Expression of CD27 and CD23 on peripheral blood B lymphocytes in humans of different ages

Abstract

Background

Due to the fact that the coexpression of CD23 and CD27 has been reported to occur in B lymphocytic leukaemic clones and that there is debate about CD23 expression on memory B cells, we evaluated the behaviour of naive B cells (CD23−/CD27−) and memory B cells (CD27+) in the peripheral blood of a large number of humans of all ages. B cells were also distinguished into B2 (CD5−) and B1-a cells (CD5+).

Methods

The cell surface expression of CD19, CD5, CD23 and CD27 was assessed on peripheral blood lymphocytes from 1,427 subjects of all ages undergoing peripheral blood immunophenotyping for a variety of reasons.

Results

The absolute number of B lymphocytes and the percentage of naive cells (CD23−/CD27−) decreased with age whereas there was an increase in memo ry cells (CD27+). A small subset of B cells co-expressing CD23 and CD27 was present in humans of all ages, although the majority of CD27+ cells were CD23−. The percentages and rate of increase with age of B1-a CD23+/CD27+ were slightly higher than those of B2 cell counterparts.

Conclusions

On the basis of our data, age-associated changes in surface markers of B cells seem to be finely balanced and probably related to functional changes after antigen encounters, while the whole peripheral blood B-cell compartment undergoes a quantitative regression.

Introduction

B-lymphocytes are characterised by the expression of CD19 surface antigen, which is present early on bone marrow progenitor cells and persists during all stages of B-cell maturation¹. This cell population does, however, include phenotypically and functionally different subgroups. The largest subgroup is formed of B2 cells, or conventional B cells, which account for about 10% of peripheral blood lymphocytes in humans². The remaining B cells are described as B1 cells and can be distinguished by the presence of CD5 into as B1-a cells (CD5⁺) and B1-b cells (CD5⁻ )^3–5.

As regards the functional differences of these B-cell populations, B2 lymphocytes are largely mediators of adaptive immunity and are located in the follicular zone; they respond to conventional protein antigens, with the help of T cells, and undergo antibody class switching and affinity maturation⁶. An additional marker on these cells, CD27, indicates the transition from naive B cells to memory cells⁷. B1 cells, on the other hand, are considered to be effectors of innate immunity; they respond to T-cell-independent antigens, mainly carbohydrates, which are common to various infectious agents. In particular, B1-a cells spontaneously secrete IgM (natural antibodies)³, while the antibody production by B1-b cells is induced and long-lasting⁸.

Another marker, CD23, is a low affinity receptor for IgE. It is located at the surface of B cells and binds IgE/antigen complexes to be presented to specific CD4⁺ cells. Thus, CD23 enhances the antibody response⁹, promotes the production of IgE in its soluble form and inhibits the production of membrane IgE. CD23 is released by stimulated B cells¹⁰. It induces B-cell proliferation¹¹ and apoptosis¹². Some authors have reported that CD23 is expressed on activated B cells^13,14 whereas others have suggested that peripheral blood CD23⁺ B cells resemble classic memory cells¹⁵.

We previously reported that the numbers of B2 and B1-a peripheral blood lymphocytes vary with age¹⁶, there being a sharp increase of B cells mainly in the first year of life, followed by a progressive decrease. In addition to the functional changes of B-cell subsets due to antigen encounters, a decrease in the absolute number of B lymphocytes in elderly men has been reported, perhaps due to precursor depletion in the bone marrow¹⁷ and altered T-cell activity¹⁸.

In this study we evaluated the behaviour of naive and memory B cells, distinguished by the expression of CD27, in the B2 cell subset and that of the B1-a cells belonging to the natural or innate immune memory compartment¹⁹. We also evaluated the co-expression of CD27 and CD23 on the two B-cell subsets during aging, since there is debate about CD23 expression on memory B cells and, moreover, the co-expression of CD23 and CD27 has been reported to occur in lymphocytic leukaemic clones.

Patients and Methods

Patients

Between January 2001 and December 2006, we assessed the cell surface expression of CD19, CD5, CD23 and CD27 on peripheral blood lymphocytes from 1,427 subjects of all ages undergoing peripheral blood immunophenotyping for a variety of reasons at the University Hospital of Verona. The median age of the patients was 37 years (range: 0–85 years). There were 675 males and 752 females (male/female ratio: 0.89).

Broadly speaking, the majority of infants were born from HIV-positive mothers; the children were affected by recurrent infections, allergies, autism and Down's syndrome; the young adults suffered from recurrent infections, allergies, fever, and autoimmune diseases; the middle-aged adults and elderly people were affected by autoimmune diseases and, to a lesser extent, by cancer.

Subjects with known HIV infection and those with evidence or a history of haematological malignancies were excluded from evaluation. On the assumption that any deviations from normality would compensate for each other given the large amount of data, no other exclusion criteria were applied²⁰.

The subjects were divided into 11 age groups: less than 1 month (group 1; 18 cases), from 1 month to less than 1 year (group 2; 60 cases), from 1 year to less than 3 years (group 3; 52 cases), from 3 years to less than 6 years (group 4; 64 cases), from 6 years to less than 12 years (group 5; 93 cases), from 12 years to less than 24 years (group 6; 124 cases), from 24 years to less than 36 years (group seven; 235 cases), from 36 years to less than 48 (group eight; 433 cases), from 48 years to less than 60 years (group nine; 207 cases), from 60 years to less than 72 (group ten; 112 cases), and from 72 years to 85 years (group eleven; 29 cases).

Blood specimens

Peripheral blood samples were collected into EDTA Vacutainers (K2E) between 9 and 12 a.m. and processed within 4 h of being taken.

Four-colour staining

Human peripheral blood leucocytes were stained as previously described²⁰. Four directly conjugated antibodies (5 μL each), labelled with CD5-CY5, CD19-APC, CD27-FITC and CD23-PE were added to 50 μL of EDTA-whole blood and incubated for 15 min in the dark at room temperature. We used a double platform based on the whole blood "Lyse/No-Wash" protocol that avoids the potential loss of cells during washing steps²¹. Full blood cell counts were performed in an automated blood analyser (Technicon H.3RTX or ADVIA 120, Bayer, Tarrytown, USA). Sample EDTA Vacutainers (K2E) tubes, FACS Lysing Solution, and monoclonal antibodies for staining the specimens were purchased from Becton Dickinson, Italy. Non-relevant isotype control monoclonal antibodies were used to determine background fluorescence levels.

Flow cytometric analysis

Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson BD Biosciences) equipped with a 488-nm argon laser and a 635-nm red-diode laser. The setting was performed with linear amplification (1024 channels) of the forward-scatter (FS) and side-scatter (SS) signals and logarithmic amplification (4 log decades) of the FL1 and FL3 signals. Compensation parameters for multiparametric analysis were adjusted at the start of sample acquisition. A total of 10,000 events in the lymphocytes gate were acquired. The immunological gating strategy used CD19 versus FS to define B lymphocytes, whereas absolute count was referred to the gate defined on morphological parameters (SS versus FS) and total leucocyte count. Data were acquired and analysed using CellQuest software (Becton Dickinson BD Biosciences).

For the assay, the threshold was set on FS in a dot plot of FS vs. SS, to exclude debris. Sequential gating strategies were established to define the distinct CD19⁺ peripheral blood lymphocytes subsets: CD19⁺/CD5, CD19⁺/CD5⁺ and CD19⁺/CD5^−/CD23⁺/CD27⁻, CD19⁺/CD5^−/CD23⁺/CD27⁺, CD19⁺/CD5^−/CD23^−/CD27⁺, CD19⁺/CD5⁺/CD23⁺/CD27⁻, CD19⁺/CD5⁺/CD23⁺/CD27⁺, CD19⁺/CD5⁺/CD23^−/CD27⁺. A complete haemogram was performed in all patients.

Results

The percentage of B lymphocytes among total lymphocytes was low at birth, doubled in the first year of life and decreased continuously thereafter. Within the B lymphocyte population, the majority of cells in neonates were CD23^−/CD27⁻, with this subset then decreasing continuously throughout life. The percentage of CD23⁺/CD27⁻ B cells increased until 12 years and then remained substantially stable, while the percentage of CD23^−/CD27⁺ B cells increased continuously with age. A small percentage of CD23⁺/CD27⁺ B cells was detected, with this subset also showing a mild tendency to increase with age (Figure 1a).

Figure 1a.

Percentage of total B cells expressing CD23 and CD27 in age

The percentage of CD5⁺ B cells (B1-a cells) decreased from birth onwards; B1-a cells were the majority of B lymphocytes at birth and the minority in the elderly. The percentage of CD23^−/CD27⁻ B1-a cells decreased progressively with age, while the percentage of CD23⁺/CD27⁻ cells increased until middle age and then slowly decreased. In contrast, the percentage of CD23^−/CD27⁺ B1-a cells increased continuously. The percentages and rate of increase with age of B1-a CD23⁺/CD27⁺ cells were slightly higher than those of the B2 cell counterparts (Figure 1b).

Figure 1b.

Percentage of B-1a cells expressing CD23 and CD27 in age

The percentage of CD5⁻ B cells (B2 cells) increased from birth onwards.

The percentage of the CD23⁺/CD27⁻ cells increased until middle age and decreased slowly thereafter. In contrast, the percentage of CD23^−/CD27⁺ B2 cells increased with age, as did the small percentage of CD23⁺/CD27⁺ B2 cells (Figure 1c).

Figure 1c.

Percentage of B2 cells expressing CD23 and CD27 in age

The absolute number of total B lymphocytes increased in the first year of life and decreased thereafter; the absolute number of B2 cells also increased until the third year of life and decreased thereafter. The absolute number of all cell subsets (CD23^−/CD27⁻, CD23⁺/CD27⁻, CD23^−/CD27⁺, and CD23⁺CD27⁺), as well as total B and B2 lymphocytes increased in the first 2 or 3 years of life and then subsequently decreased (Figure 2a, 2c).

Figure 2a.

Absolute number of total B cells expressing CD23 and CD27 in age

Figure 2c.

Absolute ngumber of B2 cells expressing CD23 and CD27 in age

Finally, the absolute number of total B1-a cells, like all the B1-a subsets (CD23^−/CD27⁻, CD23⁺/CD27⁻, CD23^−/CD27⁺, and CD23⁺/CD27⁺), increased in the first year of life and decreased thereafter (Figure 2b).

Figure 2b.

Absolute number of B-1a cells expressing CD23 and CD27 in age

As shown in Figure 3, among total B lymphocytes, the expression of CD23 antigen is alternative to the expression of CD27: only a small group of B cells co-express both antigens, while the majority of memory cells (CD27⁺) are CD23⁻.

Figure 3.

Among total lymphocytes in B cell in aging the expression of CD23 antigen is alternative to the expression of CD27 and the co-expression of both antigens is present only in a small group of B cells, while the majority of memory cells (CD27⁺) are CD23⁻.

Discussion

Peripheral blood B cells seem to undergo a dynamic process during a person's lifespan, with naive cells being activated and converted into memory cells. Although it is well established that CD27 is expressed by memory B cells, there is a debate about CD23 expression on these cells^22,23. CD23 has been described to be an acquired marker of activated B cells^15,24,25, to be cleaved after antigen interactions¹⁰, and to stimulate both B-cell proliferation²² and apoptosis²³. Our data indicate that although CD23 and CD27 surface antigens are co-expressed in a small group of B cells, the majority of memory cells (CD27⁺) are CD23⁻. Indeed, CD23 and CD27 antigens seem to be mutually exclusive; the expression of the former might promote cleavage of the latter, with both antigens possibly being only transiently expressed on the same cell. Alternatively, despite the low number of B cells bearing both antigens, CD27 and CD23 might be co-expressed in a definite subset of peripheral blood B cells or in a transient stage of maturation. Since the absolute numbers of CD23⁺/CD27⁻ B cells were higher than those of CD23^−/CD27⁺ cells in all ages, these former cells might represent transitional B cells²⁶, i.e. B cells migrating from bone marrow to secondary lymphoid tissues. The dynamics of the expression of CD23 and CD27 on peripheral blood B cells with ageing is similar when analysed in the whole B-cell compartment or in the B2 and B1-a subsets separately, indicating a uniform pathway of maturation. Our data do not confirm the lack of expression of CD23 on B1-a cells previously reported in mice²⁷ or the hypothesis that the co-expression of CD23 and CD27 on B cells, particularly on B1-a cells, occurs only in leukaemic clones^28,29. While the absolute number of B cells increased in both subsets in the first year of life and decreased subsequently, the percentage of B1-a cells among total peripheral blood B lymphocytes decreased continuously with age. In fact, while B1-a cells constitute the majority of peripheral blood B cells in the foetus, in cord blood³⁰ and in infancy, they are the minority in the elderly¹⁶. B1 cells, which include B1-a cells, are believed be the primary source of natural IgM, produced also in the absence of exogenous antigenic stimulation³. Conversely, B2 cells, or conventional B cells, are responsible for adaptive immunity, producing IgA or IgG high affinity antibodies and providing lasting protection against pathogens. As a consequence, B1 cells might play a prominent role in defence against common pathogens early in life when antigens are first encountered, but their importance progressively decreases in cases of re-exposure to the same antigen.

In conclusion, on the basis of our data, age-associated changes in surface markers of B cells seem to be finely balanced and probably related to functional changes after antigen encounters, while the whole peripheral blood B-cell compartment undergoes a striking quantitative regression.