Human Memory B Lymphocyte Subsets Fulfill Distinct Roles in the Anti-Polysaccharide and Anti-Protein Immune Response

Leen Moens, Margareta Wuyts, Isabelle Meyts, Kris De Boeck and Xavier Bossuyt
J Immunol October 15, 2008, 181 (8) 5306-5312; DOI: https://doi.org/10.4049/jimmunol.181.8.5306

Abstract

There is controversy on the role of IgM memory and switched memory B lymphocytes in the Ab response to T cell-independent and T cell-dependent Ags. We transplanted SCID/SCID mice with human B lymphocyte subsets and immunized them with heat-inactivated Streptococcus pneumoniae or with a pneumococcal vaccine. Inactivated S. pneumoniae and soluble pneumococcal capsular polysaccharides elicited an IgM anti-polysaccharide and anti-protein Ab response from IgM memory B lymphocytes and an IgG anti-polysaccharide and anti-protein response from switched memory B lymphocytes. In addition to the IgM Ab response, IgM memory B cells elicited an IgG anti-polysaccharide and anti-protein Ab response after immunization with inactivated S. pneumoniae or soluble pneumococcal capsular polysaccharides. In conclusion, our findings provide evidence for a versatile role of IgM memory B cells in T-independent and T-dependent immune responses.

In human peripheral blood, two main subpopulations of mature B lymphocytes can be distinguished: CD19+CD27− B lymphocytes and CD19+CD27+ B lymphocytes. CD27+ B lymphocytes express somatically mutated Ig V regions, whereas CD27− B lymphocytes do not (1). Therefore, CD27+ B lymphocytes have been coined memory B lymphocytes, and CD27− B lymphocytes naive mature B lymphocytes (2). Based on the relative expression of IgM and IgD, memory B lymphocytes can be further subdivided into ∼50% IgM memory (IgM+IgD+) B lymphocytes and ∼50% switched memory B lymphocytes (IgM−IgD−) (3). There is controversy on the nature of IgM+IgD+CD27+ B lymphocytes. Some authors describe these cells as IgM memory B lymphocytes (4), whereas others describe these cells as circulating splenic marginal zone B lymphocytes in charge of responses against encapsulated bacteria (5). The controversial issues have recently been reviewed by Tangye and Good (6). They discuss the following: 1) whether IgM+CD27+ memory B cells exist; 2) whether IgM+CD27+ memory B lymphocytes are generated independently of germinal centers; 3) whether IgM+CD27+ memory B lymphocytes undergo somatic hypermutation during generation of the preimmune repertoire; and 4) whether IgM+CD27+ memory B lymphocytes are exclusively involved in humoral responses to T cell-independent (TI)3 type 2 (TI-2) Ags (6). Proponents of the view that IgM+IgD+CD27+ B lymphocytes represent marginal zone B lymphocytes argue that these cells are generated independently of germinal centers, undergo somatic hypermutation during generation of the preimmune repertoire, and are exclusively involved in responses to TI Ags (3, 5, 6). The other view holds that somatic mutation in IgM+IgD+CD27+ B lymphocytes occurs in the early stages of germinal center development and that somatic hypermutation may take place in TI germinal centers, albeit at a lower frequency than in T cell-dependent (TD) germinal centers (6, 7, 8).

There is evidence that IgM+CD27+ B lymphocytes are exclusively involved in humoral immune responses to TI Ags. Also, IgM+CD27+ B lymphocytes are reduced in asplenic individuals, a condition that is associated with increased susceptibility to infection with encapsulated bacteria (3). IgM+CD27+ B lymphocytes reside in the marginal zone. They are almost not detectable in children younger than 2 years of age (3, 9) and are decreased in individuals >65 years old (10). Both age groups, young children and the elderly, are at increased risk for invasive infection with encapsulated bacteria. Moreover, HIV-infected patients are susceptible to invasive pneumococcal infections and have been reported to have decreased IgM memory B lymphocytes (11). Patients with common variable immunodeficiency suffering from recurrent infections with encapsulated bacteria had decreased IgM+CD27+ B lymphocytes, whereas common variable immunodeficiency patients not suffering from recurrent infections had normal IgM+CD27+ B lymphocytes and were able to produce IgM Abs (12).

There are a number of arguments against the view that IgM+CD27+ B lymphocytes are exclusively involved in humoral immune responses to TI Ags (2, 3, 5, 13). First, according to Tangye and Good (6), both IgM+CD27+ and IgM−CD27+ B memory lymphocytes are reduced in individuals susceptible to infection with encapsulated bacteria. Tangye and Good (6) argue that increased susceptibility results from a reduction of all memory B lymphocytes. Second, there is indirect evidence that switched memory B lymphocytes are also involved in TI immune responses. It has been reported that 15–20% of naive and IgG+CD27+ memory B lymphocytes expressed Ig specific for TI Ags (14, 15). Finally, there is evidence that IgM+CD27+ B lymphocytes contribute to TD immune response. IgM memory B cells from individuals vaccinated against hepatitis B and HIV-infected patients were able to differentiate into IgM-, IgG-, and IgA-secreting B cells in vitro in response to CD40L, IL-2, and IL-10 (6, 16, 17).

Clearly, the role of IgM+CD27+ B lymphocytes and switched memory B lymphocytes in the Ab response to TI and TD Ags is at present controversial. To resolve these controversies, we transplanted SCID/SCID mice with purified B lymphocyte subsets and studied the immune response to pneumococcal capsular polysaccharides (caps-PS), a TI-2-type Ag, and to pneumococcal surface protein A (PspA), a TD-type Ag. Caps-PS were presented to the immune system either as a soluble Ag (vaccine) or as part of a whole bacterium (heat-inactivated S. pneumoniae). This allowed us to elucidate whether IgM memory and switched memory B lymphocytes are involved in responses to soluble and particulate TI Ags and whether IgM memory B lymphocytes are exclusively involved in responses to TI Ags.

Materials and Methods

Mice

Six- to 8-wk-old SCID/SCID mice on a BALB/c background were provided by J. Mertens (Rega Institute, Leuven, Belgium). These SCID/SCID mice were kept in sterilized plastic cages and were given sterilized tap water and sterilized pelleted food. The SCID/SCID mice were held in a room with 12-h/12-h light/dark cycle. The SCID/SCID mice were tested for leakiness by analyzing the level of mouse IgG Abs according to a previously described ELISA (18). Only SCID/SCID mice with IgG concentrations less than 2 μg/ml were used in the experiments. Approval of the study was granted by the local ethics committee of Catholic University (Leuven, Belgium).

Preparation of intact heat-inactivated S. pneumoniae

S. pneumoniae serotype 3 (a gift from J. Verhaegen, Laboratory Medicine, Universal Hospital, Leuven, Belgium) was grown in Todd-Hewitt broth to mid log phase at 37°C in a CO2 incubator. Bacteria were inactivated at 60°C for 90 min. Inactivation was confirmed by blood agar culture. Bacteria were collected by centrifugation (20 min, 3000 × g), and the pellet was washed three times with sterile PBS (Life Technologies). The bacterial stock, containing 109 CFU/ml, was divided into aliquots and frozen at −80°C until used for immunization.

Isolation of human PBMC and B lymphocyte subsets

Peripheral blood buffy coat from healthy blood donors was obtained from the Blood Transfusion Centre of the Red Cross (Leuven, Belgium). Human PBMC were prepared by Ficoll-Hypaque (Axis-Shield Poc) density-gradient centrifugation. The viability of the human PBMC (>99%) was tested by tryphan blue exclusion. Four-color (anti-CD3 FITC/CD16+CD56 PE/CD45 PerCP/CD19 allophycocyanin and anti-CD3 FITC/CD8 PE/CD45 PerCP/CD4 allophycocyanin), three-color (anti-IgM FITC/IgD PE/CD27 allophycocyanin), and two-color (anti-IgG/IgA/IgE FITC and anti-CD27 allophycocyanin) (BD Biosciences) immune fluorescence analysis was performed on a FACSCalibur with CellQuest software (BD Biosciences). The healthy buffy coats (n = 10) contained 7.7 ± 2.1% B lymphocytes. The B lymphocyte fraction contained 53.0 ± 13.5% mature naive B lymphocytes and 45.0 ± 14.0% memory B lymphocytes, of which 19.6 ± 7.3% were IgM memory B lymphocytes and 24.7 ± 8.5 switched memory B lymphocytes. A representative buffy coat sample is presented in Fig. 1⇓A. For isolation of CD19+CD27−IgM+IgD+ (mature B), CD19+CD27+IgM+IgD+ (IgM, IgD memory B), and CD19+CD27+IgM−IgD− (switched memory B) B cell subsets, purified CD19+ cells (obtained through CD19 human microbeads; Miltenyi Biotec) were stained with anti-IgM FITC, anti-IgD PE, and anti-CD27 allophycocyanin or with anti-IgG/IgE/IgA FITC and anti-CD27 allophycocyanin (4°C, 30 min) and sorted on an ARIA cell sorter (BD Biosciences) under sterile conditions. A representative postsort analysis example of the different B cell subpopulations is presented in Fig. 1⇓B.

FIGURE 1.
FIGURE 1.

Flow cytometric analysis of peripheral blood cells of a healthy donor before and after isolation of the B cell subsets. A, Flow cytometric analysis of a peripheral blood sample obtained from a healthy blood donor. The left-hand panel shows CD3-FITC and CD19-allophycocyanin staining before isolation of CD19+ B cells. The right-hand panel shows surface Ig (sIg)-IgG-IgE-FITC and CD27-FITC staining on isolated CD19+ B cells. B, Flow cytometric analysis of isolated naive mature B cells, IgM memory B cells, and switched memory B cells after staining with sIgA-IgG-IgE-FITC and CD27-FITC.

Transfer of PBMC to SCID/SCID mice and immunization of SCID/SCID mice

To improve survival and functionality of the transplant, SCID/SCID mice received TMβ1 (a gift from M. Waer, Experimental Transplantation, Catholic University, Leuven, Belgium) i.p. 1 day before transplantation. TMβ1 is a rat anti-mouse anti-IL-2R β-chain mAb that depletes the murine NK cells (19). Human PBMC containing 3 × 106 B lymphocytes, or a maximum of 70 × 106 human PBMC (depending on the buffy coat), were dissolved in PBS and injected i.p. in SCID/SCID mice. The amount and purity of the B lymphocyte subsets that were transferred were 2.9 × 106 ± 0.61/mouse (purity ≈97.2 ± 2.8%) for CD19+CD27−IgM+IgD+ cells, 2.9 × 106 ± 0.69/mouse (purity ≈91.6 ± 9.8%) for CD19+CD27+IgM+IgD+ cells, and 2.8 × 106 ± 0.66/mouse (purity ≈95.5 ± 3.9%) for CD19+CD27+IgM−IgD− cells. The IgM memory B cell subsets contained 4.5 ± 4.8% CD27−IgM+IgD+B lymphocytes and 3.2 ± 2.4% CD27+IgM−IgD−B lymphocytes. The B lymphocyte subsets were transferred to the SCID/SCID mice together with PBMC depleted of CD19+ B lymphocytes (containing <0.1% CD19+ cells). The CD19/CD4+ ratio of the cells transplanted corresponded to the CD19/CD4+ ratio in the donor buffy coat. This corresponded to addition of 24.4 × 106 ± 5.8 CD4+ cells for experiments in which mature naive B lymphocytes were transplanted and addition of 22.4 × 106 ± 5.6 and 23.2 × 106 ± 4.3 CD4+ cells for experiments in which IgM memory B lymphocytes and switched memory B cells, respectively, were transplanted.

SCID/SCID mice were immunized i.p. with 2 × 108 CFU heat-inactivated S. pneumoniae serotype 3 or with 20 μl of Pneumo23 (Sanofi Pasteur MSD) on the same moment as they received the human transplant. Fourteen days later, blood was drawn by heart puncture in isoflurane (Schering-Plough Animal Health)-anesthetized mice. Mice were euthanized after isoflurane inhalation by cervical dislocation.

Determination of Abs to caps-PS serotype 3 and PspA

IgM and IgG Abs to cap-PS serotypes (20) and PspA were measured by ELISA, as previously described, but using a goat anti-human conjugate (21).

Patient with hyper-IgM type I syndrome case report

A patient was diagnosed with hyper-IgM type I syndrome at the age of 36 years, following diagnosis of hyper-IgM type I syndrome in his baby nephew. A R207T mutation in exon 5, coding for the extracellular domain of CD40L, was identified in both patients, leading to residual expression of CD40L, but deficient binding to CD40-Ig (L. Notarangelo, Istituto di Medicina Molecolare; A. Nocivelli, Clinica Pediatrica, Università di Brescia, Brescia, Italy). From his past medical history, we learned that he had been suffering from recurrent upper respiratory tract infections. He was treated for pneumonia at the age of 24. Three years later, demonstration of calcified lesions on chest-x-ray prompted a treatment with tuberculostatics; however, a diagnosis of mycobacterial infection was never confirmed. At the time of diagnosis, he was generally in a good state of health, with sporadic complaints of spontaneously resolving diarrhea and abdominal upset and fatigue. There was no recent history of significant infections. At diagnosis, IgG was 4.18 g/L (adult lower level of normal 7.51 g/L); IgA was 1.20 g/L (range 0.82–4.53 g/L); and IgM was 6.05 g/L (range 0.46–3.04 g/L). Total lymphocyte count was normal, as was proliferation in response to various mitogens and Ags. Percentage of B lymphocytes was 3%, of which 47% were CD27+ B lymphocytes and 97% of CD27+ B lymphocytes were IgM+IgD+ memory B lymphocytes. IgG memory responses to TD Ags were absent for all previous immunizations, except for polio Ags 1 and 2. A significant IgM response was detected against rubella and mumps. Immunization with Tymphim was unsuccessful at mounting an IgG response. The late diagnosis of hyper-IgM syndrome type 1 in this Ig-naive patient allowed us to study the Ab response to Pneumo23. Anti-pneumococcal Abs were quantified before and 3 wk after immunization. Written informed consent was obtained from the patient.

Results

The immune response to heat-inactivated S. pneumoniae and to Pneumo23 in humanized SCID/SCID mice

We verified that humanized SCID/SCID mice (SCID/SCID mice transplanted with unfractionated human PBMC) mounted an IgM and IgG anti-polysaccharide Ab response after immunization either with intact heat-inactivated S. pneumoniae serotype 3 or with Pneumo23. Similarly, we also verified that humanized SCID/SCID mice elicited IgM and IgG Abs to PspA after immunization with intact heat-inactivated S. pneumoniae serotype 3. The results are shown in Figs. 2⇓ and 3⇓ and indicate that humanized SCID/SCID mice mount a clear and distinct human anti-polysaccharide and anti-protein Ab response. No anti-polysaccharide or anti-PspA Abs were found in humanized SCID/SCID mice that had not been immunized (data not shown). Likewise, no Abs were detected in SCID/SCID mice transplanted with PBMC depleted of CD19+ B lymphocytes and immunized with heat-inactivated S. pneumoniae or Pneumo23 (data not shown). Immunized SCID/SCID that had not been transplanted generated no Abs (data not shown).

FIGURE 2.
FIGURE 2.

Effect of B cell subsets on the IgM and IgG Ab response to heat-inactivated S. pneumoniae. SCID/SCID mice were transplanted with different human CD19+ B lymphocyte subsets in combination with PBMC depleted of CD19+ B lymphocytes and immunized with heat-inactivated S. pneumoniae serotype 3. The immune response to caps-PS serotype 3 and PspA was measured after 14 days. The different B cell subsets were naive mature B lymphocytes (CD19+CD27−IgM+IgD+), IgM memory B lymphocytes (CD19+CD27+IgM+IgD+), switched memory B lymphocytes (CD19+CD27+IgM−IgD−), and PBMC. The results show a representative experiment of four independent experiments. The error bars show the SD. Each experiment was done with six or seven mice.

FIGURE 3.
FIGURE 3.

Effect of B cell subsets on the IgM and IgG Ab response to soluble caps-PS. SCID/SCID mice were transplanted with different human CD19+ B lymphocyte subsets in combination with PBMC depleted of CD19+ B lymphocytes and immunized with soluble caps-PS (Pneumo23). The immune response to caps-PS was measured after 14 days. The different B cell subsets were naive mature B lymphocytes (CD19+CD27−IgM+IgD+), IgM memory B lymphocytes (CD19+CD27+IgM+IgD+), switched memory B lymphocytes (CD19+CD27+IgM−IgD−), and PBMC. The results show a representative experiment of three independent experiments. The error bars show the SD. Each experiment was done with six or seven mice.

Human B lymphocyte subsets and the immune response to heat-inactivated S. pneumoniae

We studied the ability of different CD19+ B lymphocyte subsets to mount an anti-polysaccharide and anti-protein Ab response after immunization with heat-inactivated S. pneumoniae. SCID/SCID mice were transplanted with PBMC depleted of B lymphocytes and either naive mature B lymphocytes (CD19+CD27−IgM+IgD+), IgM memory B lymphocytes (CD19+CD27+IgM+IgD+), or switched memory B lymphocytes (CD19+CD27+IgM−IgD−). Subsequently, the mice were immunized with heat-inactivated S. pneumoniae serotype 3, and 2 wk later the Ab production was measured. The results are shown in Fig. 2⇑. SCID/SCID mice transplanted with naive mature B lymphocytes elicited detectable, but very low amounts of IgG and IgM Abs to caps-PS serotype 3. The Ab response in these mice was less than the Ab response in mice that had been transplanted with unfractionated PBMC. However, SCID/SCID mice transplanted with purified memory B lymphocytes were able to produce specific Abs. SCID/SCID mice that had been transplanted with IgM memory B lymphocytes produced IgM and IgG Abs to caps-PS serotype 3 and to PspA, whereas SCID/SCID mice that had been transplanted with switched memory B lymphocytes produced IgG Abs to caps-PS and PspA.

Human B lymphocyte subsets and the immune response to soluble caps-PS

Subsequently, we studied the ability of the different B lymphocyte subsets to mount an Ab response to soluble polysaccharides. SCID/SCID mice were transplanted with PBMC depleted of B lymphocytes and either naive mature B lymphocytes, IgM memory B lymphocytes, or switched memory B lymphocytes. Two weeks later, the immune response to serotypes 3, 4, 9N, 18C, and 19F was measured. The results for serotypes 3 and 9N are shown in Fig. 3⇑. SCID/SCID mice transplanted with naive mature B lymphocytes elicited small amounts of IgG and IgM Abs to caps-PS after immunization with Pneumo23. The Ab response in these mice was lower than the Ab response in mice that had been transplanted with unfractionated PBMC. SCID/SCID mice transplanted with the IgM memory B lymphocyte subset exhibited an IgG and IgM anti-caps-PS Ab response, whereas SCID/SCID mice transplanted with switched memory B lymphocytes exhibited an IgG anti-caps-PS response.

Ab response of a patient with hyper-IgM syndrome type I to a pneumococcal vaccine

Our humanized SCID/SCID model indicated that IgM memory B lymphocytes are able to elicit IgG Abs for soluble caps-PS. Based on these data, we hypothesized that a patient with a hypomorphic mutation in CD40L who has IgM memory B lymphocytes, but almost no switched memory B lymphocytes, should be able to generate considerable amounts of IgM, but also IgG anti-caps-PS Abs (Fig. 4⇓). Such a patient with a hypomorphic mutation in CD40L was vaccinated with Pneumo23, and the IgM and IgG Ab titers to serotypes 3, 4, 9N, 18C, and 19F were measured before and 3 wk after vaccination (Table I⇓). The majority (>97%) of the patient’s memory B lymphocytes were IgM memory B lymphocytes (Fig. 4⇓). The patient mounted a significant IgM Ab response for all serotypes tested. The IgG Ab response was mainly restricted to serotype 3. It was much less pronounced for serotypes 4 and 9N and almost completely absent for serotypes 18C and 19F.

FIGURE 4.
FIGURE 4.

Flow cytometric analysis of peripheral blood cells of a patient with hyper-IgM syndrome type I and a control individual. A, Peripheral blood of the patient and of a control individual stimulated with ionomycin (1 μg/ml) and PMA (25 ng/ml) and subsequently stained with CD4 FITC and CD40L (CD154) PE or CD40 murine Ig/R-PE. The right-hand panels show the results of the flow cytometric analysis from the patient, and the left-hand panels show the results from the control individual. B, Flow cytometric analysis of the B lymphocyte subsets (CD27-PE/sIgM/D-FITC) of the patient with hyper-IgM syndrome type I (right-hand panel) and of a control individual (left-hand panel).

View this table:
Table I.

Ab response of a patient with hyper-IgM syndrome type I to a pneumococcal vaccinea

Discussion

S. pneumoniae is a major cause of respiratory and meningeal infections, especially in infants, the elderly, and immunocompromised individuals. It causes significant morbidity and mortality worldwide, and the recent emergence of penicillin- and multidrug-resistant S. pneumoniae strains complicates treatment and increases the burden on public health systems (22). Phagocytes, complement, and specific Abs to caps-PS and virulent surface proteins are crucial in the host defense against S. pneumoniae (23).

Most of the knowledge on the role of B lymphocyte subsets in the immune response to S. pneumoniae has been generated in murine experimental models. Murine mature B lymphocytes can be divided into four subsets, as follows: follicular, marginal zone, B-1a (CD5+), and B-1b (CD5−) B lymphocytes. Follicular B lymphocytes recirculate and mount Ab responses against TD protein Ags. B-1 and marginal zone B lymphocytes generate TI Ab responses (24). Marginal zone B lymphocytes are localized proximal to the marginal sinus of the spleen, whereas B-1 lymphocytes are most abundant in peritoneal and pleural cavities. Guinamard et al. (25) demonstrated that Pyk-2-deficient mice, which lack splenic marginal zone B lymphocytes, are deficient in TI-2 Ab response. Haas et al. (26) reported that B-1 B lymphocytes and marginal zone B lymphocytes collaborate to combat S. pneumoniae. The authors concluded that B-1a and marginal zone B lymphocytes secrete natural polyreactive Abs that protect against infection or lower bacterial burden if infection is established, whereas B-1b B lymphocytes secrete anti-caps-PS Abs essential for the clearance of the bacteria (26, 27). In contrast to the findings of Haas et al., Wardemann et al. (28) found a correlation between the absence of B-1a B lymphocytes and poor Ab response to caps-PS. The human equivalents of the mouse B-1 lymphocytes have not been defined, and human marginal zone B lymphocytes differ from mouse marginal zone B lymphocytes in that they show signs of Ag-mediated selection, display a memory B lymphocyte phenotype, and circulate in the bloodstream (1, 5). Only few, indirect, and contradictory data are available on the role of human B lymphocyte subsets in the Ab response to S. pneumoniae. Direct identification of the human peripheral B cell subsets eliciting Abs to pneumococcal polysaccharides and proteins is missing.

In this study, we immunized humanized SCID/SCID mice with inactivated S. pneumoniae or with a pneumococcal vaccine to study the role of the various B cell subsets in eliciting Abs to pneumococcal protein and polysaccharide Ags.

In our humanized SCID/SCID model, CD27−IgM+IgD+ B lymphocytes generated very low amounts of IgM and IgG anti-caps-PS and anti-PspA Abs. This is consistent with in vitro experiments in which it was shown that CD27− B lymphocytes produced no Abs in response to Staphylococcus aureus Cowan strain (SAC) and IL-2, whereas CD27+ B cells did (26, 27). When IL-10 and T lymphocytes or anti-CD40 was added to the in vitro culture, then CD27− B cells produced Abs, but to a lesser extent than CD27+ B cells (29, 30).

A number of remarkable observations were made with respect to the role of memory B lymphocytes in the Ab response to S. pneumoniae.

First, we found that IgM anti-caps-PS Abs were produced by CD19+CD27+IgM+IgD+ memory B lymphocytes, whereas IgG anti-caps-PS Abs were produced by CD19+CD27+IgM−IgD− switched memory B lymphocytes. This was independent of the way the polysaccharide Ag was presented to the immune system, either in soluble form (as part of a vaccine) or in particulate form (as part of a whole bacterium in which a whole set of other Ags is present). This indicated that not only IgM memory B lymphocytes, but also switched memory B lymphocytes, are involved in the anti-caps-PS Ab response. Both IgM and IgG anti-caps-PS Abs are important in the immune response to encapsulated bacteria. Natural, low-affinity IgM Abs are important for the rapid first line defense responses to encapsulated bacteria, whereas IgG anti-caps-PS Abs are important because phagocytes bear receptors for the Fc portion of IgG molecules that are critical for opsonization (23, 31).

Second, we observed that IgM and IgG anti-PspA Abs were produced by CD19+CD27+IgM+IgD+ memory B lymphocytes and CD19+CD27+IgM−IgD− switched memory B lymphocytes, respectively. This revealed that IgM memory B lymphocytes are not exclusively involved in the anti-polysaccharide immune response, but also in the anti-protein immune response.

Third, a most remarkable observation was that SCID/SCID mice reconstituted with human IgM memory B lymphocytes generated IgM as well as IgG anti-caps-PS Abs after immunization with inactivated S. pneumoniae serotype 3 or Pneumo23. We hypothesized that immunization with intact S. pneumoniae or with soluble caps-PS induced IgM memory B lymphocytes to turn into IgG-producing plasma cells. We were, however, not able to substantiate this hypothesis by flow cytometric analysis of the B cell phenotype because of the very low recovery of cells after immunization. The hypothesis is consistent with the in vitro observation by Agematsu et al. and by Takizawa et al. (29, 32) that human IgM memory B lymphocytes and tonsillar B lymphocytes produce IgM as well as IgG Abs after stimulation with SAC plus IL-2 plus anti-CD40 mAb plus IL-10. It should be mentioned, however, that other authors reported that IgM memory B lymphocytes did not shift into switched memory B lymphocytes by various stimuli in vitro (33).

Fourth, we found that there was a marked serotype-specific difference in the immune response to soluble caps-PS. IgM memory B lymphocytes were able to induce IgM and IgG anti-caps-PS Abs to caps-PS serotype 3, but to a much lesser extent than to other serotypes. This observation was further reinforced by measurement of the anti-caps-PS immune response in a patient with hyper-IgM type 1 syndrome due to a hypomorphic mutation in CD40L. Hyper-IgM type 1 syndrome is characterized by an increased susceptibility to infections and decreased levels of serum IgG, IgA, and IgE, but normal or elevated levels of IgM (34). Agematsu et al. (29) found that patients with hyper-IgM type 1 syndrome lack switched memory B lymphocytes, and that this accounts for the impaired Ig production. These authors also reported that B lymphocytes of patients with hyper-IgM type 1 syndrome proliferate upon activation by anti-CD40, suggesting normal functioning (29). The patient that we present had no switched memory B lymphocytes and was able to produce large amounts of IgM anti-caps-PS Abs. However, the patient produced substantial amounts of IgG anti-caps-PS Abs to serotype 3 and detectable amounts of IgG to serotypes 4 and 9N. This finding confirmed our observations in humanized SCID/SCID mice transplanted with human IgM memory B lymphocytes and immunized with soluble polysaccharides. Serotype 3 is known to be highly immunogenic and even immunogenic at earlier age (from 3 mo of age) (35, 36 , and own unpublished data). We also previously found that an inadequate response to serotype 3 predicts unresponsiveness to other serotypes (20). This finding has also been reported by Sorensen et al. (37), who found that patients who failed to respond to serotype 3 at any age also failed to develop protective concentrations of Abs against the other serotypes.

In the present study, we actually tested the relative role of different human B cell subsets to immune responses elicited by TI and TD Ag and found that IgM memory B cells are much more versatile than proposed by other studies. Such versatile role of IgM memory B cells has been discussed by Tangye and Good (6) and is in contrast to previous correlative findings of Weller et al. (5) and Carsetti et al. (9, 12) that suggested that IgM memory B cells are the sole contributors of TI immune responses. Our findings contradict the dogma that IgM memory B cells only participate in TI immune responses.

This study encourages the identification and quantification not only of IgM memory B lymphocytes, but also switched memory B lymphocytes in patients with increased susceptibility to infections with encapsulated bacteria. Indeed, as suggested by Tangye and Good (6), the inability of some individuals to respond to encapsulated bacteria such as S. pneumoniae may result from a reduction in all memory B lymphocytes rather than from a loss of only IgM memory B lymphocytes.

Acknowledgments

We thank J.-C. Weill and S. Weller (Faculté de Médecine Necker-Enfants Malades, Université Paris, France) for helpful discussions and critical review of the manuscript. We are indebted to P. Vandervoort for expert technical assistance.

Disclosures

We have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • 1 This work was supported by a grant from the Fund for Scientific Research-Flanders. X.B. is a senior clinical investigator of the Fund for Scientific Research-Flanders.

  • 2 Address correspondence and reprint requests to Dr. Xavier Bossuyt, Laboratory Medicine, Immunology, GHB-Herestraat 49, B-3000 Leuven, Belgium. E-mail address: xavier.bossuyt{at}uz.kuleuven.ac.be

  • 3 Abbreviations used in this paper: TI, T cell independent; caps-PS, capsular polysaccharide; PspA, pneumococcal surface protein A; sIg, surface Ig; TD, T cell dependent; TI-2, TI type 2.

  • Received October 2, 2007.
  • Accepted August 1, 2008.

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