Decreased frequency and activated phenotype of blood CD27 IgD IgM B lymphocytes is a permanent abnormality in systemic lupus erythematosus patients

Abstract

Introduction: Systemic lupus erythematosus (SLE) is characterized by B cell hyper-activation and auto-reactivity resulting in pathogenic auto-antibody generation. The phenotypic analysis of blood B cell subsets can be used to understand these alterations.

Methods: The combined detection of CD19, CD27 and IgD (or IgM) by flow cytometry (FC) analysis delineates five well-defined blood B cell-subsets: naive, switched (S) memory, double negative (DN) memory and CD27 IgD IgM (non-switched memory) B lymphocytes, and plasma cells (PCs). This phenotypic study was performed in 69 consecutive SLE patients and 31 healthy controls.

Results: SLE patients exhibited several abnormalities in the distribution of these B cell subsets, including elevated levels of DN memory B cells and PCs, and decreased CD27 IgD IgM B cells. Active SLE patients also showed decreased presence of S memory B cells and increased proportions of naive B lymphocytes. Nevertheless, when the patients in remission who did not require treatment were studied separately, the only remaining abnormality was a reduction of the CD27 IgD IgM B cell-subset detectable in most of these patients. The level of reduction of CD27 IgD IgM B cells was associated with elevated values of serum SLE auto-antibodies. Further analysis of this latter B cell-subset specifically showed increased expression of CD80, CD86, CD95, 9G4 idiotype and functional CXCR3 and CXCR4.

Conclusions: The presence of a reduced blood CD27 IgD IgM B cell-subset, exhibiting an activated state and enriched for auto-reactivity, is a consistent B cell abnormality in SLE. These findings suggest that CD27 IgD IgM B lymphocytes play a role in the pathogenesis of this disease.

Figure 1

Comparative analysis of blood B cell-subsets from SLE patients and healthy controls. CD19+ cells can be distributed into different B cell-subsets according to their additional expression of CD27 and IgD. A. Representative CD27-IgD dot plots from a control and a SLE patient indicating the distribution and percentages of these B cell-subsets: CD27-IgD+ (naïve), CD27+IgD- (S memory), CD27+IgD+ (CD27 IgD IgM), and CD27-IgD- (DN memory) B lymphocytes, and CD27^highIgD^- cells (PC). B. Scatter plots represent the percentages of these B cell-subsets in 31 controls (open circles) and 69 SLE patients (closed circles). The mean of each set of values is shown as a horizontal line. P values (Student's t test) are included. C. Scatter plots represent the percentage of each B cell-subset in controls and in SLE patients distributed according to disease activity (12 active and 57 inactive), and to the treatment (54 treated and 14 untreated). Symbols representing the control and the different SLE subgroups are displayed (bottom right panel). P values were calculated for the difference between the two pairs of SLE subgroups and between SLE subgroups and the control group. P values lower than 0.05 were considered statistically different (indicated as *).

Figure 2

Comparative analysis of blood CD27 IgD IgM B cell-subsets from healthy controls and different groups of SLE patients according to the presence of autoantibodies. A. B cell subsets from healthy controls (n = 31) and SLE patients with negative or low levels (IIF titre ≤1/160; n = 23) and medium or high levels (IFF titre ≥1/320; n = 46) of ANA were compared. B and C. Comparison among healthy controls and SLE patients with anti-dsDNA negative (n = 46) or positive (n = 23) and anti-ENA negative (n = 41) or positive (n = 28) Ab, respectively. Differences between groups are indicated (* P < 0.05; ** P < 0.01).

Figure 3

Comparative phenotypic analysis of blood CD27 IgD IgM B cell-subsets (CD27+IgD+) from controls and SLE patients. This B cell-subset was examined for the surface expression of different molecules. A. Representative histograms depict the expression of IgM, CD80, CD86, CXCR3, CXCR4 and CD95 for a healthy control and a SLE patient. For each baseline plot, negative isotypic antibody controls are superimposed in dotted lines. B. Bar histograms show the percentages of positive CD27+IgD+ B cells (upper) and the mean fluorescence intensity (lower) for each marker in healthy controls (N = 10; open bars) and SLE patients (N = 10; grey bars). C. Expression of 9G4 idiotype in healthy controls (N = 6) and SLE patients (N = 22) is shown. A representative histogram is shown. Results represent the mean ± SEM. P values were calculated using the Mann-Whitney test. P values lower than 0.05 were considered statistically different (marked with an asterisk). D. A comparison is shown of the chemotaxis of CD27 IgD IgM B cells from healthy controls and SLE patients induced by CXCL12, CXCL10 and CCL3. Migration in the absence of stimuli is represented as a dotted line. Results are expressed as a migration index and represent the mean ± SEM (N = 4). P-values were calculated using the Mann-Whitney test. P < 0.05 was considered statistically significant. Asterisks represent significant differences between healthy controls and SLE patients. Hashes indicate significant differences in chemokine-induced migration with respect to medium alone.

Figure 4

Comparative phenotypic analysis of different blood B cell-subsets from controls and SLE patients. Indicated B cell-subsets were examined for the surface expression of several molecules including CD80, CD86, CXCR3, CXCR4, CD95 and 9G4. Bar histograms show the percentages of positive B cells (mean ± SEM) for each marker in healthy controls (N = 10; open bars) and SLE patients (N = 10; grey bars). P values were calculated using the Mann-Whitney U test, and statistically significant p values are marked with an asterisk (P < 0.05).