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 CD27IgM+ 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.

Streptococcus pneumoniae is a major cause of morbidity and mortality in young children, elderly adults, and immune-compromised hosts. There are currently two types of vaccines that offer protection against pneumococcal disease: conjugate vaccines for children <2 y of age and a 23-valent pneumococcal polysaccharide vaccine (PPV) for protection in adults (1). Both vaccines elicit serotype-specific opsonic Abs, which are necessary for protection (2, 3). The phenotype of the B lymphocyte population responsible for the immune response to the purified pneumococcal vaccine (Pneumovax) has been controversial. The debate centers primarily on the surface Ags expressed by the responding B lymphocytes. Recently, it has been suggested that peripheral blood CD27+IgM+ or IgM memory B lymphocytes are recirculating splenic marginal zone B lymphocytes (4, 5). These lymphocytes are believed to recognize TI-2 Ags such as pneumococcal polysaccharide by virtue of a prediversified surface IgM and respond immediately without T cell help (6, 7). This view treats CD27+IgM+ B lymphocytes as innate immune cells in the first line of defense (810).

In support of this concept, it has been shown that persons with decreased or absent IgM memory B lymphocytes, such as the splenectomized, infants <2 y of age, elderly, HIV infected, and a subgroup of common variable immunodeficiency patients, all respond poorly to polysaccharide vaccines and are highly susceptible to infections with encapsulated organisms (57, 1113). It is, however, unlikely that IgM memory B lymphocytes are exclusively responsible for anti-polysaccharide Ab production as switched memory B lymphocytes (CD27+IgM) secrete anti-pneumococcal polysaccharide (PPS) Ab following in vitro stimulation (14). Furthermore, sequence analysis of anti-PPS Abs, 5 d postvaccination, demonstrate a predominance of IgG and IgA Abs, derived from switched memory cells that have undergone somatic hypermutation (1517).

Moreover, IgM and switched memory B cells most likely play important roles in the immune response to PPV. Although several studies have demonstrated loss of IgM and/or switched memory B cells in the HIV-negative and HIV-infected populations, they did not focus on the PPS-specific cells (7, 13, 18). We have established a technique to identify PPS-specific B lymphocytes, enabling us to characterize the phenotype of PPS-specific B lymphocytes.

In this study, we have identified PPS-specific B lymphocytes using fluorescently labeled polysaccharides and analyzed the phenotype of these polysaccharide-specific B cells by flow cytometry. The results of our study demonstrate a significant increased representation of IgM memory B cells in the polysaccharide-specific B cell fraction compared with the unselected B cell fraction, providing direct evidence of the importance of IgM memory cells in the response to pneumococcal polysaccharides.

Twenty-two pneumococcal polysaccharide vaccine-naive healthy volunteers between the ages of 18 and 30 y (mean = 24) participated in the University of Toledo Institutional Review Board committee-approved study (Institutional Review Board 105137). Each individual was questioned about medications, previous illness, and present health. In addition, hepatitis B, hepatitis C, human T cell leukemia virus 1 and 2, and HIV screening were performed. Each individual was explained about study design and protocol in detail. Informed consent was obtained from all participants. Volunteers were immunized with the PPV (Pneumovax 23; Merck). Blood samples were collected prevaccination, day 7, and 4–6 wk postvaccination. Lymphocytes were obtained for flow cytometric analysis at days 0 and 7 postvaccination. Serum samples obtained at day 0 and between 4 and 6 wk were used to measure serum Ab responses and opsonophagocytic activity.

Conjugation of PPS-14 to cascade blue (CB) ethylenediamine (Invitrogen catalog C-621) or PPS-23F to 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF; Sigma-Aldrich Fluka catalogue 36565) was carried out as follows. A total of 10 mg PPS-14 or 23F (10 mg/ml in 0.1 M borate buffer [pH 9.0]) was incubated with 1.0 mg CB or 1.0 mg 5-DTAF, respectively, for 2.5 h at 4°C. The mixture was dialyzed against PBS for 24 h at 4°C with four changes of PBS (molecular cutoff = 8 kDa). Approximately 10 μl 5 M sodium cyanoborohydride was added to the dialysate, and the samples were mixed for another 30 min at 4°C in the dark. The samples were again dialyzed against PBS for 24 h at 4°C with four changes of PBS. Finally, samples were subjected to chromatography on a Sephadex G-25 column (1 cm diameter × 17 cm height) at 4°C in the dark. Fractions (150 μl each) containing PPS-14 or PPS- 23F complexed with CB or DTAF, respectively, were pooled and stored at −20°C.

Mouse hybridoma cells with specificity for pneumococcal polysaccharides 14 and 23F, used as positive control, were a gift of Pfizer (Pearl River, NY). Hybridoma cells with specificity for PPS14 and/or PPS23 were stained by incubation with 10 μg/ml fluorescently labeled pneumococcal polysaccharides 14-CB and 23F-DTAF. PPS14- and PPS23-positive B lymphocytes were identified using the following fluorescently labeled Abs: anti-CD19 PE, anti-CD27 PerCP-Cy5.5, anti-IgM allophycocyanin, anti-IgD (Alexa Fluor 700) (BD Pharmingen), and fluorescently labeled PPS 14-CB and 23F-DTAF (BioCentra, Sugar Land, TX).

PBMC were collected from immunized volunteers at days 0 and 7 postvaccination. Buffy coats were harvested and mixed 1:1 with PBS and layered onto lymphocyte separation medium (Cellgro), followed by centrifugation. Cells were then resuspended in RBC lysis buffer, followed by addition of 10 ml PBS, 2 mM EDTA, and 0.1% BSA. Cells were counted, centrifuged, and resuspended to 1 × 108 cells/ml. Before staining, cells were absorbed with 10 μg/ml cell wall polysaccharide (CPS; Statens Serum Institut, 3459; MiraVista Diagnostics, Indianapolis, IN) and PPS 22F (American Type Culture Collection) for 20 min; this step has been shown to reduce nonspecific binding in ELISA (19). For inhibition flow experiments, listed concentrations of homologous pneumococcal polysaccharide were included during the absorption step. Cells were then labeled with 10 μg/ml labeled polysaccharide, either 14-CB or 23F-DTAF, and previously mentioned markers, per manufacturer’s instructions. Cells were washed and resuspended in PBS and analyzed with three-laser FACSAria using FACSDiva software (BD Biosciences). FCS files were further analyzed using FlowJo software (Tree Star, Ashland, OR).

ELISA was performed to examine the anti-PPS–specific human Abs in all volunteers. The pneumococcal polysaccharide ELISA is a modification of the World Health Organization assay (20). Briefly, 5 μg/ml pneumococcal polysaccharide, either 14 or 23F, was absorbed onto Nunc Maxisorp microtiter plates (Nunc, Roskilde, Denmark) at 37°C overnight. Plates were then washed with PBS plus 0.1% Tween 20 (PBST). Sera was diluted 1/200 in PBST and then adsorbed with CPS (10 μg/ml) and 22F (10 μg/ml) for 30 min at room temperature. After absorption, sera were serially diluted onto the plates and incubated at 37°C for 2 h; the standard serum 89-SF was used as a positive control. Plates were washed, and bound Ab was detected using HRP-conjugated anti-human Ig (H + L) mAb (Southern Biotechnology Associates) diluted 1/3000 in 1% BSA PBST and incubated at 37°C for 1 h. After washing, plates were developed by using an o-phenylenediamine substrate, and the OD was read at a wavelength of 490 nm.

The inhibition ELISA is similar to the 22F absorption ELISA, as reported by Concepcion and Frasch (19). Briefly, 10 μg/ml purified pneumococcal polysaccharides, either 14 or 23F, were adsorbed onto Nunc Maxisorp (Nunc) high-binding microtiter plates at 37°C overnight. The plates were blocked with 1% BSA in PBST for 2 h at 37°C. Supernatants were absorbed with 10 μg/ml CPS (Statens Serum Institut, 3459; MiraVista Diagnostics, Indianapolis, IN) and 22F (American Type Culture Collection) for 30 min at room temperature. Furthermore, supernatants were blocked for 30 min at room temperature with increasing concentrations of homologous fluorescently labeled polysaccharide, either 14-CB or 23F-DTAF, to show inhibition. For negative controls, the previously described CPS- and 22F-absorbed samples were blocked with heterologous polysaccharide 23F-DTAF for polysaccharide 14 and 14-CB for 23F. Supernatants were added to the ELISA plates and incubated at 37°C for 1 h. The standard serum 89-SF was used as a positive control. Bound Ab was detected using HRP-conjugated anti-human Ig (H + L) mAb (Southern Biotechnology Associates) diluted 1/3000 in 1% BSA/PBST and incubated at 37°C for 1 h. Plates were developed by using o-phenylenediamine substrate and stopped with H2SO4, and OD was read at a wavelength of 490 nm.

Opsonophagocytic assay was performed, as previously described (21, 22), to determine functional vaccine response to pneumococcal polysaccharides 14 and 23F. Briefly, S. pneumoniae, serotypes 14 and 23F, were incubated with serial diluted heat-inactivated prevaccination and postvaccination sera. Newborn rabbit serum (Pel-Freez, Brown Deer, WI) was added as a source of complement. Differentiated HL-60 cells were added at an E:T ratio of 400:1. All sera were tested in duplicate. The opsonophagocytic titer was determined as the reciprocal of the dilution with 50% killing when compared with serum free controls and analyzed using the Opsotiter1 software program from the University of Alabama at Birmingham.

Geometric mean concentration of IgG, IgM, and IgA, and flow cell numbers, specific to PPS14 and 23F, were calculated for each group. Correlation between two groups was examined using Pearson’s correlation coefficient. Comparison between two group values was performed using unpaired t test. The p values <0.05 were considered to be significant.

To confirm pneumococcal polysaccharide-specific immune response to vaccination, we obtained prevaccination sera, day 0, and postvaccination sera, days 28–42, from healthy young adults and measured Ab responses to serotypes 14 and 23F. Ab concentrations were determined following absorption with PS22F and cell wall polysaccharide (CPS), as previous studies have demonstrated that absence of absorption with 22F and CPS results in an overestimation of polysaccharide-specific Ab concentration (19, 21). Postvaccination, donors had a significant increase in concentration of PPS14-specific IgG from 2.96 ± 2.5 μg/ml to 29.78 ± 17.07 μg/ml (p < 0.0001) and IgM from 1.37 ± 0.49 μg/ml to 28.17 ± 9.26 μg/ml (p < 0.0001). Although postvaccination PPS14-specific IgA titers were higher than prevaccination titers, no significant difference was found (p = 0.08). Similarly, postvaccination responses to PPS23F were significantly increased compared with prevaccination sera, IgG from 1.54 ± 0.71 μg/ml to 21.54 ± 13.09 μg/ml (p < 0.0001) and IgM from 0.17 ± 0.22 μg/ml to 8.86 ± 10.57 μg/ml (p < 0.0001). In our sample population, the greatest increase in pre- to postvaccination concentration was for the isotype IgG, followed by IgM for both PPS14 and PPS23F. There was a positive correlation between pre- and postvaccination PPS14-specific IgM (r2 = 0.88; p < 0.0001) and pre- and postvaccination PPS23F-specific IgM (r2 = 0.98, p < 0.0001) and IgG (r2 = 0.79, p < 0.0001) and IgA (r2 = 0.81; p < 0.001). In summary, all donors displayed an increase in serotype-specific Ab response in Ig isotypes IgG, IgM, and IgA, with the exception of donor 4, whose IgA levels remained undetectable after vaccination. This data confirm that the young healthy donors used in this study responded to Pneumovax 23. Donor Ab responses are summarized in Fig. 1A.

FIGURE 1.

Serum Ab response and opsonophagocytic activity. Healthy young volunteers were immunized with Pneumovax. Serum samples were obtained pre- and 4–6 wk postimmunization. Serum samples (n = 18) were tested for PPS14- and PPS23F-specific IgG, IgA, IgM (A), and opsonophagocytic activity (B). Serum Ab levels are expressed as μg/ml, and opsonophagocytic activity is expressed as OPI.

FIGURE 1.

Serum Ab response and opsonophagocytic activity. Healthy young volunteers were immunized with Pneumovax. Serum samples were obtained pre- and 4–6 wk postimmunization. Serum samples (n = 18) were tested for PPS14- and PPS23F-specific IgG, IgA, IgM (A), and opsonophagocytic activity (B). Serum Ab levels are expressed as μg/ml, and opsonophagocytic activity is expressed as OPI.

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The functional or opsonophagocytic response of serum Ab obtained prevaccination (day 0) and 28–42 d postvaccination against both serotype 14 and 23F PPS was determined for all donors (Fig. 1B). Data are reported as the opsonophagocytic index (OPI) or reciprocal of the Ab dilution required to obtain 50% opsonophagocytic killing by differentiated HL-60 cells. For all donors, postvaccination sera had a significant increase in OPI against serotype 14 and serotype 23F when compared with prevaccination sera. Positive correlations were found between postvaccination IgG and postvaccination OPI for both PPS14 and PPS23F (r2 = 0.8, p < 0.0001; r2 = 0.86, p < 0.0001, respectively). Moreover, the sample population displayed a functional immune response after vaccination with Pneumovax 23.

We evaluated the ability of fluorescently labeled pneumococcal polysaccharide to maintain the native epitope(s) of unlabeled polysaccharide. To this purpose, we performed an ELISA using increasing concentrations of fluorescently labeled PPS to inhibit binding of the polyclonal control serum 89-SF to wells coated with native unlabeled PPS14 or 23F. Unlabeled homologous polysaccharide was used as a positive control, and unlabeled heterologous PPS was used as a negative control. The ability of native pneumococcal polysaccharide and fluorescently labeled pneumococcal polysaccharide to inhibit 89-SF binding to homologous polysaccharides was similar, as shown in Fig. 2. At 1 μg/ml, both CB-labeled PPS14 and unlabeled PPS14 were able to inhibit 89-SF binding by >50%. Incremental addition of inhibitory PPS, up to 25 μg/ml, further increased inhibition of 89-SF binding for both native and CB-labeled PPS14. Likewise, DTAF-labeled PPS23F was able to inhibit 89-SF binding comparable to the native unlabeled PPS23F. Both labeled PPS14 and PPS23F displayed the same trend and similar magnitude of inhibitory effect as their unlabeled homologous counterparts, whereas minimal inhibition was seen in control wells where heterologous polysaccharide was used.

FIGURE 2.

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

FIGURE 2.

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

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To further confirm functionality and specificity of the labeled polysaccharides, hybridoma cells with specificity for PPS14 (α14g2b) and for PPS23F (α23F) were incubated with PPS14-CB and PPS23F-DTAF and subjected to flow cytometry. The results of these studies demonstrated that hybridoma cells α23F with specificity for PPS23F uniquely bound PPS23F-DTAF and failed to bind PPS14-CB in a concentration-dependent manner, as shown in Fig. 3. A subpopulation of small likely nonsecreting cells consistently failed to bind PPS23F-DTAF. Conversely, hybridoma cells α14g2b, with specificity for PPS14, uniquely bound PPS14-CB and failed to bind PPS23F-DTAF, demonstrating both specificity and functionality of the labeled PPS.

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 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.

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To demonstrate the specificity of binding of the fluorescently labeled PPS, an inhibition assay was performed. This was accomplished by pretreating lymphocytes isolated 7 d postvaccination, with increasing concentrations of homologous unlabeled polysaccharide before addition of fluorescently labeled polysaccharide. Inhibition of fluorescent polysaccharide binding was 87.8% at 250 μg/ml inhibitory PPS for PPS14-CB and 80.2% for PPS23F-DTAF. Inhibition occurred in a concentration-dependent manner, as shown in Fig. 4.

FIGURE 4.

Inhibition of binding to fluorescently labeled PPS. Lymphocytes isolated 7 d postvaccination were pretreated with increasing concentrations of homologous unlabeled polysaccharide before addition of fluorescently labeled polysaccharide PPS14 (A) and PPS23F (B). Fifty thousand events were recorded. Percent inhibition of binding to fluorescently labeled PPS was determined by comparison with the uninhibited cells.

FIGURE 4.

Inhibition of binding to fluorescently labeled PPS. Lymphocytes isolated 7 d postvaccination were pretreated with increasing concentrations of homologous unlabeled polysaccharide before addition of fluorescently labeled polysaccharide PPS14 (A) and PPS23F (B). Fifty thousand events were recorded. Percent inhibition of binding to fluorescently labeled PPS was determined by comparison with the uninhibited cells.

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To determine the phenotype of B lymphocytes that respond to vaccination with Pneumovax 23, 18 healthy young donors were immunized. Prevaccination (n = 9) and 7 d postvaccination (n = 18), circulating PBMC were isolated, labeled, and subjected to flow cytometry analyses by using the following fluorescently labeled Ab/Ag: CD19, CD27, IgM, IgD, PPS14, and PPS23F. The phenotype of the prevaccination- and postvaccination-specific B cells was compared with the phenotype of unselected B cells. CD19+ B lymphocytes were subdivided into four categories: naive (CD27IgM+), CD27IgM class-switched, IgM memory (CD27+IgM+), and class-switched memory (CD27+IgM) B cells.

Analysis of the unselected B cell populations obtained prevaccination showed that a large proportion, 71.2% (41.8–97.2%), of these B cells were naive or CD27. The majority (61.4%) of the CD27 B cells expressed the CD27IgM+ phenotype, whereas a minority (9.8%) of total B cells were CD27IgM. The memory or CD27+ B cell population represented 28.7% of the B cells with 14.3% expressing the IgM memory phenotype (CD27+IgM+), and 14.4% were classic switched memory B cells (CD27+IgM). Analysis of the postimmunization unselected B cell population did not differ significantly from preimmunization values (Fig. 5A, 5B).

FIGURE 5.

B cell phenotypes. The phenotype of B lymphocytes that respond to vaccination with Pneumovax 23 was determined by flow cytometry. Prevaccination (n = 9) and 7 d postvaccination (n = 18), circulating PBMC were isolated and labeled for analysis using the following fluorescently labeled Ab/Ag: CD19, CD27, IgM, IgD, PPS14, and PPS23F. The phenotype of prevaccination unselected B cells was compared with PPS-specific B cells (A), the phenotype of postvaccination unselected B cells was compared with PPS-specific B cells (B), and preimmunization PPS-specific B cells were compared with postimmunization PPS-specific B cells (C). In each sample, 75,000 events were recorded. *p < 0.0001, **p = 0.0002, #p = 0.037, ##p = 0.006.

FIGURE 5.

B cell phenotypes. The phenotype of B lymphocytes that respond to vaccination with Pneumovax 23 was determined by flow cytometry. Prevaccination (n = 9) and 7 d postvaccination (n = 18), circulating PBMC were isolated and labeled for analysis using the following fluorescently labeled Ab/Ag: CD19, CD27, IgM, IgD, PPS14, and PPS23F. The phenotype of prevaccination unselected B cells was compared with PPS-specific B cells (A), the phenotype of postvaccination unselected B cells was compared with PPS-specific B cells (B), and preimmunization PPS-specific B cells were compared with postimmunization PPS-specific B cells (C). In each sample, 75,000 events were recorded. *p < 0.0001, **p = 0.0002, #p = 0.037, ##p = 0.006.

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In the preimmunization samples, a small percentage of B cells, 0.56 ± 0.34%, stained with fluorescently labeled PPS (Table I). The PPS14- and PPS23F-specific B cells in this population demonstrated a predominance of CD27 B cells with a total of 55.4% (14–80.3%) for PPS14 and 63.8% (55.8–81.5%) for PPS23F. The CD27IgM+ phenotype represented the majority of these cells with 47.4% for PPS14 and 55.8% for PPS23F of the total B cell population. The CD27+ population represented a total of 44.6% (8.1–86.6) and 36.2% (10.3–48.2) of the PPS14- and PPS23F-specific B cells with the IgM memory component (CD27+IgM+) representing 21.8% of the total PPS14-specific B cells and 25.6% of the PPS23F-specific B cells. The remainder of the B cell population consisted of switched memory B cells (CD27+IgM), accounting for 22.8% of the PPS14-specific B cells and 10.6% of the PPS23F-specific B cells (Fig. 5A).

Table I.
The percentage of CD19+ B cells stained with fluorescently labeled PPS14 and PPS23F in peripheral blood samples obtained pre- and 7 d postimmunization
PPS-14–Specific %PPS-23F–Specific %
Preimmunization 0.56 (±0.13) 0.53 (±0.13) 
Postimmunization 2.75 (±0.56) 2.08 (±0.41) 
PPS-14–Specific %PPS-23F–Specific %
Preimmunization 0.56 (±0.13) 0.53 (±0.13) 
Postimmunization 2.75 (±0.56) 2.08 (±0.41) 

In the 7 d postimmunization samples, the percentage of PPS fluorescently labeled B cells increased significantly to 2.75 ± 1.48% for PPS14 and 2.08 ± 1.17% for PPS23F (Table I). In sharp contrast to both the unselected and preimmunization PPS-selected B cell populations, the minority of postimmunization PPS-specific B cell populations consisted of naive CD27 B cells, 27.6% for PPS14 and 20.5% for PPS23F. The naive B cell population consisted primarily of CD27IgM+ B cells, 23.8% and 17.5% for PPS14 and PPS23F, respectively. A small percentage, 3.8 and 3%, of B cells were naive, class-switched CD27IgM B cells. The majority of the PPS-selected B cells were memory B cells (CD27+), accounting for 72–79% of the total B cell population (Figs. 5B, 6). Moreover, the IgM memory population, CD27+IgM+, was significantly overrepresented compared with both unselected and preimmunization PPS-specific B cells, representing 54.2% of the PPS14-specific B cells and 66% of the PPS23F-specific B cells (Fig. 5B, 5C). In contrast, there was no significant difference in switched memory (CD27+IgM) population between postimmunization PPS14- and PPS23F-specific B cells and the unselected or preimmunization PPS-selected populations, accounting for 18.2 and 13.5%, respectively. Furthermore, there was a strong correlation between postimmunization IgM Ab concentration and postimmunization IgM memory B cell percentage for both PPS14 (r2 = 0.87) and PPS23F (r2 = 0.88). In contrast, the correlation between postimmunization IgG Ab concentration and postimmunization switched memory B cell percentage was much lower, r2 = 0.56 for PPS14 and r2 = 0.51 for PPS23F.

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 fluorescently labeled PPS14 (A, B) and 23F (C, 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) were separated into CD27+IgM+, CD27+IgM, CD27IgM+, and CD27IgM. In each sample, 75,000 events were recorded. Representative data of FACS analyses: (A and C) prevaccination; (B and D) postvaccination of PPS14 (A, B) or PPS23F (C, D).

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 fluorescently labeled PPS14 (A, B) and 23F (C, 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) were separated into CD27+IgM+, CD27+IgM, CD27IgM+, and CD27IgM. In each sample, 75,000 events were recorded. Representative data of FACS analyses: (A and C) prevaccination; (B and D) postvaccination of PPS14 (A, B) or PPS23F (C, D).

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The goal of this study was to characterize the phenotype of B cells responding to Pneumovax 23 vaccination. We specifically chose pneumococcal polysaccharides 14 and 23F as they are the most common disease-causing serotypes found in adult high-risk groups such as the elderly and HIV infected (2325).

To assess the immune competency of our volunteers, we studied pre- and postvaccination polysaccharide-specific Ig concentration and opsonophagocytic assays. All individuals responded to vaccination, displaying a significant increase in anti-polysaccharide Ab concentration, specifically IgG, with a concomitant significant increase in opsonophagocytic activity. Within our test group, there was variability in pre- and postvaccination PPS-specific Ab concentration. Variability in Ab concentrations occurred between individuals and between serotypes within individuals. Therefore, a high response to one PPS present in the multivalent vaccine did not necessarily correlate with a high response to other PPS included in the vaccine. These data are consistent with previous studies investigating the pre- and postvaccination anti-PPS Ab response in young healthy individuals to all serotypes included in Pneumovax 23 (26). Moreover, all volunteers demonstrated a 2-fold or higher increase in pre- versus postimmunization Ab concentrations. A positive correlation between pre- and postimmunization titers was noted and between postimmunization IgG and OPI. Although not the main focus of our study, these basic immunological assays demonstrate the immune competency of our study group.

The specific subset(s) of B lymphocytes responsible for immune response to polysaccharide Ags remains to be elucidated. Studies have implicated the role of IgM memory cells, by some postulated to be splenic marginal zone B cells, in response to polysaccharide Ags (5, 7, 9, 27). The nature of the B cell phenotype responsible for the production of anti-polysaccharide Abs remains to be defined.

We used fluorescently labeled PPS in conjunction with flow cytometry for identification and analysis of PPS-specific B lymphocytes. Identification of Ag-specific B lymphocytes using fluorescently labeled Ag has several advantages over previously used methods. Direct labeling of the polysaccharides allows for polysaccharide-specific B cell phenotype analysis while minimizing potential cross-reactivity to linking agents used by indirect labeling methods. Overall, this results in lower background binding and more accurate phenotype analysis. We have demonstrated the ability to identify pneumococcal polysaccharide-specific B lymphocytes using fluorescently labeled polysaccharide in conjunction with flow cytometry. The specificity of our labeled PPS is supported by their ability to inhibit binding of the control sera 89-SF to homologous unlabeled polysaccharide in ELISA and the ability to bind to PPS14- or PPS23F-specific monoclonal cell lines. Likewise, binding of the labeled PPS to postvaccination peripheral blood B lymphocytes in flow cytometry can be inhibited with addition of homologous unlabeled polysaccharide.

Flow cytometric analysis of unselected CD19+ B cells showed a predominance of naive CD27IgM+ B cells, as previously described, in the peripheral blood of healthy adults (2830). The CD27+ memory B cell population constituted ∼30% of total B cells with an equal distribution between IgM memory and switched memory B cells. We found no significant difference in phenotype distribution between pre- and postimmunization unselected samples. This is not surprising as the PPS-specific B cells represented a small fraction, between 2 and 2.75%, of the total postimmunization B cell population insufficient to cause a significant shift in overall phenotype distribution. Analysis of the preimmunization polysaccharide-specific B cells, 0.5% of the total B cell population, resulted in a phenotypic pattern very similar to that of unselected B cells with a predominance of naive CD27IgM+ B cells. There was, however, an increased percentage of CD27+IgM+ cells in the PPS-specific B cells, although this was only significant for PPS23F. In contrast, in the 7 d postimmunization PPS-specific B cells, there was a highly significant shift in cell surface expression. The majority of postimmunization PPS14- and PPS23F-specific B cells expressed the IgM memory, CD27+IgM+, phenotype with a concomitant decrease in naive, CD27IgM+, expression when compared with the unselected B cells and with the preimmunization PPS-specific B cells. It should be mentioned that we found a strong correlation between the postimmunization PPS-specific IgM and the number of PPS-specific IgM memory B cells. Notably, there was no significant change in the CD27+IgM or switched memory population postimmunization, and it correlated poorly with the IgG Ab concentration.

Isolation of Ag-specific B cells is known to be a challenging enterprise due to their limited presence in the B cell population. Overall, Ag-selected B cells represent <1–2% (3133) of total B cells, depending on technique and timing of isolation, and in line with our findings. Moreover, although a variety of techniques have been used, all have limitations in yield and purity. Both yield and purity can affect phenotype analysis performed in our studies. We employed direct fluorescent labeling of PPS in conjunction with flow cytometry/sorting. The PPS-selected population was inhibited 68–80% by unlabeled PPS, suggesting that two-thirds to 80%, depending on serotype, of our PPS-labeled B cells were indeed PPS specific. In accordance, the remaining 20–32% of our PPS-labeled cells were not PPS specific, false positive, thus likely expressing the unselected cell phenotype, that is, 14.3–15.6% CD27+IgM+, a much lower percentage than the PPS-positive B cells. Moreover, the presence of the false-positive PPS-labeled population therefore most likely diminished, not increased, virtual expression of the CD27+IgM+ phenotype in our PPS-specific population. As both unselected and PPS-selected populations expressed a similar percentage of CD27+IgM, it is unlikely that the contaminating or false-positive PPS-labeled population changed the percentage of switched memory B cells in the PPS-specific population.

These data suggest that a large portion of the PPS-responding B cells are IgM memory cells, and this concept is supported by several experimental and clinical findings. First, both asplenic individuals and children younger than 2 y of age have an undetectable or severely reduced number of IgM memory cells, are at increased risk of pneumococcal infection, and respond poorly to polysaccharide vaccines (57, 1113). Second, the percentage of IgM memory cells in common variable immunodeficiency patients correlates with incidence of encapsulated bacterial infection (13). In further support of these findings, Kruetzmann et al. (7) established a significant correlation between serum anti-polysaccharide serotype 3, 9, and 22 IgM Ab concentration and the number of IgM memory B cells in the peripheral blood of children. Several recent studies have focused on memory B lymphocyte subpopulations and immune response to pneumococcal polysaccharide vaccination particularly in HIV-positive individuals (13, 18). These studies analyzed the peripheral blood B lymphocyte population in HIV-positive individuals and correlated the results with either quantitative IgG or IgM Ab response to PPV. Moreover, both studies demonstrated a significant decrease in the switched memory B cell population in the HIV infected. However, there was no correlation found between the number of switched memory B cells and anti-PPS IgG Ab concentration. In another study, Hart et al. (13) described a highly significant loss of IgM memory B cells in HIV-positive individuals. The loss of B memory cells correlated with the decrease in the anti-PPS IgM Ab response. It should be mentioned that, in these studies, as well as others, B lymphocyte populations were not selected for PPS specificity, complicating the interpretation of the data. The results of the current study, however, support the hypothesis that IgM memory B cells play an important, if not crucial, role in the immune response to pneumococcal polysaccharides.

As stated previously, one could argue that IgM memory B cells are not solely responsible for the Ab response to pneumococcal polysaccharide Ags. First, the elderly, asplenic, and common variable immunodeficiency patients not only have reduced or absent IgM memory cells, but also significantly reduced numbers of switched memory B cells (6, 7). Second, switched memory (CD27+IgM) B cells secrete higher levels of anti-pneumococcal polysaccharide Ab than IgM memory B cells (CD27+IgM+) following in vitro stimulation (14). Third, sequence analysis of anti-pneumococcal polysaccharide Abs, obtained 5 d postimmunization, are predominately IgG and IgA isotypes (1517, 34). Fourth, recent studies performed in SCID mice transplanted with human lymphocyte subsets demonstrated that switched memory B cells produced an IgG antipolysaccharide response following vaccination with PPS (35). In support of this finding, Wardemann and colleagues (36, 37) reported that both switched memory B cells (CD27+IgG+) and naive B cells expressed Ig with specificity for T-independent Ags. In the current study, we did not find a significant quantitative difference in the switched memory B cell populations in the unselected versus selected pre- and postimmunization samples. All samples consisted of ∼10–20% CD27+IgM B cells. It should be mentioned, however, that the peripheral blood samples were obtained early, that is, 7 d postvaccination, at which time the highest number of Ab-secreting cells is found in the peripheral circulation (32, 38), whereas serum Ab response was evaluated at 4–6 wk. The number of Ab-secreting cells diminish rapidly thereafter, significantly compromising analysis of PPS-specific B cells at later time points. Moens et al. (35) demonstrated that mice reconstituted with purified CD27+IgM+ B cells produced both an anti-PPS IgM and IgG response following immunization. They hypothesized that immunization with PPS induced an isotype switch from IgM memory cells to IgG-producing plasma cells. We examined the B cell phenotype in a small (n = 4) number of individuals 4–6 wk postvaccination. These preliminary studies indicated that the number of PPS-specific B cells was similar to preimmunization values, at a mere 0.5% of total B cells. Moreover, the phenotype of the PPS-specific B cells isolated at 4–6 wk was not significantly different from the preimmunization PPS-specific B cells. These data strongly suggest that the Ab-secreting B cells are no longer present in the peripheral circulation at 4–6 wk postimmunization. Extensive analysis of the phenotype of the PPS-specific B cells present in the peripheral blood at 4–6 wk would probably result in an invalid analysis of the Ab-secreting PPS-specific B cell population, most likely present elsewhere in the B cell compartment, such as the spleen or bone marrow, at more distant time points. Thus, a large number of questions remains to be elucidated.

We are presently expanding our studies to include a 2-, 3-, and 4-wk time point postvaccination for analysis of PPS-specific B cell phenotype. In addition, immune response and B cell phenotype following Pneumovax will be compared with those generated following conjugate vaccination. Finally, although beyond the scope of the present studies, it will be interesting to determine cytokine profiles at various time points postvaccination. Recent studies have demonstrated that immunization with PPS induced secretion of IL-6 and TNF-α from macrophages attributed to the presence of TLR2 and TLR4 ligands in Pneumovax and thought to be responsible for the IgG component of the immune response to this T-independent type 2 Ag (39).

We thank Dr. P. Fernsten for constructive review and discussions and Pfizer for the generous gift of hybridoma cells. We thank all of the volunteers who participated in the study.

This work was supported by National Institutes of Health Grants RO1A081558 and RO1AG015978 (to M.A.J.W.).

Abbreviations used in this article:

CB

cascade blue

CPS

cell wall polysaccharide

DTAF

5-(4,6-dichlorotriazinyl)aminofluorescein

OPI

opsonophagocytic index

PBST

PBS plus 0.1% Tween 20

PPS

pneumococcal polysaccharide

PPV

23-valent pneumococcal polysaccharide vaccine.

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The authors have no financial conflicts of interest.