J, Bennink JR, Yewdell JW - MBio (2015)

Recombinant HA can specifically identify HA-specific GC B cells. Briefly, we treated cells with receptor-destroying enzyme for 60 min at 37°C to remove sialic acid from the cell surface to minimize nonspecific binding. We stained cells first with surface MAb, then with rHA, and finally with anti-6×His MAb to detect rHA binding. (A) Representative flow plots demonstrate that H17 L7, a PR8 HA-specific hybridoma, retains high levels of surface Ig expression. (B) Representative flow plots indicate that H17 L7 binds strongly to A/Puerto Rico/8/1934 molecular clone rHA (rHAPR8) but not to A/Vietnam/1203/2004 rHA (rHAViet 04), a serologically distinct HA. (C) Plot of percent 6×His-positive cells versus rHAPR8 concentration illustrating that H17 L7 reacts to rHAPR8 in a dose-dependent fashion. At 14 days after PR8 i.n. infection, MLNs were excised and dispersed into single-cell suspensions, and GC B cells were stained using the rHA approach. (D) representative plots depict gating strategy to observe GC B cells based on B220+ GL-7+ CD38− surface expression. Only GC B cells reacted specifically to rHAPR8. (E) B6 mice were i.n. infected with an H3N1 reassortant (J1), and MLN-resident GC B cells were stained with rHAPR8. Representative flow plots of MLN resident GC B cell 6×His staining show that J1-responding GC B cells did not react to rHAPR8 at staining concentrations up to 66 nM, providing the maximum staining concentration of rHAPR8 that can be used to identify H1 HA-specific B cells with confidence. Data represent 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Recombinant HA can specifically identify HA-specific GC B cells. Briefly, we treated cells with receptor-destroying enzyme for 60 min at 37°C to remove sialic acid from the cell surface to minimize nonspecific binding. We stained cells first with surface MAb, then with rHA, and finally with anti-6×His MAb to detect rHA binding. (A) Representative flow plots demonstrate that H17 L7, a PR8 HA-specific hybridoma, retains high levels of surface Ig expression. (B) Representative flow plots indicate that H17 L7 binds strongly to A/Puerto Rico/8/1934 molecular clone rHA (rHAPR8) but not to A/Vietnam/1203/2004 rHA (rHAViet 04), a serologically distinct HA. (C) Plot of percent 6×His-positive cells versus rHAPR8 concentration illustrating that H17 L7 reacts to rHAPR8 in a dose-dependent fashion. At 14 days after PR8 i.n. infection, MLNs were excised and dispersed into single-cell suspensions, and GC B cells were stained using the rHA approach. (D) representative plots depict gating strategy to observe GC B cells based on B220+ GL-7+ CD38− surface expression. Only GC B cells reacted specifically to rHAPR8. (E) B6 mice were i.n. infected with an H3N1 reassortant (J1), and MLN-resident GC B cells were stained with rHAPR8. Representative flow plots of MLN resident GC B cell 6×His staining show that J1-responding GC B cells did not react to rHAPR8 at staining concentrations up to 66 nM, providing the maximum staining concentration of rHAPR8 that can be used to identify H1 HA-specific B cells with confidence. Data represent 3 independent experiments.
Mentions: To identify HA-specific B cells, we used recombinant HA from A/Puerto Rico/8/34 (PR8) secreted by insect cells with its carboxy-terminal domain modified to promote trimerization and enable detection with an anti-His tag monoclonal antibody (MAb). This HA preparation (rHAPR8) is remarkably native, as shown by biochemical analysis in conjunction with a panel of MAbs to assess conformation (20). To establish the properties of rHAPR8 as a B cell probe, we screened hybridoma cell lines to identify a line that expresses cell surface Ig, which is unusual (21). H17-L7, an HA Cb site-specific hybridoma, exhibits surface IgG expression (Fig. 1A) and binds rHAPR8 in a dose-dependent fashion but does not bind to the serologically distant H5 rHAVietnam 04, demonstrating the specificity of staining with rHAPR8 (Fig. 1B and C).

Bottom Line: A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens.Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity.In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus