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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

HA-specific GC B cells exhibit different responses in local versus central lymphoid organs. At the indicated times after PR8 i.n. infection, we removed MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. Line graphs indicate the absolute number (A) and frequency (B) of GC B cells specifically binding rHAPR8 (based on a staining concentration of 66 nM) over time in the MLNs and spleens. MLN GC B cells binding rHAPR8 form earlier (7 dpi) but peak later (21 dpi) than spleen-resident GC B cells, which do not appear until after 7 dpi and peak at 14 dpi. Data are representative of 3 to 6 independent experiments.
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fig2: HA-specific GC B cells exhibit different responses in local versus central lymphoid organs. At the indicated times after PR8 i.n. infection, we removed MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. Line graphs indicate the absolute number (A) and frequency (B) of GC B cells specifically binding rHAPR8 (based on a staining concentration of 66 nM) over time in the MLNs and spleens. MLN GC B cells binding rHAPR8 form earlier (7 dpi) but peak later (21 dpi) than spleen-resident GC B cells, which do not appear until after 7 dpi and peak at 14 dpi. Data are representative of 3 to 6 independent experiments.

Mentions: Using a staining concentration of 66 nM rHAPR8 we quantified the HA-specific GC B cell response following i.n. infection in the MLNs and spleen. HA-specific GC B cells were first detected at 7 days postinfection (dpi) in MLNs and 10 dpi in the spleen. Numbers of splenic HA-reactive GC B cells peaked at 14 dpi, while their MLN-resident counterparts peaked a week later (Fig. 2A). Expressed as a fraction of total GC B cells, splenic B cells peaked at 21 dpi while MLN B cells continued to rise in frequency at 28 dpi (Fig. 2B).


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)

HA-specific GC B cells exhibit different responses in local versus central lymphoid organs. At the indicated times after PR8 i.n. infection, we removed MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. Line graphs indicate the absolute number (A) and frequency (B) of GC B cells specifically binding rHAPR8 (based on a staining concentration of 66 nM) over time in the MLNs and spleens. MLN GC B cells binding rHAPR8 form earlier (7 dpi) but peak later (21 dpi) than spleen-resident GC B cells, which do not appear until after 7 dpi and peak at 14 dpi. Data are representative of 3 to 6 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: HA-specific GC B cells exhibit different responses in local versus central lymphoid organs. At the indicated times after PR8 i.n. infection, we removed MLN or spleens, dispersed them into single-cell suspensions, and stained GC B cells with rHA. Line graphs indicate the absolute number (A) and frequency (B) of GC B cells specifically binding rHAPR8 (based on a staining concentration of 66 nM) over time in the MLNs and spleens. MLN GC B cells binding rHAPR8 form earlier (7 dpi) but peak later (21 dpi) than spleen-resident GC B cells, which do not appear until after 7 dpi and peak at 14 dpi. Data are representative of 3 to 6 independent experiments.
Mentions: Using a staining concentration of 66 nM rHAPR8 we quantified the HA-specific GC B cell response following i.n. infection in the MLNs and spleen. HA-specific GC B cells were first detected at 7 days postinfection (dpi) in MLNs and 10 dpi in the spleen. Numbers of splenic HA-reactive GC B cells peaked at 14 dpi, while their MLN-resident counterparts peaked a week later (Fig. 2A). Expressed as a fraction of total GC B cells, splenic B cells peaked at 21 dpi while MLN B cells continued to rise in frequency at 28 dpi (Fig. 2B).

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