Age-Related Trends in Pediatric B-Cell Subsets

Abstract

There are few studies of the developmental changes in B-cell subsets in children. Recent data from adult populations demonstrate that alterations to B-cell subsets have functional consequences and can be helpful diagnostically. Comparable studies in children have been hindered by the lack of normative data and by significant changes with age. This study evaluated B-cell subsets by 4-color flow cytometry in 47 children of different ages. The use of a 4-color platform is compatible with broad use in clinical laboratories. We found that there are rapid changes in the B-cell compartment in infancy and early childhood. Total B-cell numbers decline early in life, and this correlates with a decline in transitional B cells and naïve B cells. The decline is most rapid between 1 and 5 years of age, with a slower decline later in childhood. In contrast, nonswitched and switched memory B cells both increase during the 1st 5 years of life. The decline in B-cell numbers did not occur until after 1 year of age, suggesting that the period after birth is a unique developmental window. These data provide a reference set for further studies on B-cell dysfunction in pediatric disorders. The changes occurring in early childhood document the need for age-related assessments and serve to underscore the B-cell–specific kinetics of immunologic development in humans.

INTRODUCTION

Human B-cell development occurs in both the bone marrow and the peripheral lymphoid organs. In the bone marrow, B cells undergo antibody heavy chain gene rearrangement at the pro–B-cell stage, followed by light chain gene rearrangement at the pre–B-cell stage [1,2]. After the completion of antibody gene rearrangement, IgM is expressed on the cell surface. Next, IgM+ B cells are tested for self-reactivity of the IgM antibody. B cells passing this tolerance checkpoint are allowed to leave the bone marrow as transitional cells. Transitional B cells emigrate from the bone marrow to secondary lymphoid organs. In healthy individuals, transitional B cells comprise the least mature B cell subset that is readily detectable in the peripheral blood [3–5]. Follicular and marginal zone B cells comprise the major mature B cell populations and are found in secondary lymphoid organs. Activated mature B cells can undergo somatic mutation and antibody heavy chain class switching. Activated mature B cells can further differentiate into memory B cells or plasmablasts, both of which can reenter the circulation. Therefore, the sampling of B-cell subsets in the peripheral blood detects changes in a compartment characterized by transit of cells from bone marrow to secondary lymphoid organs and vice versa.

The study of peripheral blood B cells has been important in understanding B-cell defects in primary immune deficiencies, malignancies, and the effects of therapeutic interventions [6–8]. In adults, abnormal B-cell subsets are associated with autoimmune disease, as well as immune deficiency [8–10]. There are relatively few studies of aberrant B-cell subsets in pediatric disorders, and this is in part due to the difficulties in studying children, in whom the developmental changes can be significant and the related requirement for age-matched controls can make the study design unwieldy.

To apply the clinical study of B-cell subsets in a pediatric population, we made use of a simplified B-cell immunophenotyping scheme, which we have used previously in adult subjects [10,11]. In this scheme, peripheral blood B cells (CD19+ lymphocytes) are separated on the basis of CD27 and CD38 expression. This scheme is advantageous because it can be used to measure the relative frequencies of each of the major circulating B-cell subpopulations in a single tube and therefore lends itself readily to clinical and diagnostic use. Here we present data on CD27 vs CD38 expression in peripheral B-cell subsets and correlate these data with IgM, IgD, and CD5 expression in the same subjects. We also correlate the expression of CD27, CD38, CD10, IgM, and CD24 in a multicolor analysis. We show that the majority of CD5+ B cells in children less than 5 years of age have a transitional phenotype and that there is a reciprocal shift from transitional to more mature B-cell subsets with increasing age. We discuss these results in the context of other reports on B-cell subset analysis in children. This characterization of developmental changes in circulating B-cell subsets will aid in future clinical studies of pediatric patients.

MATERIALS AND METHODS

Blood samples from 47 children were analyzed by 4-color flow cytometry. A parallel complete blood cell count and differential were performed on the same specimen. Seemingly healthy children were identified in primary care outpatient clinics. Ages were recorded in months from birth to 3 years. Older children had their age recorded by year. This study was performed in accordance with a protocol approved by our internal review board.

Peripheral whole blood was prepared and stained with antibodies (BD Pharmingen, San Diego, CA), as described previously [10]. Only fresh whole blood was analyzed in this study. B cells were defined as CD19+ lymphocytes. The absolute B-cell count was obtained by multiplying the absolute lymphocyte count by the CD19+ lymphocyte fraction.

Analyses were performed on a FACSCalibur flow cytometer with CellQuest software (Version 5.2.1, Becton Dickenson, San Jose, CA). B cells were identified on the basis of CD19 expression and forward and side scatter characteristics consistent with lymphocytes. CD19+ lymphocytes were analyzed for CD27, CD38, IgM, IgD, CD24, CD5, CD20, and lambda expression. A minimum of 10 000 CD19+ events were analyzed per tube.

Validation of CD27 vs CD38 subsets was performed using 4-color and 10-color flow cytometric panels on selected samples from adult volunteers (Fig. 1 and unpublished results). Peripheral venous blood was prepared as described above and stained with commercially available fluorophore-conjugated antibodies to CD19, CD20, IgM, IgD, CD10, CD24, CD38, and CD27. A minimum of 7000 B cells were analyzed per tube (on average 10 000 B cells were analyzed per tube). Samples were analyzed on an LSR2A instrument (Becton Dickinson) for 10-color analysis and on a FACSCalibur for 4-color analysis. The data were analyzed using FloJo software v8.2.2 (Treestar Inc., Ashland, OR). For the 4-color analysis, CD19, CD27, and CD38 were shared between 3 separate tubes. The 4th channel, FL-1 (FITC), contained CD10, CD24, or IgM, allowing FITC expression levels to be overlaid and compared between tubes for each specific B-cell subset.

Figure 1.

Data validation. A. Peripheral B-cell subsets, defined by CD27 and CD38 expression (left panel), were further evaluated to confirm their identity as described in the Materials and Methods section. Events vs fluorescence intensity are shown in histogram plots for each of the gated regions. Shown are CD10 (red), CD24 (green), IgM (gold), and isotype control (blue). B. The 10-color analysis was performed on an adult patient. Cell subsets are defined based on CD27 vs CD38 expression (left panel) and analyzed for IgM vs IgD expression (right panel). The B-cell subsets are color coded: transitional (black), naïve mature (grey), mature activated (purple), resting memory (aqua). Representative data are shown. Similar results were obtained in 10 other adults for the 4-color analysis and 9 other adults for the 10-color analysis (unpublished results).

RESULTS

CD27 vs CD38: a simplified peripheral B-cell subsetting scheme

When CD19+ B cells are analyzed for the expression of CD27 (a marker of maturation) and CD38 (a marker of activation), all of the major circulating B-cell subsets can be identified. Using this scheme, we determined that transitional B cells are CD38^bright, CD27−; naïve mature cells are CD27−, CD38+; resting memory cells are CD38−, CD27+; activated memory cells are CD38+, CD27+; and plasmablasts are CD38^bright, CD27^bright. To confirm that these staining patterns correspond to these subsets, we analyzed peripheral blood from adult subjects using a 4-color and a multicolor panel and staining for CD19, CD20, CD27, CD38, CD10, IgM, CD24, IgD, and live/dead (Fig. 1 and Materials and Methods). The results from these analyses (Fig. 1) confirmed the 4-color staining strategy by correlating CD27 and CD38 expression with previously reported descriptions of CD24, IgM, and CD10 on B-cell subsets in humans [3,5,12,13]. Using the CD27 and CD38 staining pattern, we demonstrated that transitional cells expressed higher levels of CD10 and CD24 (Fig. 1A), whereas naïve mature cells appear to represent a mixed collection of cells with different levels of IgM and IgD expression (Fig. 1B). Resting memory cells have low CD10, IgM, and IgD expression, whereas mature activated cells consist of a mixed population of IgM memory and isotype-switched cells [7,10,14,15]. Taken together, these data support the CD27 vs CD38 peripheral B-cell classification scheme [11]. Others have used CD38 in combination with IgD or CD24, or CD27 in combination with IgM or IgD as peripheral B-cell subsetting schemes [3,12,16–18]. We next used the CD27 vs CD38 classification scheme to analyze B-cell subsets in infants and children of differing ages.

Absolute and relative B-cell numbers

As has been described previously [19–22], B-cell numbers declined with age. Although the percentage of B cells within the lymphocyte compartment declined slowly, the effect was much more dramatic when B-cell counts were computed. The decline in B-cell counts began after 1 year of age. In infancy (birth–12 months of age), it appeared that B-cell counts increased slightly (Fig. 2), consistent with previous reports [21].

Figure 2.

Absolute and relative B-cell numbers change with age. A. The absolute B-cell count, in cells per mL of whole blood (see the Materials and Methods section for calculation), is displayed according to age. The decline follows a logarithmic pattern. B. The percentage of B cells in the lymphocyte compartment declines throughout childhood. C. The total B-cell count appears to increase slightly in the 1st year of life. In each panel, each symbol represents a different patient.

Transitional B-cell analysis

To examine the effect of age on B-cell subsets, we initially examined transitional B cells. In the setting of bone marrow transplantation or B-cell depletion therapy, transitional B cells are usually the 1st cells to emerge in the circulation during lymphoid reconstitution [4,5,7,10,23]. As B-cell counts increase after transplantation, transitional B cells initially predominate, followed by naïve mature cells, which lack CD5 expression and express lower levels of CD38 (unpublished results) [4,5]. Based upon CD27, CD38, CD5, IgD, and IgM expression, a similar pattern is observed during the 1st 5 years of life in normal children: initially the immature B-cell subsets predominate and over time the proportion of more mature subsets increases. The relative and absolute transitional B-cell count, based on staining for CD38 (bright) and CD27 (negative), declined throughout early childhood (Fig. 3A,B). A decline in CD5 expression paralleled the decrease in transitional B cells (Fig. 3C). The majority of CD5+ B cells expressed IgD (Fig. 3D). IgD levels approached 100% in children with the highest transitional B-cell and CD5+ fractions (not shown). An alternative strategy to define maturation of the B-cell compartment utilizes surface IgM and IgD expression. IgM+IgD^bright cells declined in early childhood, whereas more mature IgM+IgD− and IgM−, IgD− cells appeared in small numbers between the ages of 2 and 5 years (Fig. 3E). These data are provided in tabular form to facilitate comparison with other B-cell populations (Tables 1,2). Taken together, these data indicate that transitional B cells predominate in infancy and are gradually overtaken by more mature B-cell fractions during childhood.

Figure 3.

Transitional B cells decline rapidly in early childhood. A. Transitional B cells, defined as CD19+, CD38^bright, CD27−, demonstrate a logarithmic pattern of decline in absolute counts as a function of age. B. The transitional B-cell fraction of the total B-cell population demonstrates a comparable decline. C. The CD5+ B-cell fraction (expressed as a percentage of B cells) decreases with increasing age. D. The CD5+ fraction (expressed as a percentage of B cells) is proportional to the transitional B-cell fraction (also expressed as a percentage of B cells). E. Analysis of B-cell subsets using IgM and IgD expression. The IgM+IgD^bright population declines in childhood with a concomitant increase in the IgM− populations.

Table 1.

CD27 vs CD38 B-cell subsets, expressed as an average percentage of B lymphocytes, are shown for different age ranges. The 2nd row of the table displays the age ranges in years. The 3rd row indicates the number (n) of children studied in each age range. The 4th row indicates the average age within the age range (given in years in parentheses). The remaining rows of the table provide the percentages of B cells in the different B-cell subsets: CD27+, CD38+ activated mature cells; CD27+, CD38− resting memory cells; CD27−, CD38+ transitional and naïve mature cells; CD27−, CD38− “double-negative” cells

	Age range (years)
	<1.0	1.0–1.9	2.0–4.9	5.0–11.9	≥12
n (average age)	7 (0.5)	4 (1.2)	5 (3)	19 (7.8)	13 (14.9)
%CD27+, CD38+	2	4	8	9	8
%CD27+, CD38−	0.2	0.3	1.2	2.6	3.5
%CD27−, CD38+	97	95	89	84	84
%CD27−, CD38-	0.4	0.6	2.4	4.5	4.1

Table 2.

IgM vs IgD expression, given as an average percentage of B lymphocytes, is shown for different age ranges. The age ranges, subject numbers, and average subject ages within each age range are as defined in Table 1. The IgM and IgD expression on different B-cell subsets is discussed in the text

	Age range (years)
	<1.0	1.0–1.9	2.0–4.9	5.0–11.9	≥12
n (average age)	7 (0.5)	4 (1.2)	5 (3)	19 (7.8)	13 (14.9)
%IgM+, IgD bright	85	83	64	72	66
%IgM+, IgD dim	11	10	20	13	15
%IgM+, IgD−	2	3	7	5	5
%IgM−, IgD+	2	2	4	3	5
%IgM−, IgD−	1	1	7	8	10

IgM+ and IgM− memory B cells

There are few studies that characterize memory B-cell subsets in children [24,25]. Human memory B-cell populations correspond less clearly to murine subsets, and there is disagreement over the functional features associated with each subset as defined by surface markers. CD27 is a marker of memory [26], but there are reports of CD27− cells with extensive somatic mutation [27]. Nevertheless, most CD27+ B cells are thought to be antigen experienced [28]. CD27+ IgM+ B cells have been described as both marginal zone B cells and as an IgM+ memory B cell [9,13,27,29,30]. CD19+ CD27+IgM+ B cells have modest levels of somatic mutation and appear to undergo somatic hypermutation in a T-cell–independent manner [9,18]. IgM+, CD27+ B cells play an important role in the defense against blood-borne pathogens and, unlike marginal zone cells in the mouse, appear to circulate [29]. IgM− CD27+ B cells represent isotype-switched cells, which are important participants in T-cell–dependent immune responses. We reasoned that evaluating the kinetics of appearance of IgM+, CD27+, and IgM− CD27+ B-cell populations could provide valuable information for ongoing discussions of their function and provenance. We observed a rapid reciprocal change in the IgM+ and IgM− subsets during early childhood. These IgM+ and IgM− CD27 subsets appear to reach a stable ratio by the age of 7 or 8 years (Fig. 4 and Table 3).

Figure 4.

Reciprocal pattern of IgM+ vs IgM− memory B-cell frequencies as a function of age. The white diamonds represent the switched memory B cells (IgM−CD27+) expressed as a percentage of the total CD27+ population. The black circles represent the IgM+ subset expressed as a percentage of the CD27+ population. The IgM+ subset declines, whereas the IgM− subset increases during childhood.

Table 3.

IgM vs CD27 expression, given as an average percentage of B lymphocytes, is shown for different age ranges. The age ranges, subject numbers, and average subject ages within each age range are as defined in Table 1. The remaining rows of the table define the peripheral B-cell subsets based on IgM and CD27 expression: IgM+, CD27− (naïve); IgM+, CD27+ (IgM memory); IgM−, CD27+ (switch memory); IgM+/−, CD27− (double negative). The majority of the “double-negative” cells have dim IgM staining. Some of these cells may represent an anergic population [39]

	Age range (years)
	<1.0	1.0–1.9	2.0–4.9	5.0–11	≥12
n (average age)	7 (0.5)	4 (1.2)	5 (3)	19 (7.8)	13 (14.9)
%IgM+, CD27−	88	82	68	67	59
%IgM+, CD27+	4	5	7	9	7
%IgM−, CD27+	1	3	7	8	9
%IgM+/−, CD27−	7	10	18	16	25

Naïve mature cells

The most common B-cell subset in the peripheral blood is the “naïve mature” B cell, herein defined as CD38+ (less bright than transitional cells) and CD27−. Naïve mature B cells are awkwardly named and appear, as their name implies, to have mixed features of naïve and more mature B cells (Fig. 1) [31]. We further examined the age-related changes in the naïve mature B-cell population (Fig. 5). This population declined with age, in parallel with the total B-cell count. We compared changes in the naïve mature B-cell subset to the other subsets defined by CD27 and CD38 staining, as a function of age group. The decline in transitional B cells (shift from CD38 bright to CD38+ naïve mature cells) (Fig. 6A) appears to coincide with the rise in memory B cells (most of which are CD38+) (Fig. 6B). Figure 6C shows the 5 major B-cell subsets, displayed as stacked bar graphs.

Figure 5.

Naïve mature B cells decline with a pattern comparable to total B cells. A. The absolute count of naïve mature B cells (CD27−CD38+CD19+) is expressed vs age. The decline follows a logarithmic pattern. B. The decline in mature naïve B cells expressed as a fraction of the total B-cell population exhibits more variability than the absolute counts but tends to decrease throughout childhood.

Figure 6.

An overview of CD38 and CD27 expression. A. CD38 expression is shown for CD27+ cells. With age, the CD38+CD27+ cells decline slightly, while the CD38−CD27+ cells increase slightly. B. There is little change in the CD38 expression within the CD27− subset with age. C. A simultaneous display of the major B-cell subsets defined by CD27 and CD38 expression. Patient data (numbered 1–47) are plotted in order of increasing age on the X-axis.

DISCUSSION

Changes in T-cell subsets have been well characterized in childhood and reflect the combined effects of thymic involution, antigenic exposure, and other developmental cues [32–34]. Changes in the B-cell compartment are less well understood. In part, cell surface markers have only recently been defined, and functional correlates of phenotypically defined populations have lagged. Recent studies of aberrant B-cell phenotypes in adult systemic lupus erythematosus patients and patients with common variable immune deficiency have suggested that better pediatric B-cell subset normative data would be valuable [8,10]. There have been comparatively very few studies of B-cell subsets in children of different ages.

Previous efforts to define pediatric B cells have included analyses of relative and absolute CD19+ lymphoctye counts, which show an increase in B-cell counts between birth and 1–2 years of age, a comparatively high B-cell fraction between 2 and 5–6 years of age, and then a gradual decline to adult levels [19,20,22]. One large analysis of apparently healthy children showed that the average absolute B-lymphocyte count increases from approximately 600/mm³ at birth to approximately 1400/mm³ at 2 years of age and then decreases, leveling off at approximately 200/mm³ in adults [19]. Similarly, in the analysis presented here, the peak total B-cell count occurred at approximately 1 year of age and declined thereafter. In a previous study, the average B-lymphocyte fraction, expressed as a percentage of cells in the lymphocyte gate, was 12% in neonates, climbed to approximately 25% by the age of 2–5 months, stayed fairly level until approximately age 5 years, and then gradually declined, returning to approximately 12% in adults [19]. Similar findings were reported in Saudi Arabian [20] and Chinese children [22]. Consistent with these studies, we observed a decline in the B-cell fraction in early childhood. The rapid decline in T-cell numbers is quantitatively similar and has been proposed to reflect thymic involution. The mechanism driving the decline in B-cell counts is not known. Our data also suggested a mild increase in total B-cell counts in the 1st year of life, and this too has been reported previously [35–37].

In this study, we correlated the changes in different peripheral B-cell subsets in children of different ages. We observed that transitional B cells and naïve mature B cells had similar patterns of decline with age, showing a logarithmic decline in absolute counts, as well as a steady decline in the percentage of cells. Conversely, IgM+ and IgM− CD27+ B-cell fractions increased with age. In one previously published analysis, IgD and CD27 were used to separate circulating B cells into naïve (CD27−, IgD+) and memory subsets (CD27+, IgD+ or CD27+, IgD−). Our subsetting schemes with IgM and CD27 and IgM and IgD are similar and, like the previous study, show that the relative proportion of CD27+ B cells increases with age in children. We also evaluated CD5 expression as a function of age and observed that CD5 levels were highest in the youngest patients, who also tended to have the highest proportions of transitional B cells. Conversely, in older children, CD5 stained both less mature and more mature B-cell subsets, based on differential IgD expression. Two large studies have evaluated CD5 expression in peripheral B cells from Italian children, most of whom were born to human immunodeficiency virus (HIV)-positive mothers and on whom their physicians had ordered immunophenotyping. In children with normal immunophenotyping profiles and no evidence of HIV infection, these authors observed a decline in CD5+ B cells with age and a shift in CD27 expression (from CD27− in younger children to CD27+ in older children). This was interpreted as maturation of the B-1a subset, although an alternative, nonmutually exclusive possibility is that most of the CD5+ B cells in the CD27− subset are transitional cells that could give rise to CD5− naïve mature and memory B-cell populations. However, these comments should be regarded as speculative, because immunophenotyping studies are static rather than kinetic measurements. It is not possible to make definitive claims on how one peripheral subset interacts with or becomes another based on immunophenotyping data alone.

Although we believe this study represents a good starting point in the establishment of normative values of B-cell subtypes, there were some limitations to the analysis. The sample size was limited and the age distribution was uneven among the various age ranges. Additionally, the children were identified as being clinically well but may have had subclinical conditions that could affect the results of the phenotyping. In addition, the study does not address the stability of the subsets over time in a given individual, a topic that will require a much more extensive analysis.

In conclusion, we were able to observe age-related trends in data for total B-lymphocyte numbers and B-cell subsets, including transitional, naïve mature, resting memory, and class-switched memory cells. There were too few plasmablasts for robust analysis. As flow cytometric analysis becomes more widely available and examination of the B-cell subsets becomes standard practice, these normative data should be useful. Potential uses include identification of primary immune deficiencies [38] and autoimmune disease [9] and the examination of consequences of prematurity.