One of the major roles of cells of the B-cell lineage is to generate antibody-secreting plasmablasts and plasma cells and also memory B cells with an enhanced capability to respond to the specific initiating antigen. These effector functions of the B lineage are well recognized and their roles in autoimmune diseases are accepted. Knowledge about the immunoregulatory role of B cells has also been substantially expanded within the last decade and their functions have been reconsidered. Historically, B cells have not been thought to play a major regulatory function in the development of autoimmunity and autoimmune diseases, although the identification of autoantibodies produced by autoreactive plasma cells and their pathogenic consequences are widely accepted. It is important to emphasize that B cells increasingly emerge as part of a tightly regulated immune activation process with numerous intimate interactions with other immunocompetent cells that have been identified. Thus, B cells are considered effector cells as well as cells with immunoregulatory potential. This review will consider B-cell involvement as both effector cells and immunoregulatory cells in the induction and maintenance of systemic autoimmunity and focus on human systemic lupus erythematosus (SLE) as a prototypic autoimmune disease.

Under normal resting conditions, B cells follow a tightly regulated life cycle (Figure 1) with a large number of check points at indicated stages (antigen-dependent and antigen-independent selection) to prevent the development of autoimmunity 1. In the bone marrow, B cells develop from stem cells through a series of precursor stages during which they rearrange their variable immunoglobulin (Ig) genes to generate a wide range of unique antigen-binding specificities. Immature CD10⁺ transitional B cells expressing surface IgM/IgD emigrate from the bone marrow into the peripheral blood and then mature into naïve B cells. In the mouse, this occurs in the spleen, although the site of maturation in humans is not known 2. After encountering antigen and T-cell help in follicles of secondary lymphoid organs, mature naïve B cells undergo germinal center (GC) reactions leading to their clonal expansion, somatic hypermutation of Ig gene rearrangements, and Ig heavy-chain class-switch recombination. Notably, these complex molecular processes are unique capacities of B cells and ensure specific higher avidity binding by the B-cell receptor (BCR) and also the production of antibodies with altered effector function. During the GC reaction, naïve antigen-specific B cells mature into either memory B cells or Ig-secreting plasma cells.

In mice, B1 B cells producing natural antibodies are important for the immediate defense against encapsulated bacteria. Whether they contribute to abnormalities of peripheral B cells in SLE 3 and primary Sjögren syndrome (pSS) 4 is not known. The reduced susceptibility of B1 B cells at mucosal sites after depletion by anti-CD20 therapy 5 suggests particular survival conditions of these cells in mice. The B1 B-cell equivalent subset and its role in human autoimmune diseases, however, remain to be delineated. Although there is an increase in CD5⁺ B cells in both SLE and pSS, these cells may represent an expended population of pre-naïve conventional B2 cells and not the human equivalent of B1 cells 6. In addition, CD5 can be regarded as a B-cell activation marker in humans and there are no convincing data providing evidence that it can be used as a lineage marker as accepted in mice.

On the other hand, B2 B cells comprise the adaptive portion of humoral immune responses. B2 cells participate preferentially in T cell-dependent GC reactions, during which they can hypermutate their IgV gene rearrangements, switch Ig classes, and differentiate into memory cells and long-lived plasma cells. However, B2 cells can also be activated during T cell-independent responses 7. B2 precursor cells are generated in the bone marrow and are subjected to central tolerance mechanisms. The immature survivors with functional BCRs leave the bone marrow and migrate to the periphery and are thought to be exposed to further selection (peripheral tolerance). Although it has been suggested that B2 B cells differentiate into either a mature follicular B-cell or a marginal zone (MZ) B-cell program 8, there are insufficient data that confirm this in humans. Alternatively, MZ B cells and B2 B cells can derive from precursor transitional 2 (T2) B cells that still need additional confirmation. Mouse models suggest that both B1 and MZ B-cell responses occur independently of T-cell help and B1 and MZ B cells are thought to be excluded from undergoing GC reactions. In conclusion, there appear to be substantial differences between mice and humans in terms of the specifics of the differentiation of B-lineage cells. Moreover, the distinct contribution of B1, B2, and MZ B cell-equivalent subsets in human systemic autoimmunity as well as the role of T cell-independent (TI) and T cell-dependent (TD) B-cell activation remain to be fully delineated. A more precise understanding of these processes in human autoimmunity would allow us a more targeted approach to treat specific autoimmune diseases.

Important for the interaction with T cells and the generation of GC reactions are a series of ligand-receptor interactions, including those mediated by CD154/CD40 and inducible co-stimulator ligand/inducible co-stimulator (ICOS-L/ICOS). Defects in these interactions have been shown to lead to hyper-IgM syndrome, resulting in impaired plasma cell and memory B-cell generation, including B lymphopenia and adult-onset common variable hypogamamglobulinemia, respectively 910. Moreover, the presence of certain cytokines, such as interleukin (IL)-6, lymphotoxin-β, IL-4, and IL-21, is required in order to facilitate specific stages of B-cell differentiation by providing essential co-stimulatory signals.

Because of the complexity of the abnormalities of immune regulation in systemic autoimmunity, a few key B-cell abnormalities will be highlighted since they provide insight into the nature of perturbations of B-cell function that could contribute to autoimmunity, either in a causative manner or as a pathway that amplifies disease. In this regard, it is not clear whether the identified abnormalities of B cells in SLE are intrinsic or secondary to the disturbed internal milieu characteristic of SLE.

Co-stimulation results from a complex mix of factors involved in effective immune activation, which includes antigen presentation, the provision of soluble and insoluble co-stimulatory factors as well as the anatomic organization of secondary lymphoid organs, including the GCs. One important function of memory B cells is antigen presentation, which is facilitated by the expression of high-avidity BCR and also major histocompatibility complex class II molecules. Under sufficient co-stimulation, memory B cells can effectively present antigen primarily to memory T cells. In mice, MZ B cells were even shown to provide co-stimulatory activating signals to naïve T cells 43. In addition, CD80/CD86 expression on B cells has been shown to be indispensable for the activation of autoreactive T cells in a murine arthritis model 44. The function of B cells as antigen-presenting cells has also been suggested by instructive data from animal models 45, in which a lupus-like disease developed when autoimmuneprone mice were reconstituted with B cells that lacked the ability to secrete Ig but not when they were deprived of B cells completely. These studies provided data on the possible role of antigen presentation by B cells and its pathogenic relevance.

Antigen presentation by B cells, in particular by memory B cells, may be important in the amplification and maintenance of autoimmunity after it has been initiated. Memory B-cell subsets in SLE 46 provide a sensitive pool of cells that immediately react to various stimuli, such as Toll-like receptor (TLR) ligands, IL-21, BAFF, IL-10, BCR activation, or co-stimulatory signals, resulting in the production of Ig-secreting cells. Although SLE memory B cells are mainly Ig-class-switched and show an activated phenotype, it remains to be determined whether these cells are also effective antigen-presenting cells. However, while the impact of individual stimuli is still a matter of debate, the decrease of IgM memory B cells 47 could represent the result of continuous memory B-cell activation by elevated BAFF, IL-21, or IL-10 levels 48495051.

Although B cells produce pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and IL-6, and immunoregulatory cytokines, such as IL-10, the immunologic relevance of B cell-derived cytokines is less clear. Furthermore, convincing evidence for a substantial production of interferon-alpha (IFNα), an important cytokine in SLE 52, by B cells has not been provided. However, it has been shown that IFNα is produced by tissue-resident plasmacytoid dendritic cells, which are reduced in the blood of SLE patients and may ultimately contribute to B-cell hyperactivity, especially in lupus nephritis. In this context, a direct effect of IFNα on B cells is less likely, but IFNα can induce BAFF/BLyS production of myeloid cells, which can result in enhanced B-cell survival and potentially activation 53. BAFF/BLyS or alternatively APRIL (a proliferation-inducing ligand), which belong to the TNF ligand family, increase survival of most B-cell subsets as well as plasma cells. Enhanced BAFF/BLyS levels could contribute to prolonged survival of autoreactive cells, which otherwise would be deleted. BAFF/BLyS and APRIL, therefore, may lead to a vicious cycle of continuous antibody-mediated inflammation and tissue destruction. Immune cells of mesenchymal origin produce functional BAFF/BLyS in response to pro-inflammatory cytokines (TNFα and IFNγ). Although BAFF transgenic mice 54 develop autoimmunity with an SLE/Sjögren phenotype, blockade of this cytokine in human SLE so far has not shown striking clinical effect but induced changes in Ig and B-cell levels. The regulatory control of BAFF production by IFNγ and TNFα 5355 in myeloid cells suggests that BAFF may act as a kind of secondary cytokine translating mainly IFN and TNF effects.

An important immunoregulatory cytokine produced by B cells is IL-10, which is able to activate dendritic cells to be more effective antigen-presenting cells and, with the help of T cells, to enhance the differentiation of memory B cells into plasma cells in the presence of IL-2 and which may likely be an important factor of the immunoregulatory function of B cells 56. Recently, it was suggested that triggering of TLRs can induce IL-10 production, which appears to play a role in protection from chronic inflammatory diseases. Therefore, immunoregulatory effects of B cells may result from their IL-10 production in autoimmune diseases. Such a regulatory function would have important implications for B-cell depletion therapies, which so far have not been substantiated by clinical trials. Whether low IL-10 levels after B-cell depletion ultimately lead to higher IFNγ and TNFα expression, translating into high BAFF levels, remains open.

With regard to the expression of co-stimulatory ligands involved in plasma cell and memory B-cell differentiation, it has been shown that T and B cells from SLE blood spontaneously express CD154, which is an indicator that GCs in this disease abnormally release activated lymphocytes into the periphery and implies the presence of overactive GCs 57 or insufficient control mechanisms allowing an egress of premature memory B cells. Similarly, CD4⁺ and CD8⁺ T cells from lupus patients show enhanced ICOS expression, whereas memory B cells downregulate ICOS-L, likely as a result of continuous interaction with T cells 58. Since these interactions of the CD28 family are important for memory B-cell and plasma cell generation but not for the formation of GCs, they are consistent with the conclusion that there is overactivated adaptive immunity in SLE and that this represents an important therapeutic target. All of these disturbances of B-cell subsets in adults and children with lupus with a predominance of memory B cells may also contribute to the increases of plasmablasts during active lupus 21, which could be sufficiently blocked by anti-CD154 therapy 57. Unfortunately, this therapy 59 had severe side effects that stopped the trials.

The role of the type I cytokine, IL-21, in the pathogenesis of SLE has been suggested by a number of recent findings. First, findings in both humans and mice have indicated an essential role for this cytokine in co-stimulating B cells to differentiate into plasma cells 29. IL-21R^-/- mice have a diminished capacity to produce IgG1 in response to immunization, whereas IL-21 transgenic mice develop hypergammaglobulinemia 60. Overexpression of IL-21 is found in the BXSByaa and the sanroche murine models of SLE 61. Finally, blocking of IL-21 activity successfully treats the manifestations of lupus in the MRL mouse, whereas crossing the BXSByaa mouse with the IL-21R^-/- mouse prevents all manifestations of SLE 62. IL-21 is produced by CXCR5⁺ follicular helper T cells, which require ICOS stimulation for their generation 61. These findings link ICOS and IL-21 in a definable pathway required for B-cell stimulation in secondary lymphoid organs and suggest that blocking IL-21 may be effective in human SLE. Formation of ectopic GCs has been identified in the kidneys of patients with SLE, in the salivary glands of patients with Sjögren syndrome, in the thymus in patients with myasthenia gravis, and in the central nervous system of patients with multiple sclerosis 45863. Although these aggregates of CD20⁺ B cells surrounded by T cells and follicular dendritic cells have been found in only a fraction of patients, it is not known whether their formation is related to disease activity or T cell-dependent or -independent activation or whether they have the full capacity to select antigen-reactive cells and delete autoreactive B cells appropriately as in typical GCs.

Even though ectopic GCs have been linked to local over-activation of autoimmune B cells and plasma cells, conclusive evidence that they are required for the development of autoimmune diseases or are secondary to the deranged internal environment characteristic of these conditions has not been provided. Thus, no firm conclusion on the differences in the nature of classic GC versus ectopic GC in autoimmunity can be drawn since even classic GCs in tonsils from SLE patients were found to be defective in selection against 9G4 B cells as an indicator of anti-dsDNA activity 16.

An area of interest within the last decade has been the role of T cell-independent activation of B cells. While this can occur in vitro, ligating one of the receptors for BAFF/BLyS and APRIL, the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) 6465666768, in only a subset of memory B cells 69, conclusive evidence concerning the role of this pathway in SLE is lacking.

Another mechanism by which B cells can be activated in the absence of T cells is via TLR activation. TLRs are also known as 'pathogen-associated molecular pattern receptors' or 'pattern recognition receptors' and are expressed by nearly every cell in the body. TLR-7, TLR-8, and TLR-9 are the most important of these with respect to B-cell activation. Bacterial DNA is the natural ligand of TLR-9, and single-stranded RNA is the ligand of TLR-7 and TLR-8. All three receptor-ligand interactions apparently lead to activation of B cells by an NF-κB (nuclear factor-kappa-B)-dependent mechanism 7071. So far, the most convincing data available for human SLE concern TLR-9 and B-cell activation. As a result of TLR-9 stimulation, B cells can differentiate into (auto)antibody-producing plasma cells or produce pro-inflammatory cytokines 71. Signaling via TLRs uniquely affects memory B cells 72. The available data, however, suggest the possibility that memory B cells in patients with SLE could easily be activated via TLR-9 without T-cell help and therefore could account for the expansion in peripheral plasmablasts.

In conclusion, a complex interplay between a constantly over-activated immune system and apparent multiple abnormalities of B-cell development can be assumed in SLE. This permanent overactivation (of whatever cause) might overwhelm all possible check capacities of the immune system. Consistent with that, early check points before antigen or T-cell influences have been identified to be defective 1415, classic GCs do not select properly 16, and ectopic GCs with a potential lack of selection are found in SLE that may permit the emergence of autoreactive cells.

After therapeutic trials of anti-CD4 therapy in RA 73 and SLE 74 failed to show substantial clinical benefit, questions about the central role of CD4⁺ T cells were raised. Recent success achieved by blocking T-cell co-stimulation with CTLA4Ig (abatacept) by antigen-presenting cells, including B cells, and the effects of blocking CD40/CD154 interactions on autoimmunity 5759 have again implied a role for the regulatory interaction of immune cells in systemic autoimmune diseases. However, in SLE, the use of anti-CD154 (BG9588) led to some safety concerns with thromboembolic complications 59 in lupus nephritis, although clear immunologic effects were seen. Moreover, blockade of ICOS-L was shown in mice to reduce lupus nephritis 75.

The success of B cell-depleting therapy in ameliorating rheumatoid inflammation and joint destruction has documented a role for B cells in RA but also in other autoimmune diseases, such as idiopathic thrombocytopenic purpura and SLE 76. Interestingly, a reduction of expressed co-stimulatory molecules such as CD80, CD86, and CD40L on T cells after B-cell depletion was observed in SLE 77, a reduced infiltration of CD68 macrophages was noted in the RA synovium 78, and an increase of regulatory T cells was seen in autoimmune thrombocytopenia 79. It is clear from these clinical trial results that B cells together with other immune cells play important roles in autoimmunity. However, their role in the induction versus maintenance phase of the disease and the specific contributions of particular B-cell subsets have not been dissected in detail. Although previous data in lupus have shown that B-cell hyperactivity and the resulting autoantibody production are central elements of the immunopathogenesis of SLE, preliminary data of the use of rituximab as an anti-CD20 antibody in non-renal (EXPLORER trial) 80 and renal (LUNAR trial) SLE have reportedly failed the primary endpoints. Although it remains possible that anti-B-cell therapy is not sufficient to suppress lupus activity, a number of other confounding variables of SLE trial design may have substantially contributed (too short trial duration, allowance of substantial glucocorticoid usage, heterogeneity of the patient population, etc). Since a number of additional early trials in SLE, such as the use of abatacept and abetimus, have not provided convincing therapeutic effects, the most likely explanation is that lupus patients are heterogeneous with regard to organ manifestations and pathophysiology and require customized therapeutic strategies. Patients need to be stratified and characterized in detail prior to the choosing of a certain therapeutic approach. Parameters like the 'interferon signature', BLyS levels, and serologic and clinical findings should be considered and validated regarding their predictive value as biomarkers. In this regard, frequently detectable autoantibodies against DNA are produced by short-lived plasma cells versus anti-extractable nuclear antigens produced by apparently long-lived plasma cells. Given that both produce pathogenic autoantibodies, therapies should target both. Future assessments will be necessary to determine whether B cell-directed therapy can be effective clinically in SLE but also to test the hypothesis that specific B-cell abnormalities are essential for the pathogenesis of this disease. A further explanation of recent trial failure could be that the British Isles Lupus Assessment Group (BILAG) scoring system is less sensitive to changes in the BILAG B, which may preclude a sufficient discrimination of therapeutic success. Overall, the results of recent lupus trials challenge the rheumatology community to improve trial approaches in SLE, a patient population in need of improved therapies.

A different B cell-directed approach is targeting CD22 on B cells; CD22 is uniquely expressed on mature B cells but not on plasma cells or pre-B cells. The humanized anti-CD22 monoclonal antibody epratuzumab causes partial B-cell reduction in the blood, inhibits B-cell proliferation, and probably interferes with intracellular signaling. Preliminary results from an open-label phase IIa study indicate that epratuzumab is efficacious in SLE 81, with BILAG index scores improving by more than 50% in 77% of treated patients at week 6. A central question yet to be answered is how partial inhibition of B-cell activation may affect the clinical risk-benefit ratio as compared with total depletion as mediated by anti-CD20 antibodies. Partial inhibition might mean that B cells can still be activated by certain pathogens in this setting while epratuzumab (anti-CD22) inhibits activation of autoreactive B cells. A preferential reduction of naïve B cells in the peripheral blood under epratuzumab treatment has been seen in clinical study. It has not been resolved whether this was caused by preferential depletion or apoptosis of naïve B cells or by enhanced migration of these cells from the blood into the tissue. In vitro data, however, suggested that this agent acts also by blocking proliferation of B cells 46, an effect that has been observed in patients with SLE but not in normal controls 46.

The current data are consistent with the conclusion that B-cell abnormalities in SLE can be targeted by cellular approaches, such as anti-B-cell therapy, but also interventions on key cytokines, such as IFNα, or blocking co-stimulation. A critical question will be to identify a common denominator of B-cell activation as a target that allows sufficient and safe immune intervention.

anti-dsDNA: anti-double-stranded DNA; APRIL: a proliferation-inducing ligand; BAFF: B cell-activating factor; BCR: B-cell receptor; BILAG: British Isles Lupus Assessment Group; BLyS: B-lymphocyte stimulator; GC: germinal center; ICOS: inducible co-stimulator; ICOS-L: inducible co-stimulator ligand; IFN: interferon; Ig: immunoglobulin; IL: interleukin; ITIM: immunoreceptor tyrosine-based inhibitory motif; MZ: marginal zone; pSS: primary Sjögren syndrome; SLE: systemic lupus erythematosus; TLR: Toll-like receptor; TNF: tumor necrosis factor.

TD declares associations with the following companies: Roche, Genentech, Immunomedics, UCB. AJ and PL declare that they have no competing interests.

The Scientific Basis of Rheumatology: A Decade of Progress

This article is part of a special collection of reviews, The Scientific Basis of Rheumatology: A Decade of Progress, published to mark Arthritis Research & Therapy's 10th anniversary.

Other articles in this series can be found at: http://arthritis-research.com/sbr