Reference values for B cell subpopulations from infancy to adulthood

Abstract

The composition of the peripheral blood lymphocyte compartment underlies developmental changes during ontogeny. Recently, several new B cell populations have been characterized which were suggested to develop in an age-dependent manner. However, age-dependent reference values for distinct B cell populations have rarely been reported. Therefore, we have characterized developmental changes in peripheral B cell populations from infancy to adulthood in order to define age-dependent reference values. Using a flow cytometric approach we analysed the frequencies as well as the absolute counts of naive, switched and non-switched memory B cells, CD27-negative memory B cells, transitional B cells as well as CD21(low) CD38(low) B cells from neonates up to the age of 50 years. Most of the B cell subsets showed age-dependent developmental changes: while the peripheral B cell pool during infancy is characterized predominantly by transitional and naive B cells, the fraction of switched and non-switched memory B cells increases gradually with age. CD21(low) CD38(low) B cells as well as plasmablasts do not exhibit developmental changes. In summary, we could demonstrate particular changes in the peripheral blood B cell compartment during ontogeny. This study provides reference values of different B cell subpopulations offering comparability for studies addressing disturbed peripheral B cell development in immunodeficiency, autoimmunity or B cell reconstitution following cell-depleting therapies.

Fig. 1

Gating strategies for the characterization of B cell subsets. The lymphocyte population was identified based on side-scatter (SSC) characteristics and CD45 expression (R1). B cells were defined further as CD19-expressing cells in the lymphocyte population (R2). CD19⁺ B cells were analysed either for the expression of IgD and CD27, CD24 and CD38 or CD21 and CD38. The following B cell populations have been delineated: CD27^-IgD⁺ (R3, naive B cells), CD27⁺IgD⁺ (R4, non-switched memory B cells), CD27⁺IgD^- (R5, switched memory B cells), CD27^-IgD^- (R6, CD27 negative memory B cells), CD24⁺⁺CD38⁺⁺ (R7, transitional B cells), CD24^-CD38⁺⁺ (R8, plasmablasts) and CD21^low CD38^low B cells (R9).

Fig. 2

Relative frequencies of different B cell subsets. The relative frequencies of total B cells (as percentage of all lymphocytes) as well as different B cell subsets (as percentage of all CD19⁺ B cells) in each individual analysed is shown dependent upon age. The thick line represents a smoothing spline from the non-parametric model and the dotted lines the 95% confidence intervals. B cell subsets have been analysed as depicted in Fig. 1.

Fig. 3

Absolute numbers of different B cell subsets. The absolute number of total B cells as well as different B cell subsets per µl of blood in each individual analysed is shown dependent upon age. The thick line represents a smoothing spline from the non-parametric model and the dotted lines the 95% confidence intervals. B cell subsets have been analysed as depicted in Fig. 1.

Fig. 4

Comparison of immunofluorescent staining approaches using isolated peripheral blood mononuclear cells or whole blood. The relative frequencies of total B cells (as percentage of all lymphocytes) as well as different B cell subsets (as percentage of all CD19⁺ B cells) obtained from staining approaches using peripheral blood mononuclear cells (PBMC) or whole blood method were compared. Spearman's r correlation values are shown and all were significant (P < 0·01).