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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 of HA-specific GC B cells can be used as a proxy for population avidity to PR8 HA. (A) Overview of AC50. GC B cell rHA staining frequency is plotted against rHAPR8 concentration. Nonspecific binding by J1-responding GC B cells occurs at concentrations above 66 nM rHAPR8. Using this dose as the maximum specific staining of HA-specific GC B cells, we estimated the half-maximal specific binding rHAPR8 concentration. This provides a molar rHAPR8 value we call the AC50, a proxy measurement of population avidity for rHAPR8. (B) Representative titration curve of H17 L7 HA-specific hybridoma using rHAPR8 (red plots) or by ELISA of purified H17 L7 antibody to plated whole virus (blue plots). (C) Bar graph depicting the calculated AC50 compared to ELISA affinity. Values are within 1.8-fold of each other. (D) Comparison of HA-specific GC B cell AC50, 7 days after PR8 i.n. infection, with mean affinity of 9 different 5-dpi MAbs. The AC50 and mean affinity are statistically indistinguishable. (E) AC50 for H17 L7 HA-specific hybridoma mixed in different proportions with naive splenocytes and assayed using rHAPR8. AC50 is independent of the proportion of specific cells. Data are representative of 2 or 3 independent experiments.
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fig3: AC50 of HA-specific GC B cells can be used as a proxy for population avidity to PR8 HA. (A) Overview of AC50. GC B cell rHA staining frequency is plotted against rHAPR8 concentration. Nonspecific binding by J1-responding GC B cells occurs at concentrations above 66 nM rHAPR8. Using this dose as the maximum specific staining of HA-specific GC B cells, we estimated the half-maximal specific binding rHAPR8 concentration. This provides a molar rHAPR8 value we call the AC50, a proxy measurement of population avidity for rHAPR8. (B) Representative titration curve of H17 L7 HA-specific hybridoma using rHAPR8 (red plots) or by ELISA of purified H17 L7 antibody to plated whole virus (blue plots). (C) Bar graph depicting the calculated AC50 compared to ELISA affinity. Values are within 1.8-fold of each other. (D) Comparison of HA-specific GC B cell AC50, 7 days after PR8 i.n. infection, with mean affinity of 9 different 5-dpi MAbs. The AC50 and mean affinity are statistically indistinguishable. (E) AC50 for H17 L7 HA-specific hybridoma mixed in different proportions with naive splenocytes and assayed using rHAPR8. AC50 is independent of the proportion of specific cells. Data are representative of 2 or 3 independent experiments.

Mentions: As initially described for hapten-specific B cells (23), the binding of rHAPR8 to GC B cells should obey the law of mass action and thereby provide a measure of Ab affinity for HA. In theory, for any B cell, Ab affinity should equal the rHAPR8 concentration that gives half-maximal binding (rHAPR850). There is no need to reach saturation to define AC50, as B cell receptor (BCR)-specific and “nonspecific” antigen binding is a continuum, where “nonspecific” means low-affinity binding of germline BCRs. However, during an immune response, Abs with high affinity will outcompete those with lower affinity. Therefore, our maximum binding is the binding at a concentration where naive BCRs do not bind. While each B cell can be tested only for binding to a single rHAPR8 concentration, at the population level, if cell surface Ig levels are equally distributed among B cell clones, the average Ab affinity will track the rHAPR8 concentration that detects half the number of HA-specific B cells. We define this as the 50% antigen concentration (AC50) (Fig. 3A). It is important to emphasize that since HA-specific GC B cells comprise a heterogeneous population of clones with various affinities and surface Ig expression, AC50 provides (at best) the mean affinity of Abs expressed by the HA-specific B cell population studied.


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 of HA-specific GC B cells can be used as a proxy for population avidity to PR8 HA. (A) Overview of AC50. GC B cell rHA staining frequency is plotted against rHAPR8 concentration. Nonspecific binding by J1-responding GC B cells occurs at concentrations above 66 nM rHAPR8. Using this dose as the maximum specific staining of HA-specific GC B cells, we estimated the half-maximal specific binding rHAPR8 concentration. This provides a molar rHAPR8 value we call the AC50, a proxy measurement of population avidity for rHAPR8. (B) Representative titration curve of H17 L7 HA-specific hybridoma using rHAPR8 (red plots) or by ELISA of purified H17 L7 antibody to plated whole virus (blue plots). (C) Bar graph depicting the calculated AC50 compared to ELISA affinity. Values are within 1.8-fold of each other. (D) Comparison of HA-specific GC B cell AC50, 7 days after PR8 i.n. infection, with mean affinity of 9 different 5-dpi MAbs. The AC50 and mean affinity are statistically indistinguishable. (E) AC50 for H17 L7 HA-specific hybridoma mixed in different proportions with naive splenocytes and assayed using rHAPR8. AC50 is independent of the proportion of specific cells. Data are representative of 2 or 3 independent experiments.
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Related In: Results  -  Collection

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fig3: AC50 of HA-specific GC B cells can be used as a proxy for population avidity to PR8 HA. (A) Overview of AC50. GC B cell rHA staining frequency is plotted against rHAPR8 concentration. Nonspecific binding by J1-responding GC B cells occurs at concentrations above 66 nM rHAPR8. Using this dose as the maximum specific staining of HA-specific GC B cells, we estimated the half-maximal specific binding rHAPR8 concentration. This provides a molar rHAPR8 value we call the AC50, a proxy measurement of population avidity for rHAPR8. (B) Representative titration curve of H17 L7 HA-specific hybridoma using rHAPR8 (red plots) or by ELISA of purified H17 L7 antibody to plated whole virus (blue plots). (C) Bar graph depicting the calculated AC50 compared to ELISA affinity. Values are within 1.8-fold of each other. (D) Comparison of HA-specific GC B cell AC50, 7 days after PR8 i.n. infection, with mean affinity of 9 different 5-dpi MAbs. The AC50 and mean affinity are statistically indistinguishable. (E) AC50 for H17 L7 HA-specific hybridoma mixed in different proportions with naive splenocytes and assayed using rHAPR8. AC50 is independent of the proportion of specific cells. Data are representative of 2 or 3 independent experiments.
Mentions: As initially described for hapten-specific B cells (23), the binding of rHAPR8 to GC B cells should obey the law of mass action and thereby provide a measure of Ab affinity for HA. In theory, for any B cell, Ab affinity should equal the rHAPR8 concentration that gives half-maximal binding (rHAPR850). There is no need to reach saturation to define AC50, as B cell receptor (BCR)-specific and “nonspecific” antigen binding is a continuum, where “nonspecific” means low-affinity binding of germline BCRs. However, during an immune response, Abs with high affinity will outcompete those with lower affinity. Therefore, our maximum binding is the binding at a concentration where naive BCRs do not bind. While each B cell can be tested only for binding to a single rHAPR8 concentration, at the population level, if cell surface Ig levels are equally distributed among B cell clones, the average Ab affinity will track the rHAPR8 concentration that detects half the number of HA-specific B cells. We define this as the 50% antigen concentration (AC50) (Fig. 3A). It is important to emphasize that since HA-specific GC B cells comprise a heterogeneous population of clones with various affinities and surface Ig expression, AC50 provides (at best) the mean affinity of Abs expressed by the HA-specific B cell population studied.

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