Short-Lived Plasmablasts Dominate the Early Spontaneous Rheumatoid Factor Response: Differentiation Pathways, Hypermutating Cell Types, and
http://www.100md.com
免疫学杂志 2005年第11期
Abstract
We used a newly validated approach to identify the initiation of an autoantibody response to identify the sites and cell differentiation pathways at early and late stages of the rheumatoid factor response. The autoimmune response is mainly comprised of rapidly turning over plasmablasts that, according to BrdU labeling, TUNEL, and hypermutation data, derive from an activated B cell precursor. Surprisingly, few long-lived plasma cells were generated. The response most likely initiates at the splenic T-B zone border and continues in the marginal sinus bridging channels. Both activated B cells and plasmablasts harbor V gene mutations; large numbers of mutations in mice with long-standing response indicate that despite the rapid turnover of responding cells, clones can persist for many weeks. These studies provide insights into the unique nature of an ongoing autoimmune response and may be a model for understanding the response to therapies such as B cell depletion.
Introduction
Systemic autoimmune diseases are characterized by activation of autoreactive B cells that leads both to autoantibody (autoAb)3 production and T cell activation (1). Whether this activation occurs because of a failure of central tolerance (2, 3, 4, 5, 6), anergy (7, 8), or other mechanisms of peripheral tolerance (9, 10, 11, 12, 13), or is due to activation of ignorant B cells (14, 15), it represents a functional loss of tolerance. After the initial loss of tolerance, tissue destruction ensues, which in turn promotes further inflammation, more extensive loss of self-tolerance, and ultimately symptoms of autoimmunity. In addition, the self-epitopes targeted early in the response may differ from those targeted later, a phenomenon known as epitope spreading. Thus, early and late stages of autoimmunity may differ in the subsets of autoreactive cells that are activated, sites of activation, and the types of effector cell that result. Immune dysregulation at the end stage of disease could be of a different nature from that which occurred during the initial loss of self-tolerance. However, because the onset of spontaneous systemic autoimmunity is not predictable from individual to individual, it has been difficult to study early events in the loss of self-tolerance and determine how they evolve into more established disease with time.
Ig-transgenic (Ig-Tg) mouse models have been instrumental in studies of normal B cell self-tolerance and its loss during disease (2, 3, 4, 5, 8). Particularly interesting in this regard are models that focus on disease-related autoantigens, which have demonstrated a variety of tolerance mechanisms and in selected cases escape from these in the context of a disease-prone genetic background (5, 8, 11, 12, 16, 17). We have been focusing on the AM14 system, an Ig-Tg model with IgG-specific (rheumatoid factor (RF)) B cells (14, 15, 18). RF autoAbs are found at high levels in rheumatoid arthritis, Sjogren’s syndrome, in some patients with systemic lupus erythematosus (SLE) (19), as well as in Fas-deficient lpr mice (20, 21). In AM14 Ig-Tg mice, the H chain confers specificity for the Fc portion of IgG2aa when paired with an endogenous or Tg-encoded V8 L chain (22). AM14 binds only to IgG2a of the "a" allele; thus, in IgH congenic mice, RF B cells can be studied in the presence or absence (i.e., in IgHb mice) of a defined and measurable autoantigen. Using this model, we initially found that, in contrast to most other models, the RF B cells develop normally and are immunocompetent in normal IgHa mice (23), a phenotype referred to as clonal ignorance.
To determine how RF B cells become activated on an autoimmune-prone background, we crossed the AM14 Tg onto the MRL/lpr strain (24). RF B cells in spleens of older H chain Ig-Tg MRL/lpr mice form clusters at the T zone-red pulp borders and undergo somatic hypermutation at this site; they are only very rarely found in germinal centers (GCs) (15). The activation of autoreactive B cells required the presence of the nominal autoantigen and did not occur on the IgHb background. Interestingly, there was great mouse to mouse variability in the onset and extent of RF B cell Ab-forming cell (AFC) induction. This is similar to the stochastic onset of appearance of various autoantibodies in non-Tg MRL/lpr and other autoimmune-prone mice (25). Such variability in the onset and nature of disease has confounded efforts to identify the earliest phases of autoreactive B cell activation. Consequently, whereas there are a number of studies of the autoimmune response in our system and others, these data mainly reflect the later stages of established autoimmunity.
To understand the initiating events in the loss of B cell tolerance, we would like to identify the site(s) of initial B cell activation in secondary lymphoid organs such as the spleen; for example, does RF B cell activation occur at the T-B border or the marginal sinus-bridging channel? In addition, we would like to know whether induction of autoimmunity occurs at other secondary lymphoid sites and whether it is synchronous with activation in the spleen. Finally, it is important to define the cell types and differentiation states found during initial RF B cell activation as well as how the response evolves over time. Recently, we developed a system to identify which mice were at the early stages of initial RF B cell activation based on the appearance of activated RF B cells in the peripheral blood (53). In the present study, we use this system to address some of the questions defined above concerning the sites, tissues, and cell types involved in the early phases of a spontaneous autoimmune response and how these evolve with time.
Materials and Methods
Hybridomas and RF affinity ELISAs
Splenocytes from an aged AM14 MRL/lpr mouse were fused to SP2/0 cells and resultant hybridomas screened for id+ Ab secretion. Two hybridomas secreting the AM14 H chain/V8 L chain pair were recovered and tested for affinity to the autoantigen IgG2a as described (29). Briefly, plates were coated with the hapten (4-hydroxy-3-nitro-2-iodo-phenyl)acetyl (NIP)26-BSA, blocked with PBS/1% BSA, and then incubated with increasing concentrations with 23.3, a NIP-specific IgG2a. After washing, plates were incubated with hybridoma or spleen cell supernatants, followed by detection with anti-IgM-alkaline phosphatase (Southern Biotechnology Associates) and development with para-nitrophenyl phosphate substrate (Sigma-Aldrich). To measure RF affinity from polyclonal AFCs in converted mice, splenocytes from aged H MRL/lpr mice were cultured in 10% complete RPMI overnight and supernatants tested as described above.
Results
The early splenic response in converted AM14 MRL/lpr mice is marked by the appearance of a CD22low population of 4-44+ B cells
We recently showed (53) that the appearance of Id 4-44+ (RF+) B cells in PBL correlated strongly with the presence of elevated numbers of RF AFC in the spleen and conversely that the absence of such cells in PBL predicted the presence of only very low numbers of RF AFC in spleen. From these data we reasoned that the initial appearance of RF+ B cells in PBL marked the onset of the initial RF autoimmune response in the spleen, a phenomenon we termed "conversion." We therefore used this approach to identify early converts and then study the nature of the response.
In early converts, 4-44+ cells accumulated and mutated primarily at the T zone-red pulp border (Fig. 1A) (15), similar to aged IgHa MRL/lpr mice (15). We used FACS and immunohistology to understand the origin and identity of these unusual mutating cells. 4-44+ cells from IgHb and nonconverted IgHa mice made up 1–2% of the splenic population and were uniformly CD22high (Fig. 1B). In recently converted mice, there was a significant expansion of a novel 4-44+, CD22low population (compare upper left quadrants in Fig. 1, B–D, summarized in Fig. 1E). This population of cells expanded further in mice that had converted at least 2 wk ago (Fig. 1E). In contrast, the CD22high population rarely expanded at any stage of conversion (Fig. 1, B–E). The CD22low population was B220low, CR1/2low, CD23low, CD44high, CD80high, CD86high, class IIint/hi, CXCR5low, and CXCR4high (Fig. 1, F–N). The expression patterns of CXCR4 and CXCR5 are consistent with localization of the CD22low population outside of follicles and in the marginal sinus-bridging channels (30). The CD22high population in both converts and nonconverts was B220high, CR1/2high, CD23 variable, class IIhigh, CXCR5high, and CXCR4low (Fig. 1, F–N). These markers, particularly the chemokine receptors, are consistent with a follicular localization (30). In converted mice, a fraction of the CD22high population reproducibly demonstrated increased expression of the activation markers CD44, CD80 and, to a slight degree, CD86. This can be seen in Fig. 1, I–K, comparing the blue, thin solid line to the dashed green one. Note that the CD22high cells from nonconverted mice (Fig. 1) lack the population of cells with higher expression of these markers; this population is also lacking in IgHb mice (not shown).
Discussion
Systemic autoimmunity is a complex process involving initial loss of B and T cell tolerance followed by engagement of multiple effector functions that lead to tissue destruction. The initial inflammation undoubtedly leads to subsequent activation of further waves of autoreactive lymphocytes and repetition of the cycle until a state of chronic inflammation ensues. Most studies of humans and mice have only been able to identify and characterize disease at this late stage. In part, this is because early stages of disease may be asymptomatic and because stochastic or environmental events play a major role in determining the onset and nature of autoimmunity—even identical twins are not always concordant, and inbred mice in the same cage can have markedly different kinetics and features of autoimmunity.
It is critical to obtain more information about the nature of early events in the process. This has been very difficult precisely because the environmental and stochastic factors that incite autoimmunity are not under experimental control, and thus it is difficult to know at what stage of disease any individual is at a given age or time point. We previously devised and validated a method to identify the onset of the RF B cell response in a cohort of autoimmune-prone mice carrying a Tg for the H chain of an RF autoantibody. These mice are destined to make an RF autoantibody response, but the time of onset varies over a period of several months (53). We also showed in this system that older mice that had made an RF response demonstrated a predominant extrafollicular Ab-forming cell response without an RF GC response. These extrafollicular B cells were quite unusual in that they were undergoing somatic hypermutation but were not further characterized (15).
B cell subpopulations that develop during the RF response
The major goal of the present work was to define the populations that were participating in the early splenic RF response, taking advantage of the ability to identify the onset of the RF response in individual animals, a process we termed conversion. We found that there were both CD22low plasmablasts and CD22high-activated B cells in converted mice. These populations were only found in mice with ongoing autoimmunity. We were surprised to clearly identify a plasmablast—as opposed to a plasma cell—as the dominant producer of RF autoAb. The normal plasmablasts that dominate early immune responses to foreign Ags are not completely characterized; however, the population we observed seemed to resemble the normal population, as evidenced by high expression of CD44 and syndecan and low expression of CD22 (43, 44). Despite this, it remains possible that there are subtle differences between plasmablasts secreting RF autoantibody in autoimmune-prone mice and those responding to foreign Ag in normal mice. An even more complete characterization of both populations is required.
The identification of the AFC as a plasmablast has important implications. These plasmablasts have characteristics that would allow presentation of Ag to T cells and thus may be active in this role. Further, plasmablasts would be expected to respond quite differently to certain therapeutic interventions; for example, their surface phenotype including the lack of typical B cell markers like CD20 and CD22 is important if one is designing Ab-mediated therapy to eliminate AFCs. Plasmablasts can interact with dendritic cells (DCs) and may require factors from DCs for their survival (45, 46). Indeed, we previously showed that RF AFCs were in contact with CD11c+ DCs in the spleens of converted mice (15). If these interactions are important for plasmablast survival then disrupting them could represent a therapeutic strategy. For example, plasmablasts in vitro respond to BAFF (46), a myeloid product (47), and this could explain the efficacy of inhibition of BAFF/APRIL in vivo in MRL/lpr mice (48). The abnormal regulation of AFCs in NZM2410 mice (49) further highlights the importance of understanding the genesis of AFCs in autoimmunity. The present study makes an important contribution by identifying the key role of the plasmablast and defining its phenotype and relationship to an activated B cell precursor.
A recent report presented a characterization of AFCs in the spleen of autoimmune New Zealand Black/White mice (54). Although this study did not determine the stage of disease we did here or focus on RF B cells, they did find a substantial proportion of short-lived plasmablasts. The authors highlighted the finding of long-lived plasma cells seen in their model, which were not observed in ours. Whether this represents a difference in strain, specificity, stage of disease, or methodology is unclear. Nonetheless, the presence of short-lived plasmablasts is a common feature.
Cell death and division in the autoimmune B cell response
Analysis of cell division and death revealed an ongoing, highly dynamic process of autoimmunity. Both the CD22high and CD22low populations were undergoing very rapid cell division and apoptosis. The revelation that the autoimmune reaction in the spleen is so dynamic, if it parallels the human situation, could explain the efficacy of cytotoxic and anti-proliferative agents in SLE as well as the rapid time course of such responses. Emerging data from humans treated with anti-CD20 to deplete CD20pos B cells (but presumably not CD20neg plasmablasts or plasma cells directly) demonstrate that some but not all autoantibody titers fall relatively quickly (50, 51). This could be explained if the plasmablast population were being renewed by a proliferating CD20+-activated B cell population such as the CD22high cells, which are evidently precursors of the CD22low plasmablasts in our system. In this context, the experiment of depleting CD20 B cells in humans with autoimmunity suggests that, as in our model, a substantial proportion of autoantibody is generated from a dynamic process of plasmablast differentiation.
Mutation and selection in the spleen
The identification of two proliferating populations raised the question of which was undergoing somatic mutation (15). However, since we found that both populations contained mutations, we still cannot distinguish whether one or both populations is mutating. Nonetheless, the BrdU and apoptosis data, along with the fact that plasmablasts normally derive from activated B cells, suggest that at least the CD22high population is mutating. This does not exclude the possibility that the plasmablasts could also be mutating. The presence of a subset of CD22low cells with substantially more mutations suggests that there may be some ongoing mutation in this population as well. However, resolving this question definitively has proved difficult.
The extent of mutation in the two populations provides insight into the dynamic nature of the response. This is based in part on the concept that the number of mutations in a given cell serves as a time clock for the number of cell divisions it has undergone. Indeed, sequences isolated from mice converted for long periods contain more mutations than those of recent converts (Fig. 4, D and E). This indicates that there are some relatively long-lived clones of RF B cells. In fact, some RF B cells have as many as 15 mutations in their V alone. Assuming a rate of 0.25 mutations/V region/division (15, 52), in these clones there were 60 or more divisions during the phase of active somatic hypermutation. BrdU labeling indicates a division rate of at least every 12 h, meaning that some clones survive for 4 wk in the actively mutating state. Thus, despite active proliferation and cell death among the plasmablast fraction, there must be a precursor population—most likely the activated CD22high cells—that can provide for clonal longevity through many cell divisions.
Finally, we addressed whether mutation during the extrafollicular RF response is accompanied by selection. Using hydridomas as well as assay of polyclonal supernatants collected from splenocytes of converted mice, we showed that much of the secreted RF has a higher affinity than the germline AM14 Ab. This is consistent with affinity maturation due to somatic hypermutation. Supporting this interpretation is the finding of certain recurrent replacement mutations in CDRs in our collection of V region sequences (Ref.15 and unpublished data). Thus, we have now shown that both mutation and selection occur outside the GC in the spontaneous RF response in MRL/lpr mice.
Conclusion
We have used the RF Tg system to develop a detailed picture of both the onset and evolution of a model autoantibody response in the spleen. Like the established response (15), the early response does not include GCs. Instead, it begins as scattered foci of extrafollicular sites of proliferation and differentiation of RF B cells (this study).4 CD22high activated B cells are very likely the precursors of CD22low plasmablasts. This event is accompanied by the appearance of a plasmablast-like cell in the PBL. The entire process is highly dynamic, particularly in the plasmablast compartment. CD22high and possibly CD22low cells are undergoing somatic hypermutation as well as selection for higher affinity. The response is progressive and includes at least some clones that live for a long period.
This picture of a dynamic ongoing response has implications for our understanding of spontaneous systemic autoimmune disease and strategies for its treatment. In future work, we would like to determine whether this scenario applies to other autoAbs and other murine models of autoimmunity as well as human disease. Given the common autoAb profiles of MRL/lpr mice and human disease, we expect that our findings will be generalizable; however, even potential differences will be interesting because they will probably reflect the range of clinical disease and autoAb profiles that vary greatly among patients with SLE.
Acknowledgments
We thank Ann Haberman, Shannon Anderson, and Martin Weigert for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant P01-AI36529.
2 Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Yale University School of Medicine, 333 Cedar Street, Box 208035, New Haven, CT 06520-8035. E-mail address: mark.shlomchik{at}yale.edu
3 Abbreviations used in this paper: autoAb, autoantibody; Tg, transgenic; RF, rheumatoid factor; GC, germinal center; AFC, Ab-forming cell; SLE, systemic lupus erythematosus; DC, dendritic cell.
Received for publication January 27, 2005. Accepted for publication March 16, 2005.
References
Lipsky, P. E.. 2001. Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat. Immunol. 2: 764-766.
Goodnow, C. C., S. Adelstein, A. Basten. 1990. The need for central and peripheral tolerance in the B cell repertoire. Science 248: 1373-1379.
Nemazee, D., D. Russell, B. Arnold, G. Haemmerling, J. Allison, J. F. A. P. Miller, G. Morahan, K. Beurki. 1991. Clonal deletion of autospecific B lymphocytes. Immunol. Rev. 122: 117-132.
Tiegs, S. L., D. M. Russell, D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177: 1009-1020.
Chen, C., M. Z. Radic, J. Erikson, S. A. Camper, S. Litwin, R. R. Hardy, M. Weigert. 1994. Deletion and editing of B cells that express antibodies to DNA. J. Immunol. 152: 1970-1982.
Gay, D., T. Saunders, S. Camper, M. Weigert. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177: 999-1008.
Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682.
Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, M. G. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349: 331-334.(Jacqueline William*, Chad)
We used a newly validated approach to identify the initiation of an autoantibody response to identify the sites and cell differentiation pathways at early and late stages of the rheumatoid factor response. The autoimmune response is mainly comprised of rapidly turning over plasmablasts that, according to BrdU labeling, TUNEL, and hypermutation data, derive from an activated B cell precursor. Surprisingly, few long-lived plasma cells were generated. The response most likely initiates at the splenic T-B zone border and continues in the marginal sinus bridging channels. Both activated B cells and plasmablasts harbor V gene mutations; large numbers of mutations in mice with long-standing response indicate that despite the rapid turnover of responding cells, clones can persist for many weeks. These studies provide insights into the unique nature of an ongoing autoimmune response and may be a model for understanding the response to therapies such as B cell depletion.
Introduction
Systemic autoimmune diseases are characterized by activation of autoreactive B cells that leads both to autoantibody (autoAb)3 production and T cell activation (1). Whether this activation occurs because of a failure of central tolerance (2, 3, 4, 5, 6), anergy (7, 8), or other mechanisms of peripheral tolerance (9, 10, 11, 12, 13), or is due to activation of ignorant B cells (14, 15), it represents a functional loss of tolerance. After the initial loss of tolerance, tissue destruction ensues, which in turn promotes further inflammation, more extensive loss of self-tolerance, and ultimately symptoms of autoimmunity. In addition, the self-epitopes targeted early in the response may differ from those targeted later, a phenomenon known as epitope spreading. Thus, early and late stages of autoimmunity may differ in the subsets of autoreactive cells that are activated, sites of activation, and the types of effector cell that result. Immune dysregulation at the end stage of disease could be of a different nature from that which occurred during the initial loss of self-tolerance. However, because the onset of spontaneous systemic autoimmunity is not predictable from individual to individual, it has been difficult to study early events in the loss of self-tolerance and determine how they evolve into more established disease with time.
Ig-transgenic (Ig-Tg) mouse models have been instrumental in studies of normal B cell self-tolerance and its loss during disease (2, 3, 4, 5, 8). Particularly interesting in this regard are models that focus on disease-related autoantigens, which have demonstrated a variety of tolerance mechanisms and in selected cases escape from these in the context of a disease-prone genetic background (5, 8, 11, 12, 16, 17). We have been focusing on the AM14 system, an Ig-Tg model with IgG-specific (rheumatoid factor (RF)) B cells (14, 15, 18). RF autoAbs are found at high levels in rheumatoid arthritis, Sjogren’s syndrome, in some patients with systemic lupus erythematosus (SLE) (19), as well as in Fas-deficient lpr mice (20, 21). In AM14 Ig-Tg mice, the H chain confers specificity for the Fc portion of IgG2aa when paired with an endogenous or Tg-encoded V8 L chain (22). AM14 binds only to IgG2a of the "a" allele; thus, in IgH congenic mice, RF B cells can be studied in the presence or absence (i.e., in IgHb mice) of a defined and measurable autoantigen. Using this model, we initially found that, in contrast to most other models, the RF B cells develop normally and are immunocompetent in normal IgHa mice (23), a phenotype referred to as clonal ignorance.
To determine how RF B cells become activated on an autoimmune-prone background, we crossed the AM14 Tg onto the MRL/lpr strain (24). RF B cells in spleens of older H chain Ig-Tg MRL/lpr mice form clusters at the T zone-red pulp borders and undergo somatic hypermutation at this site; they are only very rarely found in germinal centers (GCs) (15). The activation of autoreactive B cells required the presence of the nominal autoantigen and did not occur on the IgHb background. Interestingly, there was great mouse to mouse variability in the onset and extent of RF B cell Ab-forming cell (AFC) induction. This is similar to the stochastic onset of appearance of various autoantibodies in non-Tg MRL/lpr and other autoimmune-prone mice (25). Such variability in the onset and nature of disease has confounded efforts to identify the earliest phases of autoreactive B cell activation. Consequently, whereas there are a number of studies of the autoimmune response in our system and others, these data mainly reflect the later stages of established autoimmunity.
To understand the initiating events in the loss of B cell tolerance, we would like to identify the site(s) of initial B cell activation in secondary lymphoid organs such as the spleen; for example, does RF B cell activation occur at the T-B border or the marginal sinus-bridging channel? In addition, we would like to know whether induction of autoimmunity occurs at other secondary lymphoid sites and whether it is synchronous with activation in the spleen. Finally, it is important to define the cell types and differentiation states found during initial RF B cell activation as well as how the response evolves over time. Recently, we developed a system to identify which mice were at the early stages of initial RF B cell activation based on the appearance of activated RF B cells in the peripheral blood (53). In the present study, we use this system to address some of the questions defined above concerning the sites, tissues, and cell types involved in the early phases of a spontaneous autoimmune response and how these evolve with time.
Materials and Methods
Hybridomas and RF affinity ELISAs
Splenocytes from an aged AM14 MRL/lpr mouse were fused to SP2/0 cells and resultant hybridomas screened for id+ Ab secretion. Two hybridomas secreting the AM14 H chain/V8 L chain pair were recovered and tested for affinity to the autoantigen IgG2a as described (29). Briefly, plates were coated with the hapten (4-hydroxy-3-nitro-2-iodo-phenyl)acetyl (NIP)26-BSA, blocked with PBS/1% BSA, and then incubated with increasing concentrations with 23.3, a NIP-specific IgG2a. After washing, plates were incubated with hybridoma or spleen cell supernatants, followed by detection with anti-IgM-alkaline phosphatase (Southern Biotechnology Associates) and development with para-nitrophenyl phosphate substrate (Sigma-Aldrich). To measure RF affinity from polyclonal AFCs in converted mice, splenocytes from aged H MRL/lpr mice were cultured in 10% complete RPMI overnight and supernatants tested as described above.
Results
The early splenic response in converted AM14 MRL/lpr mice is marked by the appearance of a CD22low population of 4-44+ B cells
We recently showed (53) that the appearance of Id 4-44+ (RF+) B cells in PBL correlated strongly with the presence of elevated numbers of RF AFC in the spleen and conversely that the absence of such cells in PBL predicted the presence of only very low numbers of RF AFC in spleen. From these data we reasoned that the initial appearance of RF+ B cells in PBL marked the onset of the initial RF autoimmune response in the spleen, a phenomenon we termed "conversion." We therefore used this approach to identify early converts and then study the nature of the response.
In early converts, 4-44+ cells accumulated and mutated primarily at the T zone-red pulp border (Fig. 1A) (15), similar to aged IgHa MRL/lpr mice (15). We used FACS and immunohistology to understand the origin and identity of these unusual mutating cells. 4-44+ cells from IgHb and nonconverted IgHa mice made up 1–2% of the splenic population and were uniformly CD22high (Fig. 1B). In recently converted mice, there was a significant expansion of a novel 4-44+, CD22low population (compare upper left quadrants in Fig. 1, B–D, summarized in Fig. 1E). This population of cells expanded further in mice that had converted at least 2 wk ago (Fig. 1E). In contrast, the CD22high population rarely expanded at any stage of conversion (Fig. 1, B–E). The CD22low population was B220low, CR1/2low, CD23low, CD44high, CD80high, CD86high, class IIint/hi, CXCR5low, and CXCR4high (Fig. 1, F–N). The expression patterns of CXCR4 and CXCR5 are consistent with localization of the CD22low population outside of follicles and in the marginal sinus-bridging channels (30). The CD22high population in both converts and nonconverts was B220high, CR1/2high, CD23 variable, class IIhigh, CXCR5high, and CXCR4low (Fig. 1, F–N). These markers, particularly the chemokine receptors, are consistent with a follicular localization (30). In converted mice, a fraction of the CD22high population reproducibly demonstrated increased expression of the activation markers CD44, CD80 and, to a slight degree, CD86. This can be seen in Fig. 1, I–K, comparing the blue, thin solid line to the dashed green one. Note that the CD22high cells from nonconverted mice (Fig. 1) lack the population of cells with higher expression of these markers; this population is also lacking in IgHb mice (not shown).
Discussion
Systemic autoimmunity is a complex process involving initial loss of B and T cell tolerance followed by engagement of multiple effector functions that lead to tissue destruction. The initial inflammation undoubtedly leads to subsequent activation of further waves of autoreactive lymphocytes and repetition of the cycle until a state of chronic inflammation ensues. Most studies of humans and mice have only been able to identify and characterize disease at this late stage. In part, this is because early stages of disease may be asymptomatic and because stochastic or environmental events play a major role in determining the onset and nature of autoimmunity—even identical twins are not always concordant, and inbred mice in the same cage can have markedly different kinetics and features of autoimmunity.
It is critical to obtain more information about the nature of early events in the process. This has been very difficult precisely because the environmental and stochastic factors that incite autoimmunity are not under experimental control, and thus it is difficult to know at what stage of disease any individual is at a given age or time point. We previously devised and validated a method to identify the onset of the RF B cell response in a cohort of autoimmune-prone mice carrying a Tg for the H chain of an RF autoantibody. These mice are destined to make an RF autoantibody response, but the time of onset varies over a period of several months (53). We also showed in this system that older mice that had made an RF response demonstrated a predominant extrafollicular Ab-forming cell response without an RF GC response. These extrafollicular B cells were quite unusual in that they were undergoing somatic hypermutation but were not further characterized (15).
B cell subpopulations that develop during the RF response
The major goal of the present work was to define the populations that were participating in the early splenic RF response, taking advantage of the ability to identify the onset of the RF response in individual animals, a process we termed conversion. We found that there were both CD22low plasmablasts and CD22high-activated B cells in converted mice. These populations were only found in mice with ongoing autoimmunity. We were surprised to clearly identify a plasmablast—as opposed to a plasma cell—as the dominant producer of RF autoAb. The normal plasmablasts that dominate early immune responses to foreign Ags are not completely characterized; however, the population we observed seemed to resemble the normal population, as evidenced by high expression of CD44 and syndecan and low expression of CD22 (43, 44). Despite this, it remains possible that there are subtle differences between plasmablasts secreting RF autoantibody in autoimmune-prone mice and those responding to foreign Ag in normal mice. An even more complete characterization of both populations is required.
The identification of the AFC as a plasmablast has important implications. These plasmablasts have characteristics that would allow presentation of Ag to T cells and thus may be active in this role. Further, plasmablasts would be expected to respond quite differently to certain therapeutic interventions; for example, their surface phenotype including the lack of typical B cell markers like CD20 and CD22 is important if one is designing Ab-mediated therapy to eliminate AFCs. Plasmablasts can interact with dendritic cells (DCs) and may require factors from DCs for their survival (45, 46). Indeed, we previously showed that RF AFCs were in contact with CD11c+ DCs in the spleens of converted mice (15). If these interactions are important for plasmablast survival then disrupting them could represent a therapeutic strategy. For example, plasmablasts in vitro respond to BAFF (46), a myeloid product (47), and this could explain the efficacy of inhibition of BAFF/APRIL in vivo in MRL/lpr mice (48). The abnormal regulation of AFCs in NZM2410 mice (49) further highlights the importance of understanding the genesis of AFCs in autoimmunity. The present study makes an important contribution by identifying the key role of the plasmablast and defining its phenotype and relationship to an activated B cell precursor.
A recent report presented a characterization of AFCs in the spleen of autoimmune New Zealand Black/White mice (54). Although this study did not determine the stage of disease we did here or focus on RF B cells, they did find a substantial proportion of short-lived plasmablasts. The authors highlighted the finding of long-lived plasma cells seen in their model, which were not observed in ours. Whether this represents a difference in strain, specificity, stage of disease, or methodology is unclear. Nonetheless, the presence of short-lived plasmablasts is a common feature.
Cell death and division in the autoimmune B cell response
Analysis of cell division and death revealed an ongoing, highly dynamic process of autoimmunity. Both the CD22high and CD22low populations were undergoing very rapid cell division and apoptosis. The revelation that the autoimmune reaction in the spleen is so dynamic, if it parallels the human situation, could explain the efficacy of cytotoxic and anti-proliferative agents in SLE as well as the rapid time course of such responses. Emerging data from humans treated with anti-CD20 to deplete CD20pos B cells (but presumably not CD20neg plasmablasts or plasma cells directly) demonstrate that some but not all autoantibody titers fall relatively quickly (50, 51). This could be explained if the plasmablast population were being renewed by a proliferating CD20+-activated B cell population such as the CD22high cells, which are evidently precursors of the CD22low plasmablasts in our system. In this context, the experiment of depleting CD20 B cells in humans with autoimmunity suggests that, as in our model, a substantial proportion of autoantibody is generated from a dynamic process of plasmablast differentiation.
Mutation and selection in the spleen
The identification of two proliferating populations raised the question of which was undergoing somatic mutation (15). However, since we found that both populations contained mutations, we still cannot distinguish whether one or both populations is mutating. Nonetheless, the BrdU and apoptosis data, along with the fact that plasmablasts normally derive from activated B cells, suggest that at least the CD22high population is mutating. This does not exclude the possibility that the plasmablasts could also be mutating. The presence of a subset of CD22low cells with substantially more mutations suggests that there may be some ongoing mutation in this population as well. However, resolving this question definitively has proved difficult.
The extent of mutation in the two populations provides insight into the dynamic nature of the response. This is based in part on the concept that the number of mutations in a given cell serves as a time clock for the number of cell divisions it has undergone. Indeed, sequences isolated from mice converted for long periods contain more mutations than those of recent converts (Fig. 4, D and E). This indicates that there are some relatively long-lived clones of RF B cells. In fact, some RF B cells have as many as 15 mutations in their V alone. Assuming a rate of 0.25 mutations/V region/division (15, 52), in these clones there were 60 or more divisions during the phase of active somatic hypermutation. BrdU labeling indicates a division rate of at least every 12 h, meaning that some clones survive for 4 wk in the actively mutating state. Thus, despite active proliferation and cell death among the plasmablast fraction, there must be a precursor population—most likely the activated CD22high cells—that can provide for clonal longevity through many cell divisions.
Finally, we addressed whether mutation during the extrafollicular RF response is accompanied by selection. Using hydridomas as well as assay of polyclonal supernatants collected from splenocytes of converted mice, we showed that much of the secreted RF has a higher affinity than the germline AM14 Ab. This is consistent with affinity maturation due to somatic hypermutation. Supporting this interpretation is the finding of certain recurrent replacement mutations in CDRs in our collection of V region sequences (Ref.15 and unpublished data). Thus, we have now shown that both mutation and selection occur outside the GC in the spontaneous RF response in MRL/lpr mice.
Conclusion
We have used the RF Tg system to develop a detailed picture of both the onset and evolution of a model autoantibody response in the spleen. Like the established response (15), the early response does not include GCs. Instead, it begins as scattered foci of extrafollicular sites of proliferation and differentiation of RF B cells (this study).4 CD22high activated B cells are very likely the precursors of CD22low plasmablasts. This event is accompanied by the appearance of a plasmablast-like cell in the PBL. The entire process is highly dynamic, particularly in the plasmablast compartment. CD22high and possibly CD22low cells are undergoing somatic hypermutation as well as selection for higher affinity. The response is progressive and includes at least some clones that live for a long period.
This picture of a dynamic ongoing response has implications for our understanding of spontaneous systemic autoimmune disease and strategies for its treatment. In future work, we would like to determine whether this scenario applies to other autoAbs and other murine models of autoimmunity as well as human disease. Given the common autoAb profiles of MRL/lpr mice and human disease, we expect that our findings will be generalizable; however, even potential differences will be interesting because they will probably reflect the range of clinical disease and autoAb profiles that vary greatly among patients with SLE.
Acknowledgments
We thank Ann Haberman, Shannon Anderson, and Martin Weigert for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant P01-AI36529.
2 Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Yale University School of Medicine, 333 Cedar Street, Box 208035, New Haven, CT 06520-8035. E-mail address: mark.shlomchik{at}yale.edu
3 Abbreviations used in this paper: autoAb, autoantibody; Tg, transgenic; RF, rheumatoid factor; GC, germinal center; AFC, Ab-forming cell; SLE, systemic lupus erythematosus; DC, dendritic cell.
Received for publication January 27, 2005. Accepted for publication March 16, 2005.
References
Lipsky, P. E.. 2001. Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat. Immunol. 2: 764-766.
Goodnow, C. C., S. Adelstein, A. Basten. 1990. The need for central and peripheral tolerance in the B cell repertoire. Science 248: 1373-1379.
Nemazee, D., D. Russell, B. Arnold, G. Haemmerling, J. Allison, J. F. A. P. Miller, G. Morahan, K. Beurki. 1991. Clonal deletion of autospecific B lymphocytes. Immunol. Rev. 122: 117-132.
Tiegs, S. L., D. M. Russell, D. Nemazee. 1993. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177: 1009-1020.
Chen, C., M. Z. Radic, J. Erikson, S. A. Camper, S. Litwin, R. R. Hardy, M. Weigert. 1994. Deletion and editing of B cells that express antibodies to DNA. J. Immunol. 152: 1970-1982.
Gay, D., T. Saunders, S. Camper, M. Weigert. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177: 999-1008.
Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682.
Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, M. G. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349: 331-334.(Jacqueline William*, Chad)