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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To understand in detail the mechanisms of autoantibody production in patients with systemic lupus erythematosus (SLE), we performed a comprehensive analysis of the normal human immunoglobulin light chain Vλ repertoire and compared it with the Vλ repertoire in SLE patients.

Methods

The SLE Vλ repertoire of B cells obtained from 3 SLE patients was analyzed and compared in detail with the Vλ repertoire of IgM+ B cells obtained from 3 human fetal spleens and IgM+,CD5+ B cells obtained from 2 normal adults. Conventional IgM+,CD5– B cells obtained from normal adults were used as controls. Vλ–Jλ rearrangements were amplified from the genomic DNA of individual B cells by polymerase chain reaction.

Results

The expressed Vλ repertoire of SLE patients contained several similarities with the expressed repertoire of the fetus and the adult CD5+ B cells. The Vλ genes 3L and 1G were overexpressed in the fetus, the adult CD5+ B cells, and the patients with SLE. The selection for rearrangements with restricted junctional diversity by utilization of homology-mediated joining, together with diminished N nucleotide addition, was a prominent feature of fetal, adult CD5+, and SLE B cell repertoires. Furthermore, profound expansion of Vλ clones with identical third complementarity-determining regions was observed in the adult CD5+, fetal, and SLE B cell repertoires. Notably, significant numbers of expanded adult CD5+ B cells, fetal, and SLE Vλ clones utilized homology-mediated joining at the Vλ–Jλ junctions.

Conclusion

These data demonstrate that the SLE Vλ–Jλ repertoire manifests characteristics of normal adult IgM+,CD5+ and fetal B cell populations that are known to be enriched for the production of natural autoantibodies.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Cell preparation and sorting.

The method of cell purification, B cell staining and sorting, as well as the primer extension preamplification procedure have been published (16, 17). Briefly, SLE B cells were obtained from the peripheral blood of 3 patients with active, previously untreated SLE who fulfilled the American College of Rheumatology revised criteria for the classification of SLE (18). Experiments were carried out according to a protocol that was approved by the Ethics Committee of the University of Texas Southwestern Medical Center.

Mononuclear cells were enriched by Ficoll-Hypaque density-gradient centrifugation. The cells were then stained with a phycoerythrin-labeled anti-CD19 monoclonal antibody (Becton Dickinson, Mountain View, CA) and a fluorescein isothiocyanate–labeled anti-human IgM monoclonal antibody (Caltag, Burlingame, CA). Because of the decreased blood cell count, only CD19+ B cells were isolated from the SLE patients. The cells were sorted using a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA) outfitted with an automated single-cell–deposition unit, and 1 cell was deposited into each well of a 96-well PCR plate assembled on a microAmp base. Each well contained 10 μl of PCR buffer (50 mM KCl, 10 mM Tris HCl, pH 9.0, 0.1% Triton X-100).

Fetal sequences from 3 human fetal spleens (18 weeks of gestation) were used for analysis of the IgM+ fetal B cell repertoire. Adult sequences from 2 healthy, normal male donors (ages 26 years and 45 years) were used for analysis of adult IgM+/CD5+ and IgM+/CD5– B cell populations. Both the fetal and adult sequences have been described previously (17, 19).

PCR amplification.

PCR amplification included an initial primer extension preamplification (16) and subsequent nested PCR steps (17). Single cells in 10 μl of PCR buffer were incubated with 0.4 mg/ml of proteinase K (Sigma, St. Louis, MO) for 1 hour at 55°C, and the enzyme was inactivated by heating at 95°C for 10 minutes. Primer extension preamplification using random 15-mers and 60 rounds of amplification with Taq polymerase (Promega, Madison, WI) was performed to produce sufficient DNA for multiple subsequent amplifications. Rearranged Vλ–Jλ genes were then amplified as described previously (19).

Sequence analysis.

PCR products were separated by electrophoresis on a 1.5% SeaKem agarose gel (FMC Bioproducts, Rockland, ME) and purified using GenElute agarose spin columns (Supelco, Bellefonte, PA). Purified products were directly sequenced using the ABI Prism Dye Termination Cycle sequencing kit (Perkin-Elmer, Foster City, CA) and an automated sequencer (ABI Prism 377; Applied Biosystems, Foster City, CA). For identification of the germline Vλ and Jλ gene segments, the V BASE Sequence Directory (20) was used in conjunction with the software programs Sequencher (Gene Codes, Ann Arbor, MI) and Lasergene99 (DNAStar, Madison, WI). Vλ and Jλ gene nomenclature was adopted according to the V BASE Sequence Directory.

A rearrangement was considered productive if the Vλ–Jλ junction maintained the reading frame into the Jλ segment. Rearrangements that failed to maintain the reading frame into the Jλ gene segment and rearrangements that introduced a stop codon at the junction during the rearrangement process were considered to be nonproductive. Rearrangements that involved pseudogenes were always considered nonproductive. At the junctions, sequences were considered to be germline if at least 2 contiguous nucleotides matched the germline sequence. Junctional additions were scored as either inverted repeats at full-length coding ends (P nucleotides) or nontemplated junctional additions (N nucleotides). When junctions without N additions contained repeated nucleotides that could not be unequivocally assigned to either coding end, these were assigned as homology-mediated junctional sequences.

A summary of the λ sequences analyzed and their origin is shown in Table 1. A total of 208 SLE λ sequences, including 158 productive and 50 nonproductive rearrangements, 154 fetal λ sequences, including 84 productive and 70 nonproductive rearrangements, and 99 adult CD5+ B cell λ sequences, including 68 productive and 31 nonproductive rearrangements, were analyzed. These sequences were compared with 102 adult CD5– λ sequences (78 productive, 24 nonproductive). In this analysis, clonally identical rearrangements in the productive repertoire were considered as the same sequence and counted as only 1 sequence. All sequences are available from the GenBank Sequence database (accession nos. AY424410–AY424644, AF247204–AF247344, and AJ230234–AJ230460).

Table 1. Summary of the λ sequences analyzed*
OriginNo. productiveNo. nonproductiveTotal
  • *

    SLE = systemic lupus erythematosus.

Fetal B cells8470154
 Sample 15855118
 Sample 211516
 Sample 3151025
Adult CD5+ B cells683199
 Donor 128432
 Donor 2402767
Adult CD5− B cells7824102
 Donor 115318
 Donor 2632184
SLE B cells15850208
 Patient 111028138
 Patient 220929
 Patient 3281341

Estimation of Taq polymerase error rate.

The maximal PCR error rate for this analysis has been documented to be 1.0 × 10–4/bp (19). Thus, few, if any, of the nucleotide changes encountered in this analysis can be ascribed to PCR amplification errors.

Statistical analysis.

Chi-square tests were used to compare the gene usage, junctional nucleotide differences found in the productive and nonproductive repertoires, as well as the differences between the SLE, fetal, adult CD5+, and adult CD5– populations. The goodness-of-fit chi-square test was used to compare the actual distribution of Vλ and Jλ gene usage with the frequency that might be expected based on the number of genes in the genome. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Vλ and Jλ gene usage.

To determine whether there was restricted usage of certain Vλ and Jλ genes in the natural antibody repertoire and the SLE repertoire, individual Vλ and Jλ gene usage was analyzed. The distribution of individual Vλ gene usage is shown in Figure 1. In the productive repertoires, 4 genes were found to be different between the SLE, fetal, adult CD5+, and adult CD5– populations. The difference was statistically significant for 3L, 3R, and 3H, and there was a trend toward statistical significance for 1G. For Vλ genes 1G and 3L, the difference was caused by more frequent usage of these genes in the fetal, adult CD5+, and SLE populations compared with the adult CD5– population. In contrast, Vλ genes 3R and 3H were less frequently used in the fetal, adult CD5+, and SLE populations compared with the adult CD5– population. More frequent utilization of 1G was also found in the nonproductive repertoires of fetal, adult CD5+, and SLE B cells, suggesting that 1G appears frequently in the expressed natural antibody and SLE repertoires by preferential rearrangement rather than selection.

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Figure 1. Vλ gene usage in fetal, adult CD5+, adult CD5–, and systemic lupus erythematosus (SLE) B cells. Distributions of A, productive rearrangements and B, nonproductive rearrangements are shown. The x-axis shows Vλ genes arranged according to the chromosome position in the λ-locus, with the most Jλ-distal gene depicted as farthest to the left. ∗ = P < 0.05 by chi-square test.

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Jλ gene usage in the nonproductive repertoire showed similar patterns in the fetal, adult CD5+, SLE, and adult CD5– populations. Jλ3B was used most often, followed by Jλ2/3, Jλ7, and Jλ1. In the productive repertoire, Jλ2/3 was most frequently used, followed by Jλ3B (Figure 2).

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Figure 2. Jλ gene usage in fetal, adult CD5+, adult CD5–, and systemic lupus erythematosus (SLE) B cells. ∗ = P < 0.05 by chi-square test. P = productive; NP = nonproductive.

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To assess the Vλ–Jλ rearrangement dynamics, we analyzed Vλ and Jλ segment associations in the nonproductive repertoire (Figure 3). Preferential rearrangement (n > 3) of certain Vλ and Jλ segments was observed in the fetal, adult CD5+, and SLE populations, although such preferential rearrangement was most dominant in the fetal nonproductive repertoire. The Vλ1G–Jλ3B combination was commonly observed in the fetal (n = 7), adult CD5+ (n = 5), and SLE (n = 4) populations, whereas in the adult CD5– population, Vλ and Jλ segment association was generally random.

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Figure 3. Frequency of Vλ and Jλ gene associations in the nonproductive rearrangements of fetal, adult CD5+, adult CD5–, and systemic lupus erythematosus (SLE) B cells. The x-axis shows Vλ genes arranged according to the chromosomal position in the λ-locus, with the most Jλ-distal gene depicted as farthest to the left. The y-axis shows the number of times each Jλ is used. Solid bars show Jλ1; open bars show Jλ2/3; hatched bars show Jλ3B; lightly shaded bars show Jλ7.

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In addition, we analyzed associations between the most Jλ-distal cluster C Vλ genes and the most downstream Jλ gene, Jλ7, to assess whether multiple λ rearrangements occurred (Table 2). In the nonproductive repertoire, the frequency of cluster C Vλ–Jλ7 associations was not significantly different between the fetal, adult CD5+, adult CD5–, and SLE populations. Moreover, no significant differences were observed when the actual frequencies were compared with the predicted frequencies based on random utilization and association. In the productive repertoire, the frequency of cluster C Vλ–Jλ7 associations was significantly different between the fetal, adult CD5+, adult CD5–, and SLE populations, in that there was less frequent use of cluster C Vλ–Jλ7 associations in the fetal (0%), adult CD5+ (11.1%), and SLE (6.2%) populations compared with the adult CD5– population (50%).

Table 2. Cluster C Vλ genes and Vλ–Jλ relationships*
 FetusAdultSLEExpected frequency, %
CD5+CD5−
  • *

    Values are the number/number tested (% of total) or the number (% of total cluster C). SLE = systemic lupus erythematosus.

  • Significant differences in frequency (P ≤ 0.05) than predicted from its presence in the genome, by goodness-of-fit chi-square test.

  • Significant differences in cluster C Vλ usage (P ≤ 0.05) between the fetal, adult CD5+, adult CD5−, and SLE populations, by chi-square test.

Nonproductive     
 Cluster C Vλ gene10/70 (14.3)8/31 (25.8)4/24 (16.7)8/50 (16.0)16.7
 Cluster C Vλ–Jλ 1–310 (100)7 (85.7)3 (75.0)7 (85.7)75
 Cluster C Vλ–Jλ 70 (0)1 (14.3)1 (25.0)1 (14.3)25
Productive     
 Cluster C Vλ gene10/84 (11.9)9/68 (13.2)8/78 (10.3)16/158 (10.1)16.7
 Cluster C Vλ–Jλ 1–310 (100)8 (88.9)4 (50)15 (93.8)75
 Cluster C Vλ–Jλ 70 (0)1 (11.1)4 (50)1 (6.2)25

Junctional diversity.

Junctional diversity was analyzed by the frequency and nature of processing at the coding ends, P and N nucleotide additions, and homology-mediated joining (Figure 4). There were no significant differences in the frequencies of 5′-untrimmed ends, 3′-untrimmed ends, and 5′ or 3′ P nucleotide addition between the fetal, adult CD5+, SLE, and adult CD5– populations in either the productive or the nonproductive repertoires. However, significant differences were found in the frequencies of N nucleotide addition and homology-mediated joining.

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Figure 4. Analysis of Vλ–Jλ junctions according to the frequencies of nucleolytic processing at the coding ends, P nucleotide addition, N nucleotide addition, and homology-mediated joining. ∗ = P < 0.05 by chi-square test. B cells from the following sources are shown: fetus (hatched bars), adult CD5+ (open bars), adult CD5– (solid bars), and systemic lupus erythematosus patient (shaded bars). NP = nonproductive; P = productive.

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The frequency of N nucleotide addition found in the fetal nonproductive junctions was 29%, whereas in adult nonproductive junctions, the frequency was ∼60% (adult CD5+ 61%, adult CD5– 58%, SLE 64%). In the productive repertoire, there was a significantly decreased utilization of N nucleotide addition in the fetal (20%), adult CD5+ (47%), and SLE populations (48%) compared with the adult CD5– population (60%). In contrast, there was a significant increase in the utilization of homology-mediated joining in the productive repertoires of the fetal, adult CD5+, and SLE populations (58% versus 43% versus 37%) compared with the adult CD5– productive repertoire (27%). Notably, the increased utilization of homology-mediated joining in the productive repertoires of the fetal, adult CD5+, and SLE populations was significantly greater than that found in the nonproductive repertoires, suggesting positive selection for homology-mediated joining in these populations.

CDR3 length distributions.

The average CDR3 length in the productive repertoire was similar between the fetal, adult CD5+, SLE, and adult CD5– populations. However, in the nonproductive repertoire, the mean lengths of the CDR3 in the fetal and adult CD5+ populations were ∼3 bp shorter than those in the adult CD5– and SLE populations. No significant restrictions in CDR3 lengths were found between the 4 populations (Table 3).

Table 3. Comparison of the lengths of the third complementarity-determining region in rearranged Vλ–Jλ joins*
 FetusAdultSLE
CD5+CD5−
  • *

    Values are the mean ± SD bp. SLE = systemic lupus erythematosus.

Productive30.9 ± 2.331.5 ± 3.131.5 ± 2.732.0 ± 3.5
Nonproductive31.8 ± 4.431.2 ± 4.134.2 ± 5.9733.0 ± 5.8

Identification of clonal expansion.

When Vλ–Jλ junctional sequences from the productive rearrangements were analyzed in detail, it was noteworthy that numerous identical junctions were observed in all 4 populations (Figure 5). However, there was a significant difference between the frequencies of clonally related Vλ genes in the fetal, adult CD5+, adult CD5–, and SLE populations. In the fetal and adult CD5+ populations, respectively, 19% and 22% of the total productive rearrangements were represented more than once. In the SLE B cells, although frequencies were lower than in the fetal and adult CD5+ populations, 9% of the total productive rearrangements were represented more than once. This was in contrast to significantly lower frequencies of Vλ clones found in the adult CD5– population (6%) (Figure 6A). Notably, there was a significantly greater number of fetal, adult CD5+, and SLE Vλ clones utilizing homology-mediated joining compared with the adult CD5– Vλ clones (Figure 6B). The frequencies of homology-mediated joining found in the fetal, adult CD5+ Vλ, SLE, and adult CD5– clones were 75%, 73%, 67%, and 19%, respectively.

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Figure 5. Nucleotide sequences of Vλ clones containing productively rearranged Vλ–Jλ junctions from the fetal, adult CD5+, adult CD5–, and systemic lupus erythematosus (SLE) B cells. Sequences are aligned under prototypical germline sequences. n = number of clones isolated with particular Vλ–Jλ join. Nucleotides lost from the coding ends are indicated as hyphens. P nucleotide additions are shown in boldface. N additions are in regular type between 2 coding ends. Homology-mediated junctional sequences are boxed at the 5′-coding end.

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Figure 6. A, Frequency of Vλ clones in the fetal, adult CD5+, adult CD5–, and systemic lupus erythematosus (SLE) productive rearrangements. The numerator is the total number of clones in each population; the denominator is the total number of rearrangements analyzed from each population (in which the clone was counted only once). B, Frequency of the utilization of homology-mediated joining by Vλ clones. The numerator is the number of clones using homology-mediated joining in each population; the denominator is the total number of clones from each population analyzed. ∗ = P < 0.05 for all comparisons, by chi-square test. Arrows indicate change from the adult IgM+,CD5– population.

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In particular, 2 of the Vλ clones, Vλ1G–Jλ3B and Vλ3L–Jλ3B, were found in both the fetal and SLE repertoires. In the Vλ1G–Jλ3B clone, 4 identical versions were found in the SLE repertoire from 1 patient and 2 were found in the fetus. In the Vλ3L–Jλ3B clone, 3 identical versions were found in the SLE repertoire and 9 were found in the fetus (Figure 5). Of note, Vλ genes 1G and 3L were frequently expressed in the fetal, CD5+, and SLE productive repertoires. In addition, it was noteworthy that 7 identical versions of Vλ1G–Jλ2/3 rearrangements were detected in 1 SLE patient, and 3 of the same sequences were observed in another SLE patient (Figure 7). This clone utilized homology-mediated joining at the junction.

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Figure 7. Sequences of Vλ clones found in 2 patients with systemic lupus erythematosus. Sequences are aligned under the germline sequences. Nucleotides that are identical with the germline sequences are indicated as hyphens. Nucleotide changes made by replacement mutations are indicated as uppercase letters; silent mutations are indicated as lower case letters. Homology-mediated junctional sequences are boxed at the 5′-coding end.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES