Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen

Abstract

Splenectomized and asplenic patients have a high incidence of infections by encapsulated bacteria and do not respond to polysaccharide vaccines. To understand whether the absence of the spleen is associated with a defined B cell defect, we analyzed B cell subsets in the peripheral blood. We found that a population of B cells known as immunoglobulin (Ig)M memory is lacking in patients without spleen. The absence of IgM memory B cells correlates with an impaired immune response to encapsulated bacteria not only in splenectomized patients, but also in individuals with an intact spleen. We show that the physiological and transient predisposition to pneumococcal infections of young children (0-2 yr) is associated with the lack of circulating IgM memory B cells and of serum antipolysaccharide IgM. We also demonstrate that IgM memory B cells are undetectable in a fraction of patients with common variable immunodeficiency, who have recurrent and invasive infections by encapsulated bacteria. IgM memory B cells, therefore, require the spleen for their generation and/or survival and are responsible for the protection against encapsulated bacteria.

Figure 1.

(A) Phenotypic analysis of B cells in the human peripheral blood. Donor PBLs were stained with Abs to CD22, CD27, IgM, and IgD and analyzed by four-color flow cytometry. Data is presented as density plots. CD22⁺ B cells were separated into CD27⁺ memory B cells (60%) and CD27⁻ mature B cells (40%; left). In the right panels, IgD and IgM staining of all cells in the lymphocyte gate and of mature and memory B cells, identified as indicated in the left panel, is shown. Mature B cells are IgM⁺ IgD^bright. Two populations of memory B cells can be identified, IgM^bright IgD^dull IgM memory B cells and switched memory B cells lacking the expression of both IgM and IgD. (B) Frequency of CD5⁺ B cells in the mature and memory compartments of eight donors. The expression of CD5 in IgM and switched memory B cells was calculated based on the staining for CD22, CD27, IgM, and CD5.

Figure 1.

(A) Phenotypic analysis of B cells in the human peripheral blood. Donor PBLs were stained with Abs to CD22, CD27, IgM, and IgD and analyzed by four-color flow cytometry. Data is presented as density plots. CD22⁺ B cells were separated into CD27⁺ memory B cells (60%) and CD27⁻ mature B cells (40%; left). In the right panels, IgD and IgM staining of all cells in the lymphocyte gate and of mature and memory B cells, identified as indicated in the left panel, is shown. Mature B cells are IgM⁺ IgD^bright. Two populations of memory B cells can be identified, IgM^bright IgD^dull IgM memory B cells and switched memory B cells lacking the expression of both IgM and IgD. (B) Frequency of CD5⁺ B cells in the mature and memory compartments of eight donors. The expression of CD5 in IgM and switched memory B cells was calculated based on the staining for CD22, CD27, IgM, and CD5.

Figure 2.

(A) Frequency of IgM and switched memory B cells in 11 controls and 14 splenectomized patients. (B) PBLs from the indicated subjects were stained and analyzed as described in Fig. 1 A. On the top, the expression of CD22 and CD27 is shown. The numbers indicate the frequency of memory B cells, calculated as a percentage of CD22⁺ B cells. On the bottom, IgM and switched memory B cells were identified as described in Fig. 1 A.

Figure 2.

(A) Frequency of IgM and switched memory B cells in 11 controls and 14 splenectomized patients. (B) PBLs from the indicated subjects were stained and analyzed as described in Fig. 1 A. On the top, the expression of CD22 and CD27 is shown. The numbers indicate the frequency of memory B cells, calculated as a percentage of CD22⁺ B cells. On the bottom, IgM and switched memory B cells were identified as described in Fig. 1 A.

Figure 3.

(A) Frequency of memory B cells in children. PBLs from 0–7-yr-old healthy children were stained and analyzed as described in Fig. 1 A. The frequency of CD22⁺ CD27⁺ memory B cells as a percentage of total CD22⁺ B cells is shown. The age in months is indicated on the x axis. (B) The level of total IgM in eight children of different ages and in one adult control were measured by ELISA and compared with the concentration of IgM specific for pneumococcal polysaccharide serotype 22. Total IgM is indicated by • (scale on the right y axis in micrograms/ml) and polysaccharide-specific IgM is indicated by the columns (scale in OD on the left y axis). The age in months is shown on the x axis. (C) Flow cytometric analysis of PBL from three asplenic and three control children. Samples were stained and analyzed as described in Fig. 1 A. The density plots show the CD22 and CD27 distribution. The frequency of memory B cells (as a percentage of total B cells) and the age of the children are indicated.

Figure 3.

(A) Frequency of memory B cells in children. PBLs from 0–7-yr-old healthy children were stained and analyzed as described in Fig. 1 A. The frequency of CD22⁺ CD27⁺ memory B cells as a percentage of total CD22⁺ B cells is shown. The age in months is indicated on the x axis. (B) The level of total IgM in eight children of different ages and in one adult control were measured by ELISA and compared with the concentration of IgM specific for pneumococcal polysaccharide serotype 22. Total IgM is indicated by • (scale on the right y axis in micrograms/ml) and polysaccharide-specific IgM is indicated by the columns (scale in OD on the left y axis). The age in months is shown on the x axis. (C) Flow cytometric analysis of PBL from three asplenic and three control children. Samples were stained and analyzed as described in Fig. 1 A. The density plots show the CD22 and CD27 distribution. The frequency of memory B cells (as a percentage of total B cells) and the age of the children are indicated.

Figure 3.

(A) Frequency of memory B cells in children. PBLs from 0–7-yr-old healthy children were stained and analyzed as described in Fig. 1 A. The frequency of CD22⁺ CD27⁺ memory B cells as a percentage of total CD22⁺ B cells is shown. The age in months is indicated on the x axis. (B) The level of total IgM in eight children of different ages and in one adult control were measured by ELISA and compared with the concentration of IgM specific for pneumococcal polysaccharide serotype 22. Total IgM is indicated by • (scale on the right y axis in micrograms/ml) and polysaccharide-specific IgM is indicated by the columns (scale in OD on the left y axis). The age in months is shown on the x axis. (C) Flow cytometric analysis of PBL from three asplenic and three control children. Samples were stained and analyzed as described in Fig. 1 A. The density plots show the CD22 and CD27 distribution. The frequency of memory B cells (as a percentage of total B cells) and the age of the children are indicated.

Figure 4.

Our model on the origin and function of mature and IgM memory B cells. IgM memory B cells might be able to migrate to sites of inflammation and recognize invading pathogens through the B cell receptor (BCR) and possibly other pathogen-binding coreceptors. Switched memory B cells are generated from bone marrow–derived mature B cells after somatic mutation and class switch in the germinal centers.