Limits...
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 decrease is driven by affinity maturation. We injected B6 and AID−/− mice i.p. with UV-inactivated PR8, and 14 and 28 days later, we excised spleens and dispersed them into single-cell suspensions. We then stained GC B cells with rHA. (A and B) Titration of B6 GC B cells and AID−/− GC B cells, respectively, using rHAPR8 at 14 and 28 dpi, plotting the frequency of positive cells versus 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 indicated time points. While WT GC B cells decrease AC50 to rHAPR8 by 12-fold between 14 and 28 days postinfection, AID−/− GC B cell AC50 remains stable. Data are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526714&req=5

fig5: AC50 decrease is driven by affinity maturation. We injected B6 and AID−/− mice i.p. with UV-inactivated PR8, and 14 and 28 days later, we excised spleens and dispersed them into single-cell suspensions. We then stained GC B cells with rHA. (A and B) Titration of B6 GC B cells and AID−/− GC B cells, respectively, using rHAPR8 at 14 and 28 dpi, plotting the frequency of positive cells versus 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 indicated time points. While WT GC B cells decrease AC50 to rHAPR8 by 12-fold between 14 and 28 days postinfection, AID−/− GC B cell AC50 remains stable. Data are representative of 3 independent experiments.

Mentions: If the observed decrease in AC50 is due to affinity maturation, it should not occur in mice with activation-induced deaminase knockout (AID−/− mice), which are incapable of somatic hypermutation (25). In response to intrperitoneal (i.p.) injection of UV light-inactivated PR8, WT mice generated a robust HA-specific GC B cell response exhibiting 12-fold-decreased AC50 (5.8 nM to 0.49 nM) between 14 and 28 dpi (Fig. 5A and B). As seen in other studies, the AID−/− mice generate a large germinal-center reaction, with a robust HA-specific GC B cell response (26). However, the AID−/− response’s AC50 remained constant from 14 to 28 dpi (Fig. 5). Since Ig class switching is also abrogated in AID−/− mice, these data strongly support the conclusion that kinetic changes in AC50 reflect the selection of higher-affinity HA-specific clones by classical B cell somatic mutation and selection mechanisms.


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 decrease is driven by affinity maturation. We injected B6 and AID−/− mice i.p. with UV-inactivated PR8, and 14 and 28 days later, we excised spleens and dispersed them into single-cell suspensions. We then stained GC B cells with rHA. (A and B) Titration of B6 GC B cells and AID−/− GC B cells, respectively, using rHAPR8 at 14 and 28 dpi, plotting the frequency of positive cells versus 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 indicated time points. While WT GC B cells decrease AC50 to rHAPR8 by 12-fold between 14 and 28 days postinfection, AID−/− GC B cell AC50 remains stable. Data are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526714&req=5

fig5: AC50 decrease is driven by affinity maturation. We injected B6 and AID−/− mice i.p. with UV-inactivated PR8, and 14 and 28 days later, we excised spleens and dispersed them into single-cell suspensions. We then stained GC B cells with rHA. (A and B) Titration of B6 GC B cells and AID−/− GC B cells, respectively, using rHAPR8 at 14 and 28 dpi, plotting the frequency of positive cells versus 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 indicated time points. While WT GC B cells decrease AC50 to rHAPR8 by 12-fold between 14 and 28 days postinfection, AID−/− GC B cell AC50 remains stable. Data are representative of 3 independent experiments.
Mentions: If the observed decrease in AC50 is due to affinity maturation, it should not occur in mice with activation-induced deaminase knockout (AID−/− mice), which are incapable of somatic hypermutation (25). In response to intrperitoneal (i.p.) injection of UV light-inactivated PR8, WT mice generated a robust HA-specific GC B cell response exhibiting 12-fold-decreased AC50 (5.8 nM to 0.49 nM) between 14 and 28 dpi (Fig. 5A and B). As seen in other studies, the AID−/− mice generate a large germinal-center reaction, with a robust HA-specific GC B cell response (26). However, the AID−/− response’s AC50 remained constant from 14 to 28 dpi (Fig. 5). Since Ig class switching is also abrogated in AID−/− mice, these data strongly support the conclusion that kinetic changes in AC50 reflect the selection of higher-affinity HA-specific clones by classical B cell somatic mutation and selection mechanisms.

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