Phenotypic perturbation of B cells in the Wiskott-Aldrich syndrome

Abstract

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency/platelet disease due to mutations of WASP, a cytoskeletal regulatory protein of blood cells. Patients exhibit a range of immune defects generally attributed to defective T-cell function, including poor response to immunization, skewed immunoglobulin isotypes, eczema, recurrent infections, autoimmune disease and increased frequency of malignancies. Here we show a deficit of total B-cells in WAS patients of various ages and identify phenotypic perturbations involving complement receptors and CD27. Whereas B-cells of normal healthy donors are overwhelmingly CD21/CD35-positive, B-cells expressing these receptors are significantly reduced in number in WAS patients, and their paucity may cause suboptimal antigen capture and presentation. The frequencies of IgD(-) and IgG(+) patient B-cells were not different from healthy donors (although absolute numbers were decreased), indicating that isotype switching is occurring. In contrast, the frequency of cells positive for CD27, the marker of post germinal centre B-cells, was significantly decreased even among isotype-switched cells, and B-cells resembling germinal centre progenitors (CD10(+)CD27(-)CD38(bright)) were more frequent in adult patients, suggesting impaired germinal centre maturation/differentiation. The documentation of these phenotypic perturbations and deficit of total cells suggest that defects intrinsic to B-cells contribute to the impaired humoral immunity that characterizes this disease.

Fig. 1

Peripheral blood B cell counts. Number of B cells (CD19⁺ cells) ( × 10⁶/ml) in peripheral blood of normal healthy individuals (○) and WAS patients (•) grouped according to age: infants, 0–36 months; children, 3–17 years; and adults, 18–55 years. Bars indicate mean cell counts. Note that the scale for adults differs from that for infants and children. These data are a composite of previously published [15] and new values. The difference in B cell counts for patients versus normal was statistically significant (P < 0·05) within each age group (Student's unpaired t-test).

Fig. 2

Increased frequency of complement receptor negative B cells in WAS patients. Frequency of CD21^–cells (a) and CD35^– cells (b) as a percent of total B cells (CD19 gated cells) for normal healthy individuals (○) and WAS patients (•). (c) Representative dot plots (from analysis of 11 patients) of CD35 and CD21 expression on B cells of a normal individual (an adult) and a WAS patient (12 years). (d) Correlation of CD21-negativity and CD35-negativity of B cells of 19 patients.

Fig. 3

Characterization of CD21^– patient B cells. (a–c) Dot plots of gated B cells of a normal healthy individual (left, 7 months) and a WAS patient (right, 18 months) stained for CD21 and (a) CD10 (b) CD62L, and (c) CD38. The majority of patient CD21^– B cells are negative for CD10, a marker of immature bone marrow B cells, and negative for CD62L and CD38, markers of blood plasma cells. (d) Representative histogram showing larger size of patient CD21^– B cells compared to CD21⁺ B cells.

Fig. 4

Immunoglobulin isotype expression. Isolated PBMCs of normal healthy individuals and WAS patients, 15 years of age or older, were stained for IgD and IgG. (a) Representative dot plots gated for CD19⁺. (b) Frequencies of IgD^– (left) and IgG⁺ (right) B cells.

Fig. 5

Deficit of CD27⁺ postgerminal centre B cells in WAS patients. (a) Frequency of CD27⁺ B cells of normal healthy individuals (○) and WAS patients (•). Whole blood staining results are shown. Note the age-dependent increase in CD27⁺ B cells for the normal healthy group but not for WAS patients. (b,c) Expression of CD27 as a function of surface IgD and IgG on PBMCs of normal healthy individuals and WAS patients 15 years and older. (b) Representative dot plots of CD19⁺ gated cells stained for CD27 and IgD. (c) Decreased frequencies of CD27⁺ B cells within IgD^– and IgG⁺ B cell subsets. CD27⁺ B cells were also significantly decreased within the IgD⁺ population (15 ± 2·5% for normal donors versus 6·6 ± 2·3% for patients; data not shown).

Fig. 6

Frequency and characteristics of CD10⁺ B cells in adult WAS patients. (a) Frequency of CD10⁺ B cells of normal individuals (○) and WAS patients (•). The frequency of these cells is significantly increased in adult patients. (b) Representative dot plots of B cells of a normal healthy adult (left, 40 years old) and an adult patient (right, 40 years old) stained for CD27, CD38, and CD10. CD10⁻ cells are shown as red dot events. CD10⁺ cells, shown as blue dot events, are seen as CD27^– and CD38^bright. The near absence of CD27⁺ B cells is conspicuous in the patient. Similar results were obtained for two additional comparisons of adult patients and normal healthy adults.