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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

AC50 characterization of HA-specific GC B cells. At indicated times after PR8 i.n. infection, we excised MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict MLN resident GC B cell reactivity to graded concentrations of rHAPR8. GC B cells at 28 dpi react more strongly to lower staining concentrations than those at 14 dpi. (B) Titration curves of MLN-resident GC B cells to rHAPR8 following i.n. PR8 infection. Data represent the frequency of positive cells plotted against rHAPR8 concentration. (C) AC50 was calculated for each titration curve and is presented as the concentration (nanomolar) of rHAPR8 required to reach 50% maximal binding at the indicated time points. MLN-resident GC B cells decrease AC50 to rHAPR8 29-fold between 7 and 28 days postinfection, or a 140% increase each day. (D) AC50 of spleen-resident GC B cells at 14 and 21 dpi following i.n. infection. Despite different frequencies and kinetics of response, HA AC50 is similar to that of MLN GC B cells. (E) At 14 and 28 dpi, MLNs were excised, dispersed into single-cell suspensions, and stained with anti-IgG Fcγ MAb. The graph illustrates normalized MFI levels of IgG Fcγ expression of GC B cells relative to naive B cells that express no IgG (set as baseline MFI) at 14 and 28 dpi. No significant difference in per-cell-surface IgG expression occurs between 14 and 28 days postinfection. All data are representative of 3 to 7 replicate experiments.
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fig4: AC50 characterization of HA-specific GC B cells. At indicated times after PR8 i.n. infection, we excised MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict MLN resident GC B cell reactivity to graded concentrations of rHAPR8. GC B cells at 28 dpi react more strongly to lower staining concentrations than those at 14 dpi. (B) Titration curves of MLN-resident GC B cells to rHAPR8 following i.n. PR8 infection. Data represent the frequency of positive cells plotted against rHAPR8 concentration. (C) AC50 was calculated for each titration curve and is presented as the concentration (nanomolar) of rHAPR8 required to reach 50% maximal binding at the indicated time points. MLN-resident GC B cells decrease AC50 to rHAPR8 29-fold between 7 and 28 days postinfection, or a 140% increase each day. (D) AC50 of spleen-resident GC B cells at 14 and 21 dpi following i.n. infection. Despite different frequencies and kinetics of response, HA AC50 is similar to that of MLN GC B cells. (E) At 14 and 28 dpi, MLNs were excised, dispersed into single-cell suspensions, and stained with anti-IgG Fcγ MAb. The graph illustrates normalized MFI levels of IgG Fcγ expression of GC B cells relative to naive B cells that express no IgG (set as baseline MFI) at 14 and 28 dpi. No significant difference in per-cell-surface IgG expression occurs between 14 and 28 days postinfection. All data are representative of 3 to 7 replicate experiments.

Mentions: We next used AC50 to characterize maturation of specific GC B cell responses in MLNs and spleens during IAV infection. GC B cells at 28 dpi had a significantly higher frequency of HA reactivity at lower rHAPR8 concentrations than 14-dpi counterparts (Fig. 4A), with a clear increase in HA-specific GC B cell frequency and a decrease of AC50 over 4 weeks following infection (Fig. 4B). AC50 decreased approximately 30-fold, from 10 nM at 7 dpi to 0.36 nM at 28 dpi (Fig. 4C).


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)

AC50 characterization of HA-specific GC B cells. At indicated times after PR8 i.n. infection, we excised MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict MLN resident GC B cell reactivity to graded concentrations of rHAPR8. GC B cells at 28 dpi react more strongly to lower staining concentrations than those at 14 dpi. (B) Titration curves of MLN-resident GC B cells to rHAPR8 following i.n. PR8 infection. Data represent the frequency of positive cells plotted against rHAPR8 concentration. (C) AC50 was calculated for each titration curve and is presented as the concentration (nanomolar) of rHAPR8 required to reach 50% maximal binding at the indicated time points. MLN-resident GC B cells decrease AC50 to rHAPR8 29-fold between 7 and 28 days postinfection, or a 140% increase each day. (D) AC50 of spleen-resident GC B cells at 14 and 21 dpi following i.n. infection. Despite different frequencies and kinetics of response, HA AC50 is similar to that of MLN GC B cells. (E) At 14 and 28 dpi, MLNs were excised, dispersed into single-cell suspensions, and stained with anti-IgG Fcγ MAb. The graph illustrates normalized MFI levels of IgG Fcγ expression of GC B cells relative to naive B cells that express no IgG (set as baseline MFI) at 14 and 28 dpi. No significant difference in per-cell-surface IgG expression occurs between 14 and 28 days postinfection. All data are representative of 3 to 7 replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: AC50 characterization of HA-specific GC B cells. At indicated times after PR8 i.n. infection, we excised MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. (A) Representative flow plots depict MLN resident GC B cell reactivity to graded concentrations of rHAPR8. GC B cells at 28 dpi react more strongly to lower staining concentrations than those at 14 dpi. (B) Titration curves of MLN-resident GC B cells to rHAPR8 following i.n. PR8 infection. Data represent the frequency of positive cells plotted against rHAPR8 concentration. (C) AC50 was calculated for each titration curve and is presented as the concentration (nanomolar) of rHAPR8 required to reach 50% maximal binding at the indicated time points. MLN-resident GC B cells decrease AC50 to rHAPR8 29-fold between 7 and 28 days postinfection, or a 140% increase each day. (D) AC50 of spleen-resident GC B cells at 14 and 21 dpi following i.n. infection. Despite different frequencies and kinetics of response, HA AC50 is similar to that of MLN GC B cells. (E) At 14 and 28 dpi, MLNs were excised, dispersed into single-cell suspensions, and stained with anti-IgG Fcγ MAb. The graph illustrates normalized MFI levels of IgG Fcγ expression of GC B cells relative to naive B cells that express no IgG (set as baseline MFI) at 14 and 28 dpi. No significant difference in per-cell-surface IgG expression occurs between 14 and 28 days postinfection. All data are representative of 3 to 7 replicate experiments.
Mentions: We next used AC50 to characterize maturation of specific GC B cell responses in MLNs and spleens during IAV infection. GC B cells at 28 dpi had a significantly higher frequency of HA reactivity at lower rHAPR8 concentrations than 14-dpi counterparts (Fig. 4A), with a clear increase in HA-specific GC B cell frequency and a decrease of AC50 over 4 weeks following infection (Fig. 4B). AC50 decreased approximately 30-fold, from 10 nM at 7 dpi to 0.36 nM at 28 dpi (Fig. 4C).

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