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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: A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens.Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity.In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies.

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: A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens.Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity.In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies.

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