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

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical manifestations characterized by B lymphocyte hyper-reactivity and formation of pathogenic auto-antibodies (Ab). The nature of the immune alterations causing this disease remains elusive. The occurrence of marked B cell activation and auto-reactivity leads to consideration that one, or more, of the physiological checkpoints controlling the self-antigen recognizing B cell receptor (BCR) repertoire, and the triggering and maturation of B lymphocytes, might be affected [1]. In an attempt to gain a deeper knowledge on the patho-physiology of human SLE, blood B lymphocyte subsets have been extensively explored. In this regard, when blood B cells are stained with labeled-monoclonal Ab (mAb) anti-CD19, -CD27 and -IgD (or -IgM), and analyzed by three-color flow cytometry (FC), five well-defined B cell-subsets can be identified [2]; these are: 1) naive B cells (CD19+ CD27- IgD+); 2) non-switched memory B cells, also termed CD27 IgD IgM B cells (CD19+ CD27+ IgD+); 3) conventional or switched (S) memory B cells (CD19+ CD27+ IgD-); 4) a small proportion of double negative (DN) memory B cells (CD19+ CD27- IgD-); and 5) plasma cells (CD19+ CD27++ IgD-). From a comparison of these circulating B cell-subsets between healthy controls and SLE patients, the existence of several B cell alterations in this disease has been determined. These alterations include B cell lymphopenia, increased proportion of plasma cells (PC) and DN memory B cells, decreased naive B lymphocytes and, more recently, decreased CD27 IgD IgM B cells [3–7]. The present study reveals that a marked decrease of this latter B cell-subset is a consistent and permanent blood B cell aberration in SLE patients. Further results indicate that this circulating cell-subset is enriched for cells exhibiting auto-reactivity and activation features.

The present study demonstrates that the distribution of blood B cell-subsets in SLE patients exhibits a variety of alterations including elevated proportions of DN memory B lymphocytes and PC, and decreased CD27 IgD IgM B lymphocytes. In addition, active patients show decreased S memory and increased naïve B lymphocytes, indicating that patients in more active phases exhibit more abnormalities in the distribution of these B cell-subsets, and these abnormalities are more pronounced. These data are consistent with previous reports [3, 5–7] and, taken together, indicate that, in SLE patients, the B cell compartment undergoes profound alterations and imbalances that are noticeable in the distribution of the normal B cell-subsets present in the circulation. Moreover, the results observed in the group of patients in remission (untreated patients, Table 2) show that a decreased proportion of CD27 IgD IgM B lymphocytes is the only B cell-subset alteration persisting in the majority of these cases. Serum SLE auto-Ab are relevant clinical parameters in this disease. Present data reveal that the level of elevation of these auto-Ab correlates with the intensity of reduction of CD27 IgD IgM B lymphocytes in this disease. Further analysis indicates that CD27 IgD IgM B cells from SLE patients exhibit higher expression of CD95, CD80, CD86, CXCR3 and CXCR4. Chemotaxis assays confirm that the increased expression of CXCR4 and CXCR3 observed in SLE CD27 IgD IgM B cells is functional, as the migration capacity to the appropriate ligands exhibited by these lymphocytes is higher in SLE patients' than in healthy controls. Increased CD95 expression has been previously reported in blood DN memory B cells from SLE patients [11]. Present results show that this observation can be also extended to CD27 IgD IgM B cell-subset (Figures 1 and 4). In addition, increased expression of CD80 and CD86 in certain SLE circulating B lymphocytes has been previously reported [12], although the use of a B cell marker selection profile different from that employed in the present study makes the comparison of these results difficult. Present data are consistent with the notion that the SLE patients' CD27 IgD IgM B cells distinctively are in an activated state, exhibiting a potentially higher capacity to migrate and to interact with T cells. Taken together, the present findings indicate that a decreased proportion of blood CD27 IgD IgM B lymphocytes appears to be an alteration that is permanent in SLE patients, irrespective of disease activity; in addition, these cells exhibit an activated phenotype. These observations suggest that CD27 IgD IgM B cell-subset might play a role in the patho-physiology of SLE.

Previous studies have established that the repertoire of mature naïve B lymphocytes is enriched for self-reactive clones in SLE patients [13, 14]. This observation indicates that SLE patients exhibit defective B cell tolerance checkpoints present in normal subjects [15] and, in consequence, the emergence of an increased frequency of these auto-reactive naive B cells is probably relevant in the pathogenesis of SLE. However, a possible role of CD27 IgD IgM B lymphocytes in the state of auto-reactivity characteristic of this disease has been less studied. This is, at least in part, due to the fact that the origin, nature and functional significance of normal CD27 IgD IgM B cells remain subject of debate [7, 9, 10]. These lymphocytes were defined as non-switched (IgM+ IgD+) memory B cells based on the findings that they express the putatively memory B cell marker CD27 [2], and harbor IGV genes exhibiting a low but detectable number of somatic mutations, an event classically restricted to post-germinal center (GC) memory B cells [2, 9]. Nevertheless, the observation of normal quantities of mutated CD27 IgD IgM B lymphocytes in immunodeficient patients that lack GC formation and conventional switched memory B cells, indicates that the generation of the cell-subset under study can be GC-independent; hence, the IGV gene somatic mutations present in this B cell-subset are thought to represent a pre-immune diversification process [9, 16]. Human CD27 IgD IgM B lymphocytes have been associated with the spleen marginal zone, and they appear to be involved in the production of natural antibodies [9, 16]. The mechanisms that determine the somatic mutations occurring in CD27 IgD IgM B cells remain to be clarified, although it has been recently shown that this B cell subset can express activation-induced cytidine deaminase (AID), either during a transient post-natal phase [17], or upon Toll Like Receptors (TLR)-engagement [18]. Under this latter condition, CD27 IgD IgM B cells are capable of differentiating into IgG-secreting plasma cells [18]. Interestingly, normal human CD27 IgD IgM B lymphocytes exhibit a frequency of auto-reactive BCR much lower than that observed in naive B lymphocytes, suggesting that an additional checkpoint for preventing self-reactivity exists at this level [19]. In this context, it is reasonable to think that a failure of this BCR control process would necessarily lead to the emergence within this B cell-subset of auto-reactive clones. It is well-established that the immunoglobulin (Ig) idiotype recognized by the 9G4 mAb is enriched in auto-reactive B cell populations, including anti-DNA Ab-bearing B cells occurring in SLE patients [20]. Accordingly, the presence of B lymphocytes containing the 9G4 idiotype in their surface Ig was examined in the blood CD27 IgD IgM cell-subset. Present data reveal that the frequency of 9G4+ cells detected in this B cell-subset was, on average, five times higher in SLE than in controls. This result indicates that SLE patients show a defective control on the appearance of auto-reactive clones within the circulating CD27 IgD IgM B cell-subset. The cause of this failure remains unknown. It is conceivable that these auto-reactive cells, after appropriate self-antigen recognition, maybe in combination with TLR-activation [18], would undergo activation and migration toward lymphoid tissue where they could progress into further differentiation, giving rise to auto-reactive switched memory B cells and PCs. This explanation is consistent with the findings reported here. Thus, a permanent decrease of circulating CD27 IgD IgM B cells could be the result of self-antigen activation of auto-reactive B cells contained in elevated proportions in this subset, and their subsequent recruitment into lymphoid tissues. In fact, 9G4+ activated B cells and PCs have previously been detected in lymphoid tissues (spleen, tonsil GC and bone marrow) from SLE patients, but not from normal subjects [21]. In conclusion, CD27 IgD IgM B lymphocytes might play a role in the complex B cell alteration causing SLE.

This study shows that the presence of a reduced blood CD27 IgD IgM B cell-subset, exhibiting an activated state, an increased capability to migrate towards CXCR4 ligand and enriched for auto-reactivity is a prominent B cell abnormality in SLE. In addition, higher levels of ANA, anti-dsDNA and anti-ENA Ab were associated with lower numbers of CD27 IgD IgM B cells in SLE patients. These findings suggest that CD27 IgD IgM B lymphocytes play a role in the pathogenesis of this disease.