Phenotypic analysis of pneumococcal polysaccharide-specific B cells

Abstract

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.

Figures

Figure 1

Serum antibody response and opsonophagocytic activity. Healthy young volunteers (n=18) were immunized with Pneumovax®. Serum samples were obtained pre- and 4–6 weeks post-immunization. Serum samples were tested for PPS14 and PPS23F specific IgG, IgA, IgM (A) and opsonophagocytic activity (B). Serum antibody levels are expressed as μg/mL and opsonophagocytic activity is expressed as opsonophagocytic index.

Figure 2

Inhibition ELISA with fluorescent-labeled PPS14 and PPS23F. Various amounts of labeled PPS14 or PPS23F were pre-incubated with a 1:1000 dilution of standard serum 89SF. Percent inhibition was calculated compared to uninhibited samples.

Figure 3

Specific staining of hybridoma cells. Hybridoma cells with specificity for PPS14 (α14g2b) (A) or PPS23F (α23F) (B) were incubated with various amounts of PPS23F-DTAF and PPS14-CB, and subjected to flow cytometry. PPS14 hybridoma cells specifically bound PPS14-CB in a dose dependent manner and failed to stain with PPS23F-DTAF. PPS23F hybridoma cells specifically bound PPS23F-DTAF in a dose dependent manner and failed to stain with PPS14-CB. Fifty thousand events were recorded.

Figure 4

Inhibition of binding fluorescently labeled PPS. Lymphocytes isolated 7 days post-vaccination were pre-treated with increasing concentrations of homologous unlabeled polysaccharide before addition of fluorescently labeled polysaccharide A=PPS14 and B=PPS23F. Fifty thousand events were recorded. Percent inhibition of binding to fluorescently labeled PPS was determined by comparison to the uninhibited cells.

Figure 5

B cell phenotypes. The phenotype of B lymphocytes that respond to vaccination with Pneumovax23® was determined by flow cytometry. Pre-vaccination (n=9) and seven days post-vaccination (n=18) circulating PBMC were isolated and labeled for analysis using the following fluorescently labeled antibodies/antigen: CD19, CD27, IgM, IgD, PPS14 and PPS23F. The phenotype of pre-vaccination unselected B cells were compared to PPS-specific B cells (A), the phenotype of post-vaccination unselected B cells were compared to PPS-specific B cells (B) and pre-immunization PPS-specific B cells were compared to post-immunization PPS-specific B cells (C). In each sample 75,000 events were recorded. *p

Figure.6

Phenotype analyses of B cells in the human peripheral blood. Healthy donor PBMC sample was stained with Abs to CD19, CD27, IgM, and fluorescent labeled PPS14 (A and B) and 23F (C and D). CD19+ B cells (shown in histogram, dotted line=isotype control) were gated on PPS23F or PPS14. PPS14 or 23F specific B cells (CD19+PPS14+, CD19+PPS23F+) and PPS14 or 23F negative B cells (CD19+PPS14−, CD19+PPS23F−) cells were separated into CD27+IgM+, CD27+IgM−, CD27−IgM+ and CD27−IgM-. In each sample 75,000 events were recorded. Representative data of FACS analyses; (A and C) pre-vaccination, (B and D) post-vaccination of PPS14 (A and B) or PPS23F (C and D).