The data presented herein are consistent with the presence of anergy in a significant fraction of human naïve B cells as documented by low Ca++
flux, attenuated proliferation, and substantially decreased ability to secrete antibody in response to both BCR crosslinking and BCR-independent stimulation (CD40L + IL-21). These cells are characterized by low surface expression of IgM, absence of activation markers, increased autoreactivity, and abnormal expression of inhibitory receptors as compared to control naïve B cells, which express higher levels of sIgM and respond more vigorously to BCR cross-linking. Of note the hypo-response observed to BCR stimulation was not due solely to the decreased levels of sIgM since similar attenuated responses were obtained in response to anti-IgD stimulation. Rather, our results indicate that, as was observed in mouse anergic B cells (29
), the level of sIgM determines the threshold of response to stimulation.
Interestingly, intracellular staining detects similar levels of cytoplasmic IgM in IgMlo
cells (supplemental fig. 3A–C
) indicating that decreased sIgM levels are not due to decreased transcription (as also supported by the sustained level of sIgD). Instead, these observations are consistent with increased IgM internalization and/or defective transport to the cell surface reported by Bell et al.
) These mechanisms have also been invoked to explain low sIgM levels in anergic transgenic B cells, a phenotype that can be reversed when the responsible self-antigen is removed from the system. Our results indicate that similarly, human IgMlo
cells are able to up-regulate sIgM after resting for 48 hours in culture medium in the absence of stimulation (supplementary figure 3C
). These conditions would also preclude IgMlo
cells from engaging self-antigens that might have been present in vivo, other than those present in the surface of naïve B cells themselves or those released from dead or apoptotic cells. Given that we observed negligible levels of cell death/apoptosis in the 48-hours culture (not shown), our results suggest that down-regulation of sIgM may be secondary to chronic engagement of self-antigen and may be reversed by interrupting this interaction.
As we and others have established (7
), transitional cells share some of the properties assigned here to anergic naïve B cells (including attenuated BCR responses, hypo-proliferation to TLR9 stimulation, and increased autoreactivity), and the differentiation between these populations may be difficult because the surface phenotypic markers typically used (IgM, IgD, CD24, CD38 and CD10) are expressed as a continuum in transitional and naïve populations. The mature nature of IgMlo
cells, however, is supported by their competency to extrude rhodamine or mitrotracker, a property characteristic of mature naïve B cells owing to their expression of the ABCB1 transporter (15
maturity is also supported by the higher expression of CD22 and by the differential up-regulation of this marker in response to BAFF stimulation. Collectively, our results are consistent with the recent description of An1 anergic B cells in wild-type mice rather with a T3 transitional phenotype (6
CD22 is a critical inhibitory receptor that becomes operational in mature follicular B cells and is reduced upon B cell activation (22
). In this compartment, CD22 determined the threshold for BCR-stimulated activation and is critical for the enforcement of B cell tolerance. In keeping with this model, CD22 deficiency may result in autoimmunity (32
). Thus, our finding of increased CD22 levels in IgMlo
cells strongly indicates that, also in keeping with signaling, proliferation and antibody secretion studies, these cells have a higher activation threshold as compared with other naïve cells with lesser degree of autoreactivity. IgMlo
cells expressed lower levels of CD19 and CD21, lacked expression of activation markers, and displayed attenuated up-regulation of some early activation markers in response to in vitro
stimulation. Collectively, this phenotype suggests that in healthy subjects IgMlo
cells represent cells chronically stimulated in vivo
, presumably by self antigens, leading to decreased levels of IgM and co-stimulatory BCR molecules. Also of interest, IgMlo
cells express lower level of CD32b suggesting that in these cells low levels of BCR signaling fail to recruit this important inhibitory receptor for the feedback inhibition of B cells engaged in productive antigen-specific responses (34
). These features also argue against the alternative possibility that sIgM down-regulation could reflect the recent BCR internalization in response to acute B cell activation. Instead, our data suggest that IgMlo
cells from SLE patients may represent acutely activated cells as indicated by increased CD95 and decreased CD22 expression.
While the relative decrease in CD19 and increase in CD22 should impose an enhanced threshold for activation of IgMlo
cells, thereby contributing to enforcing tolerance in autoreactive B cells, the up-regulation of CD80/86 observed in vitro
indicates that the state of unresponsiveness in IgMlo
cells is reversible; therefore, these cells represent a dangerous reservoir of potentially pathogenic autoreactive B cells. Although some transgenic models suggest that this danger can be minimized by the shortened lifespan of anergic B cells (35
), our data highlight the risk created by the persistence of these cells. Consistent with their lack of CD95 expression, IgMlo
cells did not appear to be pro-apoptotic, and in the absence of stimulation, on average 90% of the 18 hours cultured cells survived, which is similar to the survival of control cells (data not show).
Similar scenarios indicating the ability of anergic B-cell to be activated by both T-dependent and independent stimulation are well described in different animal models (17
). Antigen-activated, but not resting, naïve B cells can up-regulate co-stimulatory molecules and effectively serve as antigen-presenting cells (APC) that mediate cognate activation of T cells (40
). B cell APC activity may critically contribute to the initiation of T cell-mediated autoimmunity (43
), and therefore, failure to up-regulate critical T cell co-stimulatory molecules such as CD80 and CD86 has tolerogenic effects beyond the censoring of the B cells themselves. However, the ability of anergic B cells to express these co-stimulatory molecules has been inconsistent in different autoreactive transgenic models (17
). The unaltered up-regulation of CD86 in vitro
culture suggests that the APC activity of IgMlo
cells could be intact, as shown in the anti-insulin T125tg B cells (17
), and thus the pathway leading to anergy of IgMlo
cells may be distinct from the pathway leading to becoming an APC.
This manuscript expands the spectrum of anergic naïve B cells identified in humans. Indeed, our initial report on this tolerance mechanism identified anergic behavior in autoreactive 9G4-pos cells that represent 5–10% of all human naïve B cells in healthy subjects, and that display markedly depressed Ca++
responses upon BCR stimulation despite expressing intermediate levels of sIgM (46
). While other autoreactivities may also be involved in the censoring of 9G4 B cells (Jenks et al.
, in preparation), the main antigenic target of 9G4-pos B cells is represented by the blood group i antigen, which is also expressed by a CD45/B220 glycoform preferentially up-regulated in naïve B cells (47
). More recently, another population of human anergic naïve B cells has been reported. These cells, termed BND
, lack expression of surface IgM, account for about 2.5% of all B cells and are likely to represent the IgM-negative tail of the population studied in our work. Given that naïve cells represent 50–80% of all peripheral blood B cells, BND
cells would account for approximately 3–5% of all naïve cells. Of interest, BND
cells demonstrate significant autoreactivity against nuclear and cytoplasmic antigens in anti-nuclear antibody (ANA) testing as well as polyreactivity against two or more antigens (ssDNA, dsDNA, insulin, and LPS) when tested by ELISA (9
). A third type of human naïve anergic B cell has also been reported during the preparation of this manuscript. These cells, characterized by poor BCR responsiveness and autoreactivity similar to the one described above for BND
cells, are distinguished by the down-regulation of CD21 (CD21lo
cells). Of note, these anergic, autoreactive CD21lo
cells were identified in patients with CVID and in a subset of Rheumatoid Arthritis patients who may be deficient in receptor editing mechanisms. However, CD21lo
anergic cells did not seem to represent a substantial fraction of naïve B cells in healthy subjects (48
). Thus, previously reported anergic populations represent relatively small fractions of all naïve B cells, and therefore, could not account for the large fraction of autoreactive cells consistently identified in the healthy naïve B cell compartment (9
). Our data help fill this gap by identifying a larger subset of autoreactive naïve B cells with an anergic phenotype that are consistently identified in healthy subjects. Accordingly, this work contributes to the growing body of evidence identifying anergy as mechanism of tolerance enforcement in mature naïve cells that is present universally in healthy subjects. While our preliminary results suggest that this mechanism may be defective in SLE patients, larger and more detailed studies will be required to assess the participation of defective anergy in this and other autoimmune diseases.