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Original Article |
mark.shlomchik{at}yale.edu
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Key Words: autoimmunity • systemic lupus • B cell tolerance • autoantibody • IgG
Acharacteristic of systemic autoimmune diseases is the production of high-titer autoantibodies (autoAbs)1 to a variety of self-constituents 1. These autoAbs are important diagnostic markers of disease, and their patterns are specific for particular autoimmune diseases 1. They are also important because autoAbs can be pathogenic under certain circumstances 234. The pathogenic consequences of activated autoreactive B cells are not limited to autoAb production, as B cells also promote T cell activation 5.
Thus, it has been of great interest to understand the origins of autoreactive B cells in autoimmune animals and, conversely, how they are controlled in normal animals. It is possible that intrinsic B cell defects 678 leading to B cell hyperactivity 910 account for the production of autoAbs. In this view, autoAbs are the result of nonspecific B cell activation. On the other hand, an early clue to an important role of autoantigen (autoAg) in the genesis of such B cells in autoimmune animals came from the study of autoAb hybridomas. These were found to be somatically mutated and clonally related 111213141516171819. In many cases, the somatic mutations had high ratios of replacement to silent mutations in the CDRs, and in some instances, mutations led to higher affinity for the nominal autoAg. These data were interpreted as evidence that autoAg played a critical role in driving the expansion and selection of autoreactive B cells. These interpretations were only indirect; the analysis of autoAb hybridomas could not provide direct proof that autoAg drives autoreactive B cells, and this issue remains controversial 20.
A second issue is at what stage tolerance must fail in order to allow for the developmental progression of self-reactive B cells. Transgenic (Tgic) mice have been invaluable tools in efforts to address this question. The earliest models, using model antigens such as hen egg lysozyme (HEL) or class I, revealed deletion, receptor editing, and anergy as basic mechanisms of B cell self-tolerance 21222324. When the anti-HEL or anti–class I models were crossed onto the Fas-deficient background, tolerance was generally found to be intact 2526. Studies of model autoAgs do not permit a clear extrapolation to the situation in disease. This is because particular autoAgs, such as DNA, chromatin, or self-IgG are targets in autoimmune diseases 1; tolerance to many other autoAgs remains intact even in systemic autoimmune disease. Why these autoAgs are preferred targets is still unclear. Nonetheless, the regulation of autoreactive B cells with disease-related specificities must be unique and could not be readily predicted by the behavior of B cells specific for model autoAgs that are not actual disease targets.
Recognizing this, several groups have extensively studied Tgic mice expressing anti–single-stranded (ss)DNA and/or –double-stranded (ds)DNA 272829303132333435363738. Such B cells may be regulated by receptor editing, deletion, and anergy, possibly depending on the fine specificity of the cell. In contrast to the artificial autoAg systems, anti-DNA B cells that use a 3H9 H chain transgene (Tg) are activated in Tgic mice on the MRL.Faslpr background 39. This is an extremely important observation because it demonstrates that in an autoimmune mouse, B cells with disease-related specificities may indeed be regulated differently from B cells specific for arbitrary "self" antigens. However, it is not yet clear how these activated DNA-specific B cells arise, whether by defeating central tolerance, anergy, or both.
The regulation of rheumatoid factors (RFs), another typical yet less-studied autoAb, may again be different. Our group has investigated a model based on an RF, AM14, that was originally isolated from an MRL.Faslpr mouse 40. Because AM14 binds only to IgG2a of the "a" allotype (IgG2aa), we have the opportunity to study the Tgic B cells in the presence and absence of autoAg in both normal and autoimmune-prone backgrounds. This capability is similar to the design of the model autoAg systems but is not possible in other Tgics expressing authentic autoAbs. We have previously shown that AM14 B cells are clonally ignorant. They are not tolerized by deletion, editing, or anergy in normal BALB/c mice, nor do they appear activated in the absence of intentional immunization 41.
We suggested based on these results that the precursors of autoAb-secreting cells were not necessarily tolerized in normal animals and became selectively activated in autoimmune animals due to increased propensity to activate the otherwise quiescent cells and/or a failure to downregulate them once activated. This hypothesis, inferred from the phenotype of normal animals, has never been tested in autoimmune mice. It remained possible that such cells would also be ignored in the autoimmune-prone animal. Alternatively, RF B cells might be activated in the autoimmune-prone background regardless of whether autoAg is present.
To investigate these possibilities, we crossed the separate H and L chain Tgs that comprise the AM14 RF specificity onto both the C57B6.MRL.Faslpr (B6/lpr: IgHb, Ag–) and B6/lpr/IgHa background. B6/lpr mice produce large numbers of autoAbs, including RF and antichromatin and have a mild autoimmune disease 7424344, owing chiefly to their Fas deficiency but also to some B6-derived background genes 45. The B6/lpr strain was a particularly attractive model, as congenic strains were available that both had B6/lpr/IgHa and lacked the autoAg 7. We studied the activation state of RF B cells and T cells in vivo in age-matched sets of congenic mice. These studies have provided insights into the role of autoAg in driving B cell autoimmunity, the identity of the RF Ag, where tolerance breaks down to permit autoAb production, and the role of Fas in this process.
PCR Genotyping.
Antibodies.
Cell Isolation and FACSTM Analysis.
ELISA and Enzyme-linked Immunospot Assay.
Statistics.
Spleen Cell Number.
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Mice.
The following three strains of mice were constructed from our BALB/c-based Tgic lines: AM14Vh/B6/lpr (H chain Tgics), AM14V
/B6lpr, and AM14V/B6/lpr/IgHa (L chain Tgics). These were derived by continuous backcrossing to either B6/lpr (originally obtained from The Jackson Laboratory) or B6/lpr/IgHa mice 7 (a gift of Dr. Robert Eisenberg, University of Pennsylvania, Philadelphia, PA). B6/lpr and B6/lpr/IgHa mice were also maintained by intercrossing at Yale University. At each generation, Tgic mice were identified by PCR (see below) for breeding to the next generation. At BC1, mice were typed for homozygosity for the Faslpr mutation by PCR 5, which was confirmed at BC2. The IgHa genotype was also confirmed at BC1 by an allele-specific PCR assay for IgG2aa versus IgG2ab. From BC4 (97% B6 genes) and beyond, AM14Vh/B6/lpr mice were crossed with AM14Vl/B6/lpr mice to create Ag– double-Tgic controls (HLb mice) or to AM14Vh/B6/lpr/IgHa mice to create Ag+ double-Tgic experimental mice (HLab). All other transgenotypes (H, H chain only; L, L chain only; and N, non-Tgic) were also obtained in these crosses and were analyzed as additional controls (see Results). Age-matched IgHb or IgHab mice 40 that were wild type at the Fas locus were available on the BALB/c background and were analyzed as controls. All mice were housed in the same room in our specific pathogen–free barrier colony.
PCR to genotype for H and L Tgs, IgH genotype, and the lpr mutation was performed as described 40. PCR to genotype the IgG2a locus was performed as previously described 46.
The selection, preparation, and labeling of antibodies was as described 4041. Anti-CD3–biotin was obtained from PharMingen.
These were performed essentially as described 41 with the following modifications. Spleens were harvested, weighed, and then divided, with a portion being quick-frozen in OCT for later immunohistochemical analysis. The other portion was weighed again and then processed into a single-cell suspension. Red cells were then lysed with ammonium chloride/Tris solution. Cell number was determined by counting in a hemocytometer, and a corrected total number of cells in the spleen was derived by considering the fraction by weight of the total spleen that was used to create the cell suspension. In most experiments, cells were preincubated with saturating concentrations of 2.4G2 (rat anti–mouse FcR) to block nonspecific binding. Stained cells were analyzed on a FACSCaliburTM (Becton Dickinson Immunocytometry Systems). When possible, live gating using propidium iodide was used to exclude dead cells. When four labeled mAbs were used, forward and side scatter profiles were used to exclude most dead cells and RBCs. At least 30,000 events were collected for two- and three-color analysis, and 50,000 events were collected for four-color analysis.
The assays were performed as described 41.
Data were not distributed normally, mandating the use of Mann-Whitney nonparametric tests (two-tailed) to compare groups. Tests were performed using StatView 4.5 (Abacus Concepts, Inc.).
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Protocol.
Age-matched cohorts of AM14 Tgic mice were established on the B6/lpr (IgHb) and B6/lpr/IgHa backgrounds. As these were generated by intercrossing H and L single-Tgic mice, all possible genotypes were created, among which H, HL, and N were extensively studied. In addition, similar age-matched cohorts were generated on the BALB/c (IgHa) and congenic CB.17 (IgHb) backgrounds. These strains were available as nonautoimmune, Fas-sufficient controls. Mice were allowed to age to 4–7 mo to allow spontaneous autoimmunity to develop, at which point they were killed and analyzed as described below.
The number of total splenocytes was greater in the HLab Tgics (which had the AM14 autoAg) compared with the corresponding HLb Tgic mice, which lacked the autoAg (Table ; P = 0.0003). Interestingly, a similar difference was observed in the H chain Tgic mice (P = 0.0017). This was not due to differences between B6/lpr and B6/lpr/IgHa per se, as no such difference was seen in the N controls (Table ; P = 0.53). Notably, this difference was also not observed in the comparison between BALB/c and CB.17 HL Tgics of similar age (P = 0.76).
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8 L chains that are identical to or resemble the germline-encoded Tgic V8. This interpretation is supported by the fact that among LPS hybridomas isolated from BALB/c H chain Tgics, both of the Id+ cell lines that were isolated had the same endogenously derived V8 sequence as the Tg L chain (Shlomchik, M., unpublished data). If indeed these Id+ cells in H-only mice reconstitute the RF specificity of the original AM14 HL pair, finding higher percentages in the Hab mice compared with the Hb mice would be consistent with an active, autoAg-driven process in which rare RF B cells are substantially expanded. RF ELISpot data (see below) is also consistent with this interpretation; however, confirmation of it will require isolation of the cells from Hab mice and sequencing of their L chains.
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The existence of low-affinity "natural" autoantibodies in normal individuals has been known for a long time 5556. These B cells are likely to be clonally ignorant, as suggested by the phenotype of AM14 B cells in normal mice. What has remained controversial is whether such B cells had any relevance or relationship to pathologic autoantibodies produced in systemic autoimmune disease 5657. Our results indicate that clonally ignorant cells can indeed be relevant precursors for pathologic autoantibodies.
Evidence that Proliferation and Differentiation of Autoreactive B Cells In Vivo Depends on AutoAg.
A second issue that our data addresses is the role of autoAg-stimulated versus nonspecific B cell activation in the induction of autoAbs. Several studies had shown that antibodies to a wide variety of antigens can be detected in autoimmune animals, suggesting that polyclonal activation was at work 585960. On the other hand, the oligoclonal nature of B cell hybridomas specific for IgG, DNA, nucleosomes, or Sm isolated from autoimmune animals suggested an antigen-driven process 1113141516171819. Notably, a nonrandom pattern of somatic mutations and the presence of certain mutations that increased the affinity for Ag suggested that Ag was playing a role in driving at least some autoimmune responses. These conflicting interpretations were based on indirect inferences. Moreover, neither argument could establish whether autoAg was required or was simply altering or exacerbating an underlying polyclonal process.
The data in this report provide more direct evidence that autoAg is required for the activation of autoreactive B cells. In the presence of autoAg, substantial accumulations of RF plasma cells were observed in the spleens of Tgic mice compared with those mice that lacked the autoAg. Remarkably, only in the presence of the autoAg did H chain Tgic mice efficiently select rare endogenous L chains that reconstruct both the idiotype and RF specificity of the original HL pair that comprised AM14. This phenomenon provides an additional strong argument for the role of autoAg in driving RF B cells.
These conclusions are based on direct ELISpot data and splenic histology; we did not systematically measure and do not present serum RF data that are at best an indirect measurement of B cell activation and differentiation. Serum RF data are widely gathered in humans, where such direct measurements as ELISpot are not feasible. In our system, serum RF levels would be influenced by Ag competition and increased clearance rates of immune complexes as well as by competition by IgG2a naturally occurring in serum. These confounding features would be present in IgHa mice but not in IgHb mice, precluding any meaningful comparison. Furthermore, it would be difficult in such assays to distinguish RF secreted by B cells expressing endogenous Ig genes. ELISpot and histology assays are not subject to these concerns.
The Nominal AutoAg IgG2a Is the In Vivo Antigen Driving RF B Cells.
These observations raise the related issue of the identification of the true autoAg. It has been controversial whether the nominal Ags assayed in vitro as targets of autoAbs are actually those that drive autoreactive B cell clones in vivo 6162. Perhaps the strongest evidence in favor of nominal Ags as actual Ags came from a single study in which the clonality and specificity were simultaneously determined for a large number of hybridomas isolated from a single autoimmune mouse 63. Nearly all of the expanded clones could be assigned to one of a relative few nominal autoAb specificities, again suggesting that the nominal antigens were driving most of the clonal expansion. As congenic strains were compared in this study, the only important difference between the two strains is almost certainly the IgH locus allotype and, in particular, the presence or absence of IgG2aa, which is the only ligand for AM14 that is encoded by the IgH locus (Shlomchik, M., unpublished data). Thus, IgG2aa is sufficient in vivo to drive B cell clonal expansion and to recapitulate the original expansion that must have occurred in the MRL/lpr mouse from which AM14 was isolated as a hybridoma. No other autoAg on the B6/lpr (IgHb) background was capable of driving this activation.
T Cell Activation Is Promoted by the RF B Cell Antigen.
A surprising observation was that T cell activation, as reflected by the accumulation of B220+ T cells, was affected by the presence of the B cell Ag. This means that the extent of B cell activation of one particular clone was sufficient to affect the activation of polyclonal T cells. The mechanism for this is unclear but intriguing. It could be an indirect effect due to cytokines produced by B cells 64656667. More likely, it is a direct effect of T–B cell collaboration. Recently, our group has shown that the absence of B cells affects T cell activation 5. Sobel and colleagues also showed in mixed BM chimeras that Fas-deficient B cells are largely responsible for promoting T cell activation 52 and that cognate interactions are required for autoAb elicitation 68. Our recent data 69 demonstrate that this effect on T cell activation is independent of secreted antibody, again arguing for a direct, cognate interaction. To explain the effect of the B cell Ag on T cell activation we observed here, one would have to suppose that AM14 B cells could activate a substantial enough fraction of T cells to make this pathway apparent by bulk FACSTM analysis. It is unlikely that there is a high frequency of T cells specific for self-IgG2a, the AM14 antigen. However, RF B cells can take up immune complexes and present a wide variety of autoAgs, provided they were complexed with self-IgG 70. This is expected to be the case in B6/lpr mice, which produce a variety of IgG2a autoAbs, including antichromatin 68. Indeed, this feature of RF B cells—the ability to present many autoAgs to T cells and thus garner T cell help—may explain why RF is a predominant specificity in Fas-deficient mice 63 as well as in several autoimmune diseases. It is worth noting that although the origin of B220+ T cells in lpr mice is unclear, most "double-negative" T cells arise from a CD8+ T cell precursor 7172. If the generation of B220+ T cells is related to the same process, then an effect of B cells on CD8+ T cells may be playing a role. In this regard, activated and memory phenotype CD8+ T cells fail to accumulate in MRL/lpr mice in the absence of B cells 5. Further work will be required to expand on this unexpected observation and to identify the T cells promoted by RF B cells and the autoAgs that are recognized.
Implications for the Role of Fas/FasL in Regulating Autoimmunity.
Fas deficiency is the major determinant of autoAb production in B6/lpr mice 4445. Because of constraints and logistics of animal breeding as well as the desire to compare results to those previously obtained on the BALB/c background 4041, we have only been able to compare Fas-deficient mice on the B6 background to Fas-sufficient mice on the BALB/c background. Thus, we cannot formally rule out a role for background genes in the B6 strain. However, this seems very unlikely, as neither strain background is associated with autoimmunity or RF production and especially because many of the phenotypes we show—high numbers of RF ELISpots, activated B cells at the T–B interface and in the red pulp, and accumulation of B220+ T cells—are distinctive to the Fas-deficient phenotype. With this caveat in mind, our results bear on how Fas normally prevents autoAb production. Fas is expressed throughout B cell ontogeny, including in developing B cells, newly anergized B cells, newly activated B cells, germinal center (GC) B cells, and plasma cells 73747576777879. In principle, Fas deficiency could be critical at multiple stages of B cell development and tolerance in promoting autoimmunity. Our data demonstrate that Fas deficiency need not act at early stages of tolerance (editing, deletion, and anergy) to promote autoreactivity. In the presence of functional Fas, AM14 B cells develop beyond these stages and are quiescent in the presence or absence of antigen. Fas must therefore play an important role in the regulation of RF B cells during or after autoAg-specific activation, because when Fas is deficient, these B cells expand, persist, and make autoAb, but only in the presence of autoAg. It is also possible that Fas is acting more indirectly, e.g., by causing increased IgG2a autoAg levels. In our case, this is doubtful, as the average serum IgG2a level in a small group of 4–5-mo-old HLab mice is 40 ± 19 µg/ml (± 1 SD), levels that are similar to those in BALB/c mice. More work needs to be done to pinpoint at which stage(s) after the initial Ag activation event Fas is playing a role. It is notable in this regard that although Fas is expressed in GC B cells, GC reactions appear relatively normal in Fas-deficient mice 8081. We observed that RF Tgic B6/lpr mice accumulated large numbers of RF AFCs with few GCs (data not shown) and had a relative B lymphopenia. This raises the possibility that Fas on plasma cells is playing at least one critical role.
Fas deficiency is likely not the cause of human systemic autoimmunity. However, given the central role of Fas/FasL and possibly other homologues in the TNFR/TNF family in immune system homeostasis, it does seem likely that subtle defects in apoptotic pathways could underlie human disease 8283. In this regard, pure Fas deficiency may be an excellent model for understanding the in vivo pathogenic mechanisms of such deficiencies. The overall impact of this idea will only be known when genes that promote lupus are identified 8485 and the Fas and Fas-related signaling pathways are better elucidated.
AM14 B6/lpr Mice Have a Novel Phenotype Compared with Other AutoAb Tgic Mice.
Previous studies had investigated how autoimmune-prone genetic backgrounds affected the regulation of B cells that would normally be tolerant rather than ignorant. In the case of mice that edited or deleted B cells specific for model self-antigens such as Class I or HEL, it was generally found that regulation was intact even in autoimmune-prone backgrounds 2526. These data suggested that Fas and Fas/MRL defects did not grossly impair central tolerance at this level for these autoAgs.
Regulation of B cells specific for either HEL or DNA that are anergic in normal mice has also been investigated on the B6/lpr or MRL/lpr backgrounds. In the case of HEL, induction of anergy was essentially intact 25. In related experiments, Rathmell et al. transferred anergic anti-HEL B cells along with activated CD4+ T cells and demonstrated that anergic cells are sensitive to elimination; this did not occur when the T cells were deficient in FasL, indicating a role for this pathway in the elimination of anergic cells when T cell help was also being delivered 86. This remains a potential pathway by which autoreactive B cells could escape regulation, although unmanipulated Fas- or FasL-deficient mice did not show gross defects in self-tolerance, as discussed above. The situation is different in our model, as the B cells are not anergic and should be capable of rescue by surface Ig cross-linking 87; thus, the Fas pathway must function during additional regulatory steps (see below).
The situation is more complex for anti-DNA B cells; their fate may depend on the specificity/affinity of the DNA-specific B cell. In normal mice, anti-ssDNA B cells appear anergic, albeit with a somewhat different phenotype from the anti-HEL anergic B cells 2733. Some dsDNA-specific B cells may be subject to receptor editing/deletion 28293088, but others may persist in the periphery localized at the T–B interface, where they turn over rapidly 3234. In autoimmune MRL/lpr mice Tgic for the 3H9 H chain, which can generate a variety of anti-DNA depending on the endogenous L chain 89, anti-DNA is seen in the serum and hybridomas secreting antinuclear Abs with homogenous nuclear staining are readily detected 3139. These autoAbs may arise through several pathways; they may result from loss of central tolerance, though recent detection of anti-dsDNA B cells in BALB/c spleen by Roark et al. 34 suggests that rescue from anergy may also be possible. Recent work by Weigert and colleagues on anti-ssDNA site-directed Tgic mice also suggests that rescue from anergy in MRL/lpr mice can occur 90. As there cannot be an antigen-free anti-DNA Tgic, determination of the ontogeny of the autoreactive B cells and, in particular, proving that this is a specific process is less straightforward than in the RF system. In any case, the anti-DNA models represent a different scenario from ours in that the precursors of anti-DNA Abs are thought to escape from either deletion or anergy, whereas in the case of AM14, ignorant B cells are being positively selected by Ag.
Conclusion.
We have shown that B cells that would be ignored in a normal mouse are driven to activation, expansion, and secretion in an autoimmune-prone Fas-deficient mouse. This process is specific and requires the presence of autoAg as well as the Fas defect. Because this is the only autoAb Tgic system, to our knowledge, that demonstrates Ag-specific activation of defined, nontolerant mature B cells, it provides a unique opportunity to study the temporal and spatial course of the initiating events of Fas-deficient B cell autoimmunity and perhaps ultimately other scenarios of B cell autoimmunity. It may also shed light on the induction of autoreactive T cells and may be a system for their generation and study in vitro and in vivo.
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Acknowledgments |
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This work was supported by National Institutes of Health grant P01 AI36529.
Submitted: 7 April 1999
Revised: 11 June 1999
Accepted: 28 June 1999
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