Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of multiple autoantibodies against nuclear antigens and the production of certain autoantibodies that appear to cause damage to specific organs (1, 2). Despite the evidence for the pathogenic role of autoantibodies, the mechanisms that lead to their production remain largely unknown.
Natural antibodies are autoantibodies occurring physiologically in the sera of healthy humans and animals that are thought to serve beneficial functions, including clearance of senescent and apoptotic cells and provision of immediate responses to pathogenic bacteria (3–5). They are produced largely by CD5+ B cells, which are abundant in the fetal stage of development, but contract into a minor fraction in the adult, suggesting that adult CD5+ B cells are a remnant of a distinct fetal differentiation pathway (6). The characteristic feature of the natural antibody repertoire is multireactivity that results in low-affinity binding to many self antigens. Studies of the structure and function of natural antibodies suggest that distinct molecular processes involved in the generation of the Ig repertoire provide a structural basis for multireactivity. Polyreactive antibodies frequently rely on developmentally selected Ig variable-region genes of fetal and neonatal origin, possibly in unmutated configuration (7–9). In addition, junctional sequences of the third complementaritydetermining region (CDR3) have been suggested to play a crucial structural role in multiple antigen binding (10, 11).
Because of the reactivity of natural antibodies with various self antigens and their role in apoptotic cell clearance, it has been suggested that natural antibodies may serve as a template for high-affinity autoantibodies emerging in patients with SLE. Several VH and VL genes used by human IgG anti-DNA hybridomas were found to be expressed in the natural antibody repertoire (12). A human SLE-related anticardiolipin/single-stranded DNA autoantibody was found to be encoded by a somatically mutated variant of the developmentally restricted VH gene, 3–23 (13). Through structural modifications, such as somatic mutation, and subsequent selection processes during abnormal immune responses, the physiologic natural antibody repertoire may ultimately encode monoreactive, high-affinity, and potentially pathogenic autoantibodies characteristic of SLE (14, 15). The conclusions from these data indicate that there may be an important contribution of the natural antibody repertoire in the development of pathogenic autoantibodies in patients with SLE. However, a comprehensive analysis of the human natural antibody repertoire in comparison with the SLE Ig repertoire has not previously been reported.
To understand in detail the mechanisms of autoantibody production in patients with SLE, we characterized the Vλ repertoire of patients with SLE and compared it with the natural antibody repertoire obtained from human fetal spleen and normal adult CD5+ B cells. A detailed Ig λ gene repertoire was generated from genomic DNA obtained from individual B cells by polymerase chain reaction (PCR). This technique favors the identification of both the productive and the nonproductive repertoires and allows the introduction of only minimal bias by lymphocyte activation. Using this approach, we found that the expressed Vλ repertoire of SLE patients manifests characteristics of the natural antibody repertoire. The Vλ genes 3L and 1G, which are not commonly used in the conventional adult CD5– B cell population, were more frequently observed, and the Vλ genes 3R and 3H were less frequently observed. Junctional diversity was limited by selection for sequences with limited N nucleotide addition as well as selection for homology-mediated joining. There was also profound expansion of Vλ clones, especially those utilizing overexpressed Vλ genes 3L and 1G, and homology-mediated joining at the junctions. These observations imply that the fetal antibody repertoire with restricted antigen specificities can be conserved through development via CD5+ B cells and, during abnormal immune responses, can potentially give rise to SLE-associated autoantibodies.
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- MATERIALS AND METHODS
Self-reactive B cells are negatively affected during development, either being induced to enter an anergic state or being deleted upon encounter with self antigen (21, 22). If self-reactive B cells are not eliminated by negative selection, editing of the B cell receptor operates to eliminate the self reactivity (23, 24). In SLE, there is a major dysfunction in the self-tolerance mechanism, and B cells that somehow escape tolerance produce pathogenic autoantibodies directed against various nuclear antigens.
Escape of self tolerance can also occur in a physiologic state. A unique subset of CD5+ B cells can escape tolerance and express B cell receptors with self reactivity and produce physiologically autoreactive natural antibodies (4). Both SLE B cells and CD5+ B cells can express self reactivity. Whereas natural autoantibodies produced by CD5+ B cells are low affinity and multireactive, autoantibodies produced by SLE B cells are high affinity and monospecific. It is possible, therefore, that pathogenic autoantibodies in patients with SLE could originate from the structurally restricted natural antibody repertoire.
Previous analyses of the natural autoantibody repertoire using hybridoma models suggested some connection between the natural antibody repertoire and the pathogenic autoantibodies of SLE (7–11). In addition, increased numbers of CD5+ B cells have been found in the peripheral blood of patients with SLE (25, 26). However, a comprehensive analysis of the human natural antibody repertoire in comparison with the SLE Ig repertoire has not been performed previously.
In this study, we analyzed the fetal IgM+ B cell repertoire and the adult IgM+,CD5+ B cell repertoire to determine the characteristics of the human natural antibody repertoire and compared them with those of the SLE B cell repertoire. The λ L chain was chosen for analysis since germline-encoded genetic elements of the L chains were found to be critical in conferring DNA binding specificities (15, 27), and anti-DNA–associated idiotypes are reported to be encoded by the VλII family genes (28).
Analysis of the Vλ gene usage clearly demonstrated recurrent usage of 2 Vλ genes, 1G and 3L, in both the natural antibody and the SLE repertoires. Since the recurrent usage of these 2 genes was found in the productive as well as the nonproductive repertoires, this finding suggests that the frequent expression of these 2 genes occurs not because of selection but, rather, because of preferential rearrangement during the somatic recombination process. Both Vλ genes, 1G and 3L, are not commonly used in the conventional CD5– B cell repertoire. Gene 1G was found to be the sixth most frequent of the commonly rearranged genes, and 3L was not detected in the productive repertoire of conventional IgM+,CD5– B cells (17). In addition, 1G is normally negatively selected, but less so in SLE B cells.
It is not clear why these 2 Vλ genes are preferentially rearranged in the autoreactive state. One possibility is that these genes contain CDR1 and CDR2 sequences that might lead to autoantigen-binding properties, such as germline-encoded charged residues (29–32). In this regard, 1G contains an additional Arg residue in the CDR2 region, as compared with other functional Vλ genes. Notably, 3L, which is positively selected in SLE, also contains an Arg in all 3 CDR regions. It is also noteworthy that 1G and 3L contain 20 and 23 RGYW/WRCY motifs, respectively, which are known targets of the somatic hypermutation mechanisms and may provide frequent targets for mutations.
To assess the Vλ–Jλ rearrangement dynamics in the autoreactive state, preferential joining of certain Vλ and Jλ gene segments and the use of multiple λ rearrangements by receptor editing were analyzed. The preferential recombination of certain Vλ and Jλ gene segments was found in the natural antibody and the SLE nonproductive repertoires, whereas in the adult CD5– population, the Vλ and Jλ recombination was generally random. Most notably, the Vλ1G–Jλ3B association was frequently observed in the natural and the SLE repertoires. Preferential recombination of certain Vλ and Jλ gene segments can contribute to the development of autoreactivity by resulting in restriction of CDR3 diversity (30, 33–35). Although a previous report suggested the possibility of increased receptor editing of the Vλ locus in a patient with SLE (36), we could not find evidence of multiple λ rearrangements in our analysis.
The frequency of association of the most Jλ-distal cluster C Vλ genes and the most downstream Jλ gene, Jλ7, was not significantly different compared with the expected frequency in both the productive and the nonproductive repertoires. However, the usage of cluster C Vλ–Jλ7 associations in the fetal, CD5+, and SLE nonproductive repertoires appeared to be less frequent compared with the expected frequency, although the difference did not reach statistical significance. In the productive repertoires, cluster C Vλ–Jλ7 associations were significantly less frequent in fetal, CD5–, and SLE B cells compared with the adult CD5– population. This finding suggests that in both the natural antibody and SLE repertoires, proximity of gene segments in the locus plays a role in the somatic recombination process that limits the utilization of the most-downstream Jλ gene, Jλ7. Evidence of increased receptor editing in the fetal, CD5+, and SLE populations was not found. In fact, the data could be interpreted as being compatible with less receptor editing in these populations.
Analysis of the Vλ–Jλ junctions revealed severe restrictions in junctional diversity in the natural antibody repertoire, and this restriction was also found in the SLE repertoire. Fetal and neonatal junctional diversity is known to be limited by a lack of N nucleotide addition (34, 35, 37, 38) and predominance of homology-directed junctional sequences (35, 39). The results of such restrictions would be significantly limited heterogeneity in the antigen-binding CDR3, which could contribute to the likelihood of multireactivity, and low-affinity binding to self antigens (40, 41).
We observed that the frequencies of N nucleotide additions in the human fetal Vλ–Jλ junctions were ∼20%. Although not to the level of the fetus, utilization of N nucleotide addition at the adult CD5+ and SLE productive Vλ–Jλ junctions was significantly restricted compared with the 60% frequency of the conventional CD5– productive Vλ–Jλ junctions containing N nucleotides. However, this restriction was not found in the nonproductive Vλ–Jλ junctions, suggesting that the restrictions in utilization of N nucleotide addition are related to selection, rather than the molecular mechanisms of somatic recombination. Further restriction in the Vλ–Jλ junctions was observed in the productive repertoires of both the natural antibody and the SLE repertoires by increased utilization of homology-mediated joining. Notably, this increased utilization of homology-mediated joining was observed only in the productive repertoire, suggesting that limitation of junctional diversity by homology-mediated joining is a result of an active positive selection process. The developmental selection for homology-mediated joining was also shown in an analysis of the muscovy duck Ig repertoire (42).
One of the remarkable findings of this study was the identification of numerous identical Vλ–Jλ junctions in both the natural antibody and the SLE repertoires. Although identical Vλ–Jλ junctions were also found in the conventional CD5– B cell repertoire, the frequencies were significantly lower. It was striking that significant numbers of identical Vλ–Jλ junctions found in the fetal, adult CD5+, and SLE repertoires contained junctional sequences by homology-mediated joining. The frequent occurrence of identical Vλ–Jλ junctions utilizing homology-mediated joining in the natural antibody and SLE repertoires could imply clonal expansion of such B cells (36). However, since in a previous analysis of the fetal λ repertoire, we observed numerous Vλ “clones” with disparate heavy-chain rearrangements, the frequent occurrence of identical Vλ–Jλ junctions may be a result of extreme selection caused by VH–VL pairing constraints (17). In particular, 2 Vλ clones, Vλ1G–Jλ3B and Vλ3L–Jλ3B, were found in both the fetal and the SLE repertoires. Genes 1G and 3L were the most frequently expressed Vλ genes in the natural antibody and SLE repertoires observed in this study. When clones were analyzed in detail, fetal clones were in unmutated germline configuration, whereas SLE clones contained several somatic mutations. Although not found in the fetal repertoire, 1 particular Vλ clone, Vλ1G–Jλ3B, was found in 7 identical versions from 1 SLE patient and in 3 versions from another patient, suggesting clonal expansion driven by a common autoantigenic influence.
In summary, there are similarities between the fetal, adult CD5+, and SLE Vλ repertoires, and future studies should focus on the possibility that the bulk of the Ig repertoire of patients with active SLE is “fetal like.” Moreover, the results are consistent with the conclusion that somatic mutations in B cells expressing natural antibodies may lead to their expansion and predispose to their eventual transformation to pathogenic autoantibodies (43, 44).