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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: As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate.Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses.This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.

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: As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate.Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses.This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.

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