To determine the distribution of Vλ and Jλ as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vλ and Jλ or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vλ and VH was noted among the productive rearrangements. The distribution of productive Vλ rearrangements was skewed, with significantly greater representation of the Jλ distal cluster C Vλ genes and the Vλ distal Jλ7 element, consistent with the possibility that there was receptor editing of the Vλ locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vλ1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.

Systemic lupus erythematosus (SLE)3 is an autoimmune disease characterized by the production of multiple autoantibodies, especially anti-dsDNA Abs (1). Despite intensive study, the factors that lead to the production of autoantibodies in SLE remain unknown. The possibility that there are abnormalities in the Ig repertoire of patients with SLE has not been completely examined. It is not known whether an aberrant V(D)J recombination process itself predisposes to the generation of autoreactive Abs, as has been suggested for A30/Jκ2 rearrangements (2), or, alternatively, whether abnormalities in somatic hypermutation and/or subsequent selective influences can lead to the generation of autoantibodies.

Recently, we found that the VκJκ recombination process, as judged by analysis of nonproductive VκJκ rearrangements in an untreated SLE patient, appeared to be comparable to that in normal subjects (3). However, striking differences in the productive Vκ repertoire of this patient were noted, with enhanced usage of the Jκ distal Vκ genes and a marked increase in the usage of Jκ5, the most Vκ distal Jκ gene. These data suggested that the replacement of primary Vκ rearrangements by subsequent rearrangements (receptor editing) was more frequent in this SLE patient than had been observed in normal subjects (4). Although these results were obtained from a single patient, they were so strikingly different from a previous VκJκ repertoire analysis in normal subjects (4) that a more extensive examination of this patient was conducted. Specifically an analysis of the Vλ and VH repertoire was conducted because of the possibility that Vλ and VH receptor editing in SLE might also be abnormal.

The distribution of Vλ/Jλ and VH/JH rearrangements in the normal Ig gene repertoire has been delineated recently (5, 6, 7). Therefore, to determine whether there was increased receptor editing of Vλ and VH genes in SLE or other abnormalities in the V gene repertoire, the current study analyzed and compared the usage of Vλ/Jλ and VHDJH gene elements in this same untreated SLE patient with the repertoire of normal donors. The distribution of VH and Vλ genes in the nonproductive repertoire of this SLE patient was compared with that in normal subjects, suggesting that there were no major molecular abnormalities in VλJλ recombination. Striking abnormalities in the distribution of Vλ genes were noted in the productive repertoire, however, consistent with accentuated receptor editing of Vλ genes. In addition, although no evidence of increased receptor editing of VH genes was found, skewing of the expressed VH repertoire, increased somatic hypermutation, and clonal expansion was detected. These results are consistent with the conclusion that there is marked overactivity of B cells in early SLE that could contribute to the production of autoantibodies.

The method of cell purification, B cell staining and sorting, as well as the primer extension preamplification procedure have been reported in detail recently (6, 7). Briefly, B cells were obtained from a 54-yr-old Hispanic man with SLE, who was previously undiagnosed. Features of SLE included a typical butterfly rash, hyperkeratotic lesions of SCLE, increased fatigue, intermittent episodes of fever, and arthralgias of the proximal interphalangeal joints. The antinuclear Ab titer was 1:2560 (speckled pattern), and anti-Ro, -La, and -RNP Abs were present, whereas anti-dsDNA Abs were repeatedly absent. The patient did not have hypergammaglobulinemia. It should be noted that titers of these autoantibodies remained comparable for the next 1.5 yr. Clinically, disease manifestations began about 4 wk before the B cells were sorted, as evidenced by the onset of a typical rash. The patient had noted photosensitivity for many years. At this particular time point, there were no signs or symptoms of another connective disease. A reduction of complement factors C3 (62.6 mg/dl; normal, 65–203) and C4 (<10 mg/dl; normal, 16–54) was also found. The white blood cell count was 3.8 × 103/μl, with 20% lymphocytes. Because of the decreased blood cell count, only CD19+ B cells were isolated. The patient fulfilled the revised criteria for classification of SLE (8).

Altogether 276 individual CD19+ B cells were sorted into wells of 96-well plates (Robbins Scientific, Sunnyvale, CA) using a FACStar Plus flow cytometer with an automated single cell deposit unit (Becton Dickinson, Mountain View, CA) as described previously (4, 5, 6, 7). Twelve wells (four per plate) that received no cells were used as negative controls. Rearranged VλJλ and VHDJH genes were then amplified as described recently (5, 6, 7). The PCR amplification included a primer extension preamplification (7) and subsequent nested PCR steps (5, 7). After column purification of PCR products (GenElute Agarose Spin Column, Supelco, Bellefonte, PA), all PCR products were directly sequenced using the ABI Prism Dye Termination Cycle Sequencing Kit (Perkin-Elmer, Palo Alto, CA) and analyzed with an automated sequencer (ABI Prism 377, Perkin-Elmer). Sequences were analyzed using the V BASE Sequence Directory (9) to identify the respective germline gene. For the identification of the underlying germline segments, the software programs GeneWorks (release 2.45; IntelliGenetics, Mountain View, CA) and Sequencher (Gene Codes, Ann Arbor, MI) were employed.

The usage of VH and JH genes as well as Vλ and Jλ rearrangements segments from two healthy normal male donors (26 and 45 yr old) that had been published previously (5, 6) served as a comparison. Both the nonproductive and productive repertoires of these two normal, age-disparate donors exhibited an overall similar usage of V and J gene elements.

The maximal PCR error rate for this analysis has been documented to be 1.2 × 10−3 mutations/bp, and the minimal error to be 1 × 10−4 (5, 10).

Sequences were analyzed with the χ2 test to compare the differences in the distribution of particular gene segments as well as mutational frequencies between the VλJλ and VHDJH rearrangements of the SLE patient and the normal subjects. The goodness of fit χ2 test (11) was used to compare the actual distribution of Vλ and Jλ as well as the VH and JH family gene usage in the SLE patient to the frequency that might be expected based upon the number of genes in the genome (6). P < 0.05 was considered statistically significant.

A total of 104 productive and 58 nonproductive VλJλ rearrangements were analyzed. The distribution of individual Vλ families is shown in Table I. Of importance, no major differences in Vλ gene usage between the nonproductive repertoire of the SLE patient and that of the normal subjects was found. Some minor differences between the nonproductive repertoire of normal subjects and that of the SLE patient were noted. For example, Vλ6 genes were found significantly more often in the nonproductive repertoire of the SLE patient (9%) than expected by chance alone, whereas this Vλ gene family was not over-represented in the nonproductive repertoire of the normal subjects (3.6%).

Table I.

Comparison of Vλ gene family usage in an SLE patient and in normals

Gene FamilyFunctional Genes (% of total)SLENormals
Nonproductive (NP) No. (% of total)Productive (P) No. (% of total)Comparison NP/P in SLENonproductive (NP) No. (% of total)Productive (P) No. (% of total)Comparison Normals NP/P
Vλ1 5 (16.7) 11 (19) 30b (29) NS 14 (25.4) 50b (29.1) NS 
Vλ2 5 (16.7) 14 (24) 27b (26) NS 17b (30.9) 57b (33.1) NS 
Vλ3 8 (26.7) 1b (2) 8 (8) NS 3 (5.4) 2 (15.7) 0.051 
Vλ4 3 (10.0) 12 (21) 0 (0)a 0.001 10b (18.2) 10 (5.8)a 0.005 
Vλ5 3 (10.0) 5 (9) 2 (2) 0.05 4 (7.3) 6 (3.5) NS 
Vλ6 1 (3.3) 5 (9) 18b (17)a NS 2 (3.6) 6 (3.5)a NS 
Vλ7 2 (6.7) 2 (3) 2 (2) NS 1 (1.8) 7 (4.1) NS 
Vλ8 1 (3.3) 4 (7) 14b (13)a NS 1 (1.8) 2 (1.2)a NS 
Vλ9 1 (3.3) 3 (5) 3 (3) NS 1 (1.8) 1 (0.6) NS 
Vλ10 1 (3.3) 1 (2) 0 (0) NS 2 (3.6) 6 (3.5) NS 
        
Total 30 58 104  55 172  
Gene FamilyFunctional Genes (% of total)SLENormals
Nonproductive (NP) No. (% of total)Productive (P) No. (% of total)Comparison NP/P in SLENonproductive (NP) No. (% of total)Productive (P) No. (% of total)Comparison Normals NP/P
Vλ1 5 (16.7) 11 (19) 30b (29) NS 14 (25.4) 50b (29.1) NS 
Vλ2 5 (16.7) 14 (24) 27b (26) NS 17b (30.9) 57b (33.1) NS 
Vλ3 8 (26.7) 1b (2) 8 (8) NS 3 (5.4) 2 (15.7) 0.051 
Vλ4 3 (10.0) 12 (21) 0 (0)a 0.001 10b (18.2) 10 (5.8)a 0.005 
Vλ5 3 (10.0) 5 (9) 2 (2) 0.05 4 (7.3) 6 (3.5) NS 
Vλ6 1 (3.3) 5 (9) 18b (17)a NS 2 (3.6) 6 (3.5)a NS 
Vλ7 2 (6.7) 2 (3) 2 (2) NS 1 (1.8) 7 (4.1) NS 
Vλ8 1 (3.3) 4 (7) 14b (13)a NS 1 (1.8) 2 (1.2)a NS 
Vλ9 1 (3.3) 3 (5) 3 (3) NS 1 (1.8) 1 (0.6) NS 
Vλ10 1 (3.3) 1 (2) 0 (0) NS 2 (3.6) 6 (3.5) NS 
        
Total 30 58 104  55 172  
a

Significant difference between the productive repertoire of the SLE patient compared to the productive normal repertoire (Vλ3, p = 0.053; Vλ4, p = 0.012; Vλ6, p < 0.001; Vλ8, p < 0.001; Vλ10, p = 0.054).

b

, p ≤ 0.05, significantly higher frequency than predicted from its presence in the genome (goodness of fit χ2 test); and †, p ≤ 0.05, significantly lower frequency than predicted from its presence in the genome (goodness of fit χ2 test).

Of note, however, significant differences were detected when the productive repertoires of the SLE patient and those of the normal subjects were compared. Thus, Vλ4 family members occurred significantly more often in the productive repertoire of the normal subjects than in that of the SLE patient (5.8 vs 0%; p = 0.012), whereas members of the Vλ6 (p < 0.001) and Vλ8 (p < 0.001) families were more frequently employed in the productive repertoire of the SLE patient. These differences suggest that factors dependent on expression of a productively rearranged Vλ gene may have influenced the distribution of Vλ genes in the SLE patient.

The distribution of rearranged Jλ elements in the SLE patient is summarized in Table II. Of note, the usage of Jλ2/3 and Jλ7 was significantly different between the nonproductive repertoire of the SLE patient and that of the normal subjects. Whereas Jλ2/3 genes were employed more often in the patient (58.6%) than in normal subjects (34.5%; p = 0.01), Jλ7 was found more frequently in normal subjects (60.0%) than in nonproductive rearrangements of the SLE patient (36.2%; p = 0.011). In general, the usage of Jλ1 was significantly less than expected by chance alone regardless of whether nonproductive or productive rearrangements were analyzed.

Table II.

Distribution of Jλ genes in individual B cells from an SLE patient and in normals

Expected Frequency per Jλ Segment (%)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
Jλ1 25 3 (5.2) 4 (3.8) 3 (5.5) 12 (7.0) 
Jλ2/3 50 34 (58.6)a 30†‡ (28.8) 19 (34.5)a 67 (39.0) 
Jλ7 25 21b (36.2)a 70b (67.3)a 33b (60.0)a 93b (54.1)a 
      
Total  58 104 55 172 
Expected Frequency per Jλ Segment (%)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
Jλ1 25 3 (5.2) 4 (3.8) 3 (5.5) 12 (7.0) 
Jλ2/3 50 34 (58.6)a 30†‡ (28.8) 19 (34.5)a 67 (39.0) 
Jλ7 25 21b (36.2)a 70b (67.3)a 33b (60.0)a 93b (54.1)a 
      
Total  58 104 55 172 
a

Significant difference between the frequency of usage of the Jλ genes in the nonproductive and productive repertoire, respectively, between the SLE patient and the normals (χ2 test) (p = 0.01 for Jλ2,3; p = 0.011 for Jλ7 in the nonproductive repertoires; p = 0.03 for Jλ7 between the productive repertoires).

b

, p ≤ 0.05, significantly higher frequency than predicted from its presence in the genome (goodness of fit χ2 test); †, p ≤ 0.05, significantly lower frequency than predicted from its presence in the genome (goodness of fit χ2 test); ‡, p < 0.001 significant difference between the frequency of usage of the nonproductive compared to the productive repertoire in the SLE patient; no significant differences between the frequency in the productive and nonproductive repertoires was found by the χ2 test in the normals.

When the productive repertoires of the patient and the normal subjects were compared, a significantly greater usage of Jλ7 was found in the SLE patient than in normal subjects (67.3 vs 54.1%; p = 0.03). Moreover, the frequency of Jλ7 was significantly greater in the productive than in the nonproductive repertoire of the SLE patient (67.3 vs 36.2%; p < 0.05).

As shown in Table III, there were no differences in the distribution of individual Vλ genes in the nonproductive repertoire of the SLE patient and the normal donors. When the distribution of particular Vλ gene segments in the productive repertoire of the SLE patient was compared with that in the normal subjects, four Vλ genes, 2A2 (p = 0.001), 3H (p = 0.045), 1B (p = 0.001), and 4B (p = 0.012), were found significantly less often in the SLE patient. Moreover, a significant over-representation of 3L (p = 0.025), 1G (p = 0.04), 6A (p = 0.001), and 8A (p = 0.001) was found in the productive repertoire of the SLE patient. In general, the over-represented Vλ genes in the productive repertoire of the SLE patient tended to be Jλ distal. To analyze this in greater detail, the use of Vλ genes in the three major gene clusters was assessed (Table IV). The usage of the Vλ gene clusters, A, B, and C, was similar in the nonproductive repertoires of the patient and the normal subjects. In the productive repertoires, however, the usage of Vλ genes of the most Jλ proximal cluster, A, was significantly less frequent in the SLE repertoire (33.7%) than in the normal repertoire (48.8%; p < 0.05). Moreover, genes belonging to the most Jλ distal cluster, C, were found significantly more frequently in the SLE patient (30.8%) than in normal subjects (14%; p < 0.001). Thus, the productive rearrangements of the SLE patient employed the Jλ distal Vλ gene cluster C as well as the Vλ distal Jλ7 gene element significantly more frequently than those of the normal subjects.

Table III.

Distribution of individual Vλ gene usage in an SLE patient and normals

GeneSLENormalsDifferences Between the Productive Repertoires, p Valuea
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
3R 3 (2.9) 1 (1.8) 11 (6.4)  
4C 3a (5.5)  
2A1(ψ) 2 (3.4) 6 (10.9)  
3A2(ψ) 1 (1.8)  
2C 6 (5.8) 5 (2.9)  
3J 2 (1.2)  
2E 2 (3.4) 5 (4.8) 1 (1.8) 7 (4.1)  
3I(ψ) 1 (1.8)  
2A2 1 (1.7) 4 (3.8) 5 (9.1) 30 (17.4) 0.001 
3L 3 (2.9) 0.025 
3H 1 (1.7) 2 (1.9) 0a 13a (7.6) 0.045 
2B2 9 (15.5) 12 (11.5) 5 (9.1) 15 (8.7)  
3M 1 (0.6)  
7C(ψ) 1 (1.8)  
1A 1 (1.0)  
5E 1 (0.6)  
5A(ψ) 1 (1.7) 2 (3.6)  
1E 1 (1.0) 7 (4.1)  
7A 2 (3.4) 2 (1.9) 4 (2.3)  
1C 4 (6.9) 12 (11.5) 3 (5.5) 12 (7.0)  
5C 2 (3.4) 2 (1.9) 1 (1.8) 5 (2.9)  
7B 3 (1.7)  
1G 7 (12.1) 16 (15.4) 9 (16.4) 13 (7.6) 0.04 
5D(ψ) 2 (3.4)  
9A 3 (5.2) 3 (2.9) 1 (1.8) 1 (0.6)  
1B 2 (3.6) 18 (10.5) 0.001 
5B 1 (1.8)  
10A 1 (1.7) 2 (3.6) 6 (3.5)  
6A 5 (8.6) 18 (17.3) 2 (3.6) 6 (3.5) 0.001 
8A 4 (6.9) 14 (13.5) 1 (1.8) 2 (1.2) 0.001 
4B 12a (20.7)a 0a 7 (12.7) 10 (6) 0.012 
      
Total 58 104 55 172  
GeneSLENormalsDifferences Between the Productive Repertoires, p Valuea
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
3R 3 (2.9) 1 (1.8) 11 (6.4)  
4C 3a (5.5)  
2A1(ψ) 2 (3.4) 6 (10.9)  
3A2(ψ) 1 (1.8)  
2C 6 (5.8) 5 (2.9)  
3J 2 (1.2)  
2E 2 (3.4) 5 (4.8) 1 (1.8) 7 (4.1)  
3I(ψ) 1 (1.8)  
2A2 1 (1.7) 4 (3.8) 5 (9.1) 30 (17.4) 0.001 
3L 3 (2.9) 0.025 
3H 1 (1.7) 2 (1.9) 0a 13a (7.6) 0.045 
2B2 9 (15.5) 12 (11.5) 5 (9.1) 15 (8.7)  
3M 1 (0.6)  
7C(ψ) 1 (1.8)  
1A 1 (1.0)  
5E 1 (0.6)  
5A(ψ) 1 (1.7) 2 (3.6)  
1E 1 (1.0) 7 (4.1)  
7A 2 (3.4) 2 (1.9) 4 (2.3)  
1C 4 (6.9) 12 (11.5) 3 (5.5) 12 (7.0)  
5C 2 (3.4) 2 (1.9) 1 (1.8) 5 (2.9)  
7B 3 (1.7)  
1G 7 (12.1) 16 (15.4) 9 (16.4) 13 (7.6) 0.04 
5D(ψ) 2 (3.4)  
9A 3 (5.2) 3 (2.9) 1 (1.8) 1 (0.6)  
1B 2 (3.6) 18 (10.5) 0.001 
5B 1 (1.8)  
10A 1 (1.7) 2 (3.6) 6 (3.5)  
6A 5 (8.6) 18 (17.3) 2 (3.6) 6 (3.5) 0.001 
8A 4 (6.9) 14 (13.5) 1 (1.8) 2 (1.2) 0.001 
4B 12a (20.7)a 0a 7 (12.7) 10 (6) 0.012 
      
Total 58 104 55 172  

a There were no significant differences in the frequency of gene usage between the nonproductive repertoire of the SLE patient compared to that of normals.

a

, p < 0.05, significant differences between the frequency in the productive and nonproductive repertoires (χ2 test) of the SLE patient and of the normals, respectively.

Table IV.

Distribution of Vλ gene clusters from individual B cells in an SLE patient compared to normals

ClusterKnown Functional Genes per VL Gene Cluster (% of total)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
14 (46.7) 22 (37.9) 35 (33.7)a 15 (34.9) 84 (48.8)a 
11 (36.7) 21 (36.2) 37 (35.6) 16 (37.2) 64 (37.2) 
5 (16.7) 15 (25.9) 32b (30.8)a 12b (27.9) 24 (14.0)a 
      
Total 30 58 104 43 172 
ClusterKnown Functional Genes per VL Gene Cluster (% of total)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
14 (46.7) 22 (37.9) 35 (33.7)a 15 (34.9) 84 (48.8)a 
11 (36.7) 21 (36.2) 37 (35.6) 16 (37.2) 64 (37.2) 
5 (16.7) 15 (25.9) 32b (30.8)a 12b (27.9) 24 (14.0)a 
      
Total 30 58 104 43 172 
a

Significant differences between the frequency in the productive and nonproductive repertoires, respectively, between the SLE patient and the normals (χ2 test) (nonproductive: p < 0.05 for cluster C; productive: cluster A, p = 0.04; cluster C, p = 0.001).

b

, p ≤ 0.05, significantly higher frequency than predicted from its presence in the genome (goodness of fit χ2 test); †, p ≤ 0.05, significantly lower frequency than predicted from its presence in the genome (goodness of fit χ2 test).

The mutational frequencies of nonproductive and productive Vλ rearrangements from the SLE patient were 3.12 and 3.38%, respectively (Table V). Thus, there were no major differences between the mutational frequencies of the productive and nonproductive repertoires. Moreover, the nonproductive Vλ rearrangements using Jλ7 exhibited a mutational frequency of 3.05% compared with a mutational frequency of 3.04% in the productive repertoire. Thus, the distribution of mutations in rearrangements using the most 3′-proximal Jλ element was comparable in productive and nonproductive repertoires. This compares with a mutational frequency of 3.43% for nonproductive Vλ rearrangements using Jλ1–3 and 3.53% for productive rearrangements using Jλ1–3. Analysis of mutational frequencies in Vκ genes in this patient indicated that productive Vκ rearrangements were mutated significantly less than nonproductive Vκ rearrangements (2.80 vs 3.60%; p < 0.01). Moreover, productively rearranged Vκ genes using the distal Jκ5 element were less mutated (1.99%) than rearrangements using Jκ1–4 (3.08%; p < 0.001) (3).

Table V.

Comparison of the overall mutational frequencies between productive and nonproductive Vλ and Vκ gene rearrangements of the SLE patient

RearrangementsNonproductiveProductive
Mutations (n)Total bpMutational frequency (%)Mutations (n)Total bpMutational frequency (%)
Vλ 412 13,194 3.12 807 23,819 3.38 
Vκ 145 4,054 3.60a 379 13,569 2.80a 
RearrangementsNonproductiveProductive
Mutations (n)Total bpMutational frequency (%)Mutations (n)Total bpMutational frequency (%)
Vλ 412 13,194 3.12 807 23,819 3.38 
Vκ 145 4,054 3.60a 379 13,569 2.80a 
a

, p < 0.01, significant difference between the mutational frequency (χ2 test); †, p < 0.002, significant difference between the mutational frequency (χ2 test).

Further analysis sought to compare N nucleotide addition at the joins of Vλ and Jλ elements as an estimate of TdT activity operative on the rearrangements (Table VI). Remarkably, in the nonproductive repertoire, there were no Jλ7 rearrangements that failed to contain N additions, implying that TdT activity was active during the rearrangement of these gene segments. In the productive repertoire, 20% of rearrangements employing Jλ7 exhibited no evidence of TdT activity. The opposite tendency was noted when rearrangements employing Jλ1–3 were analyzed, with a higher frequency exhibiting no TdT activity in the nonproductive compared with the productive repertoire. Since TdT activity decreases during B cell ontogeny (12), these results suggested that the productive repertoire may be enriched in Jλ7-containing rearrangements that were generated later in ontogeny, as might be anticipated if central receptor editing accounted for their introduction.

Table VI.

N additions in Vλ gene rearrangements of the patient with SLE

Rearrangements EmployingNumber of Nonproductive RearrangementsNumber of Productive Rearrangements
Without N addition (%)With N addition (%)Without N addition (n)With N addition (n)
Jλ7 0a (0) 21 (100) 20a (29) 50 (71) 
Jλ1-3 20†‡ (54) 17 (46) 9 (26) 25 (74) 
Rearrangements EmployingNumber of Nonproductive RearrangementsNumber of Productive Rearrangements
Without N addition (%)With N addition (%)Without N addition (n)With N addition (n)
Jλ7 0a (0) 21 (100) 20a (29) 50 (71) 
Jλ1-3 20†‡ (54) 17 (46) 9 (26) 25 (74) 
a

, p < 0.006, significant difference between the N additions (χ2 test); †, p < 0.009, significant difference between the N additions (χ2 test); ‡, p < 0.001, significant difference between the N additions (χ2 test).

A total of 41 productive and six nonproductive VHDJH rearrangements were analyzed. The comparison of the productive VH repertoires between the SLE patient and the normal subjects revealed differences, in that there was a striking over-representation of VH3 family members (82.9 vs 54.9%; p < 0.001) and under-representation of the VH4 family (7.3 vs 22.0%; p < 0.03) in the peripheral B cell repertoire of the SLE patient (Table VII). Of note, members of the VH5, -6, and -7 families were not found in the productive repertoire of the SLE patient, whereas only one VH1 family member (2.4%) and three members of the VH2 family (7.3%) were found.

Table VII.

Comparison of VH gene family usage in an SLE patient and in normalsa

Gene FamilyFunctional Genes (% of total)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
VH11 (21.6) 0 (0) 1 (2.4) 7 (13.0) 54 (15.4) 
VH3 (5.9) 1 (16.7) 3 (7.3) 3 (5.6) 7 (2.0) 
VH22 (43.1) 4 (66.7) 34c (82.9)b 22 (40.7) 192c (54.9)b 
VH11 (21.6) 1 (16.7) 3 (7.3)b 17 (31.5) 77 (22.0)b 
VH2 (3.9) 0 (0) 0 (0) 3 (5.6) 8 (2.3) 
VH1 (2.0) 0 (0) 0 (0) 2 (3.7) 7 (2.0) 
VH1 (2.0) 0 (0) 0 (0) 0 (0) 5 (1.4) 
      
Total 30 41 54 350 
Gene FamilyFunctional Genes (% of total)SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
VH11 (21.6) 0 (0) 1 (2.4) 7 (13.0) 54 (15.4) 
VH3 (5.9) 1 (16.7) 3 (7.3) 3 (5.6) 7 (2.0) 
VH22 (43.1) 4 (66.7) 34c (82.9)b 22 (40.7) 192c (54.9)b 
VH11 (21.6) 1 (16.7) 3 (7.3)b 17 (31.5) 77 (22.0)b 
VH2 (3.9) 0 (0) 0 (0) 3 (5.6) 8 (2.3) 
VH1 (2.0) 0 (0) 0 (0) 2 (3.7) 7 (2.0) 
VH1 (2.0) 0 (0) 0 (0) 0 (0) 5 (1.4) 
      
Total 30 41 54 350 
a

The total number of sorted B cells was 276 from the SLE patient and 736 from the normals.

b

Significant difference between the productive repertoire of the SLE patient compared to the productive normal repertoire (VH3, p < 0.001; VH4, p < 0.03).

c

, p ≤ 0.05, significantly higher frequency than predicted from its presence in the genome (goodness of fit χ2 test); †, p ≤ 0.05, significantly lower frequency than predicted from its presence in the genome (goodness of fit χ2 test).

Despite obtaining only six nonproductive rearrangements from the SLE patient, the distribution of VH families (Table VII) in the nonproductive repertoire was similar to that in normal subjects. Of note, the ratio of nonproductive/productive rearrangements (0.15%) was identical in the SLE patient and the normal subjects.

As shown in Table VIII, the distribution of JH genes did not differ between the SLE patient and the normal subjects. In general, JH4 and JH6 dominated the repertoire of productive rearrangements in the SLE patient (53.7 and 22.0%, respectively) and the normal subjects (54.0 and 25.1%, respectively).

Table VIII.

Distribution of JH genes in individual B cells from an SLE patient and in normalsa

SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
JH0 (0) 1 (2.4) 2 (3.6) 3 (0.9) 
JH0 (0) 2 (4.9) 4 (7.1) 10 (2.9) 
JH1 (16.7) 5 (12.2) 4 (7.1) 25 (7.1) 
JH1 (16.7) 22 (53.7) 22 (39.3) 189 (54.0) 
JH1 (16.7) 2 (4.9) 9 (16.1) 35 (10.0) 
JH2 (33.3) 9 (22.0) 15 (26.8) 88 (25.1) 
     
Total 41 56 350 
SLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
JH0 (0) 1 (2.4) 2 (3.6) 3 (0.9) 
JH0 (0) 2 (4.9) 4 (7.1) 10 (2.9) 
JH1 (16.7) 5 (12.2) 4 (7.1) 25 (7.1) 
JH1 (16.7) 22 (53.7) 22 (39.3) 189 (54.0) 
JH1 (16.7) 2 (4.9) 9 (16.1) 35 (10.0) 
JH2 (33.3) 9 (22.0) 15 (26.8) 88 (25.1) 
     
Total 41 56 350 
a

One JH segment could not be ascribed to one of the JH gene segments in the nonproductive repertoire of the SLE patient as well as in the nonproductive repertoire of the normals.

There was a significantly increased usage of VH3-11 in the productive repertoire of the SLE patient (p < 0.001), whereas the frequency of occurrence of all other genes did not differ compared with that in normal subjects. This indicated a preferential expansion of B cells using the VH3-11 element in this patient (see below). Of note, the VH3-11 gene segment has previously been documented to be negatively selected in the normal peripheral B cell repertoire (7).

Detailed analysis of individual gene usage in the nonproductive repertoire of the SLE patient revealed that VH3-08 (p < 0.002), VH3-11 (p < 0.001), and VH3-64 (p < 0.002) were detected significantly more often than in normal subjects (Table IX). However, the small number of nonproductive rearrangements from the SLE patient does not permit a firm conclusion, although the significantly increased detection of these three VH genes suggests the possibility of preferential gene usage not seen in normal subjects.

Sequence analysis of the 8 VH3-11 rearrangements obtained from the SLE patient revealed a high degree of sequence homology in six cases (92.2–99.2%; Fig. 1,B) consistent with the possibility that B cells expressing this VH3 gene may have derived from of a single B cell precursor. These rearrangements used VH3-11, JH6 and the D elements LR5 and inverse D12/9. The CDR3 length was 45 bp (15aa) and with the exception of putative mutations was identical in each member of the clone. In addition, a productive Vλ light chain rearrangement employing Vλ1G/Jλ7 (Fig. 1, A and C) was found in five of these same B cells. The CDR3 length of this Vλ rearrangement was 33 bp (11 aa) and was also identical in each clone member, with the exception of putative mutations. In one of the six potential members of this clone, the light chain could not be amplified (D2-2g3E5). The two remaining VH rearrangements using VH3-11 differed from the clone in that two of them used JH4 genes (D2-1g3D11 and D2-1g3E8) with CDR3 lengths of 14 and 8 aa, respectively.

FIGURE 1.

A, VH and Vλ sequences of the putative progenitor cell of the clone. Bold and underlined letters refer to complementarity-determining regions (CDR) 1, 2, and 3 of each V rearrangement. Each member of the clone expressed the same CDRs, albeit with mutations. B, Mutational tree of clonally related B cells expressing VH3-11. Five sequences employing VH3-11 and JH6 exhibited a striking homology and used the same light chain rearrangement (B) consistent with a clonal relationship. In addition, gene D2-2g3E5/∗ exhibited VH3-11 sequence homology with the clone, but the light chain could not be amplified. The number in each B cell indicates the total number of mutations, of which the number of mutations shown in the immediate precursor was shared. C, Mutational tree of five sequences employing the Vλ1 family member 1G and Jλ7 detected in the same cells as the VH sequences shown in A. All rearrangements shared the two mutations detected in D2-2L1 B1.

FIGURE 1.

A, VH and Vλ sequences of the putative progenitor cell of the clone. Bold and underlined letters refer to complementarity-determining regions (CDR) 1, 2, and 3 of each V rearrangement. Each member of the clone expressed the same CDRs, albeit with mutations. B, Mutational tree of clonally related B cells expressing VH3-11. Five sequences employing VH3-11 and JH6 exhibited a striking homology and used the same light chain rearrangement (B) consistent with a clonal relationship. In addition, gene D2-2g3E5/∗ exhibited VH3-11 sequence homology with the clone, but the light chain could not be amplified. The number in each B cell indicates the total number of mutations, of which the number of mutations shown in the immediate precursor was shared. C, Mutational tree of five sequences employing the Vλ1 family member 1G and Jλ7 detected in the same cells as the VH sequences shown in A. All rearrangements shared the two mutations detected in D2-2L1 B1.

Close modal

The nonproductive VH rearrangements of the SLE patient exhibited a mutational frequency of 6.5% (87 mutations/1330 bp) compared with 4.4% for the productive VH rearrangements (446 mutations/10,172 bp; p < 0.001, by χ2 test). Of note, there were only two productively rearranged VH genes (one VH3 and one VH4 family member, respectively) that did not contain nucleotide substitutions, indicating that VH genes of the vast majority of the CD19+ B cells analyzed had undergone somatic hypermutation. The clonally related VH3-11 genes (see Fig. 1 B) acquired 82 mutations/1446 bp for a mutational frequency of 5.67%. The mutational frequency of the clonally related VH3-11 genes was significantly higher compared with that of the remaining genes (3.7%; p < 0.001).

The current study analyzed the Vλ and Jλ as well as the VH and JH gene usage in a patient with SLE and documented that there are few molecular differences apparent in the peripheral B cell repertoire of this patient compared with that of normal controls as indicated by the distribution of Vλ and heavy chain genes in the nonproductive repertoires. Analysis of genomic DNA made it possible to obtain a representative number of nonproductive gene rearrangements and thereby permitted the comparison with productive rearrangements.

Marked differences were noted between the productive VλJλ gene repertoire of the SLE patient and that of the normal subjects and between the productive and nonproductive repertoires of the SLE patient. These differences could have been attributed to a variety of influences that are dependent on expression of a Vλ gene product, including selection and receptor editing.

Detailed analysis of the productive Vλ repertoire revealed significant deviations in the distribution of both Vλ and Jλ elements in the SLE patient compared with normal subjects, in that the frequencies of both the Jλ distal Vλ elements and the Vλ distal Jλ segment were increased. In detail, there was an under-representation of the Jλ proximal genes, 2A2, 3H, 1B, and 4B in the SLE patient, whereas the Jλ distal genes 3L, 1G, 6A, and 8A were over-represented compared with those in normal subjects. This contributed to a significantly different usage of the Jλ proximal gene cluster A and the Jλ distal Vλ gene cluster C between the SLE patient and the controls. The number of Vλ genes that exhibited biased representation as well as their locations were most consistent with the conclusion that receptor editing of the Vλ locus might have been frequently used in shaping the productive repertoire of the SLE patient. The over-representation of Jλ7 in the productive repertoire of the SLE patient was consistent with augmented receptor editing. Analysis of the Vκ repertoire of the same patient also revealed evidence of extensive receptor editing of the Vκ locus (3). In both circumstances, receptor editing was significantly increased in degree in the SLE patient compared with that in the normal controls.

Despite the evidence that receptor editing of Vκ and Vλ genes was increased in this SLE patient compared with that in normal subjects, the mechanisms appeared to be different. The data suggested that Vκ receptor editing was most marked in the periphery of this SLE patient based on the higher mutational frequency of productive rearrangements employing Jκ1–4 compared with those using Jκ5 (3). This finding implies that receptor editing of Vκ in SLE occurs in the periphery after somatic hypermutation has been initiated. The current analysis supports this conclusion, since productively rearranged Vκ genes exhibited a lower frequency of mutations than nonproductive Vκ rearrangements. This is markedly different from the situation in normal subjects, in which the mutational frequency of productive Vκ rearrangements is significantly greater than that for the nonproductive repertoire (4). This finding implies that receptor editing of Vκ rearrangements after mutation is sufficiently robust to result in an overall decrease in the mutational frequency of productive Vκ rearrangements in this SLE patient.

The distribution of Vλ genes also implied that receptor editing of Vλ had occurred in this patient. As opposed to the Vκ genes, however, analysis of the mutational pattern suggested that the dominant influence was central, and not peripheral, receptor editing of Vλ genes. Thus, there was an increase in the usage of 5′ Vλ genes and the 3′ Jλ7 segment, but there was no decrease in the mutational frequency of productive Vλ rearrangements using these elements or of the entire productive Vλ repertoire. These results imply that the bulk of Vλ receptor editing in this SLE patient occurred before the mutational machinery had been activated and therefore most likely occurred in the bone marrow during B cell ontogeny. This contention was supported by an analysis of apparent TdT activity exerted on rearrangements employing Jλ1–3 vs Jλ7. As TdT activity diminishes during B cell ontogeny (12), it would be anticipated that productive rearrangements that were introduced later in B cell development as a result of receptor editing would contain fewer additions because of waning TdT activity. The increased number of Jλ7 containing productive rearrangements with no N segment additions is consistent with this conclusion. The combination of Jλ7 containing rearrangements with fewer N nucleotides but a comparable mutational frequency is most consistent with the conclusion that their over-representation in the productive repertoire resulted from central receptor editing.

Whether additional peripheral editing of Vλ genes also occurred cannot be determined from this analysis, although it should be noted that if such a process occurred, it was of insufficient magnitude to be detected by this approach. Similarly, we cannot determine whether central receptor editing of Vκ rearrangements occurred because peripheral editing was so dominant.

It is noteworthy that recombined Vλ4B and Jλ2/3 genes were found exclusively in the nonproductive repertoires of both the normal subjects and the SLE patient, suggesting that they were eliminated from the productive gene repertoire of each comparably. This implies that some elements of negative selection or receptor editing operated normally in the SLE patient. Similarly, A30/Jκ2 was exclusively found in the nonproductive repertoire of this SLE patient (3). Productively rearranged A30/Jκ2 genes have been shown to bind dsDNA in their germline configuration (2, 13). Although the binding specificity of 4B/Jλ2/3 gene rearrangements has not been delineated, it was detected only in the nonproductive and not the productive repertoires, suggesting that it might bind an autoantigen. Its elimination from the productive repertoire might, therefore, result from negative selection and/or receptor editing. Whatever the mechanism of elimination, this process appears to be intact in this SLE patient and comparable to that in normal subjects.

Data from Vλ transgenic mice have shown that central receptor editing can operate to replace light chains of B cells expressing autoantibodies (14), although there are no previous examples of central receptor editing of Vλ chains. There is, however, no conceptual reason that central receptor editing of Vλ chains could not occur if these Vλ gene products encoded autoantigen recognition. This suggests that emergence of Vλ-containing autoantibodies during B cell ontogeny may have been the stimulus for central Vλ receptor editing in this SLE patient. In this context, Vλ genes have been shown to be critical parts of a number of human autoantibodies, including Abs to dsDNA (15, 16, 17, 18), Abs to La/SS-B and Ro/SS-A (19, 20), rheumatoid factor (21, 22, 23, 24), and Abs to laminin (25), phospholipids (26), collagen, and histone 2A (27). It should be noted, however, that there are insufficient data on the light chain usage of autoantibodies to conclude that receptor editing differentially effects the use of Vλ or Vκ.

In contrast to analyses of VL gene usage in this same patient, the current examination of the VH gene usage revealed no evidence of increased VH receptor editing, but detected other differences in the VH gene repertoire that could contribute to autoantibody formation.

Receptor editing of the VH gene locus has been observed in a site-directed manner in a transgenic mouse model (28, 29). Defects in receptor editing have been suggested to play a role in retaining autoreactive B cell receptors (BCRs) in autoimmune diseases (29, 30, 31, 32, 33). Despite this, no evidence of abnormalities in receptor editing of VH genes was detected in the current analysis by assessment of the distribution of VH genes, but the possibility that this process is impaired in this SLE patient cannot be completely excluded. Only defects in receptor editing of sufficient magnitude to alter the distribution of VH genes in the productive repertoire were detected. It remains possible that a defect in site-directed receptor editing of VH genes could contribute to autoimmunity in this SLE patient. Of note, however, evidence of enhanced receptor editing of VL chains was easily detected in this patient, making it unlikely that there was an overall defect in the receptor-editing process in this SLE patient.

Comparison of the VH gene usage in the productive repertoire provided evidence that the gene segment VH3-11 was found significantly more often in this SLE patient than in normal subjects. This over-representation was accounted for by the above noted preference to rearrange this germline gene in this patient as well as by the expansion of a B cell clone expressing this VH gene segment. Moreover, other B cells expressing VH3-11 rearranged to other JH segments were also over-represented, suggesting that the negative selection of rearrangements employing VH3-11 observed in normal subjects (7) was disturbed in this patient. Negative selection of VH3-11 in normal subjects has previously been suggested in other studies (34, 35) regardless of the genetic background of the donor. The over-representation of VH3-11 in this SLE patient, therefore, is unusual and mandates an analysis of other SLE patients to determine whether this is a consistent feature of this disease. The possibility that this patient manifested a generalized enhancement in positive selection of VH3-expressing B cells was suggested by the analysis of the entire VH3 family, as well as of the VH3-23 (DP-47) gene. VH3-23 (DP-47) is the most frequently used VH3 family member (6, 7, 34, 35), accounting for 12–14% of the normal repertoire (6). In this SLE patient, VH3-23 was even more frequently used, being expressed by 22% of the B cells. Of importance, VH3-23 has previously been noted to encode anti-DNA Abs frequently, especially the 16/6 Id (36). Whether an abnormal mechanism, such as B cell superantigen stimulation (37, 38), is causing expansion of VH3-expressing B cells in this SLE patient will require carefully analysis of other patients.

One of the remarkable findings of this study is the identification of six B cells that expressed BCRs using VH3-11 and in five cases Vλ1G rearrangements with a high degree of sequence homology. Of note, unique patterns of mutations and, with the exception of putative mutations, identical CDR3s were identified, suggesting that these resulted from clonal expansion and Ag-mediated selection. The usage of VH3-11 and Vλ1G by this clone requires emphasis, since both genes have been reported to be negatively selected in normal subjects (5, 7). Although proof of autoreactivity of these resulting BCRs is lacking, these data are consistent with the conclusion that clonal expansion of B cells can occur in the initial stages of SLE, suggesting an overwhelming antigenic stimulus. Studies in mice have extensively documented that clonal expression of autoreactive B cells occurs in early lupus (39, 40). In addition, this clone used two D elements, one of which was employed in an inverted orientation. Although the use of inverted D segments has been suggested to increase the frequency of arginines in CDR3s and thereby contribute to the development of anti-dsDNA Abs (41, 42), inverted D elements have also been detected in normal subjects (6, 7). The current data do not allow a firm conclusion about whether an enhanced rate of inverted D elements is a hallmark of clonally expanded B cells in SLE or whether these clones encode anti-DNA Abs. However, the expansion of a B cell clone in the initial stages of SLE in this patient is consistent with findings noted in autoimmune-prone mice (40, 43).

Analysis of mutations provided further insights into the generation of diversity in this SLE patient. The marked degree of somatic hypermutation of the VH rearrangements of this untreated SLE patient is obvious. The mutational frequencies of the CD19+ B cells from the SLE patient (6.5% for nonproductive and 4.4% for productive rearrangements) were significantly greater than those found in normal subjects (CD19+ peripheral B cells from a female Caucasian donor exhibited mutational frequencies of 3.8% for nonproductive and 3.3% for productive rearrangements) (44, 45). Thus, both the nonproductive (p < 0.001) as well as the productive repertoire (p < 0.001) were significantly more mutated in the SLE patient than in normal controls (45). Since previous analyses provided evidence that age influences the number of mutations in memory B cells, we compared the mutations in the 54-yr-old SLE patient with that previously reported in a 45-yr-old male donor (6, 45). The mutational frequency in the SLE patient significantly exceeded that in the nonproductive (p < 0.001) and the productive repertoire (p < 0.005) of this normal donor (nonproductive: 245 mutations/6528 bp; mutational frequency, 3.8%; productive: 1601 mutations/47872 bp; mutational frequency, 3.3%). Since mutational activity, in general, is induced in response to T-dependent Ags, and the frequency of mutations in the nonproductive repertoire reflects the activity of the mutational machinery without subsequent selection (44), the B cells of this patient appear to have been stimulated in a T cell-dependent manner more intensively or more persistently than in normal subjects. Whether this reflects the intensity or persistence of stimulation or a defect in apoptosis of B cells expressing mutated BCRs, as has been suggested (46, 47), remains to be determined. Preliminary data analyzing the mutational frequency of nonproductive Vκk rearrangements revealed a marked increase compared with that in normal subjects (3.6 × 10−2 vs 4.8 × 10−3; p < 0.001). As the mutational frequency of the nonproductive rearrangements is an indication of the immediate impact of the mutator without the subsequent influence of selection or B cell survival, these results are most consistent with the conclusion that the mutational machinery was overactive in this patient.

The difference in the frequency of mutations in the productive and nonproductive repertoires reflects the influence of selection, with elimination of mutation-generated defective BCR normally more evident than positive selection of those with increased avidity (6, 44, 45). This process seems to be generally intact in this SLE patient, even though the overall resulting frequency of mutations in the productive repertoire is much greater than normal.

In summary, skewing of the VH repertoire toward utilization of VH3 genes, clonal expansion of B cells, and a generalized increase in somatic hypermutation may all contribute to the emergence of autoimmunity in this SLE patient. These data are most consistent with the conclusion that extreme B cell overactivity is found in the initial stages of SLE, leading to remarkable changes in peripheral V gene usage and, despite extensive light chain receptor editing, predisposes to the emergence of autoimmunity.

Table IX.

Distribution of individual VH gene usage in an SLE patient and normalsa

GeneSLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
2-05 DP-76 0 (0) 1 (2.4) 1 (1.8) 5 (1.4) 
2-70 DP-28 1 (16.7) 2 (4.9) 2 (3.5) 2 (0.6) 
1-24 DP-5 0 (0) 1 (2.4) 0 (0) 4 (1.1) 
4-31.1 DP-65 0 (0) 1 (2.4) 0 (0) 0 (0.0) 
3-30.3 DP-46 0 (0) 2 (4.9) 1 (1.8) 22 (6.3) 
4-34 DP-63 1 (16.7) 2 (4.9) 4 (7) 13 (3.7) 
3-07 DP-54 0 (0) 2 (4.9) 3 (5.3) 20 (5.7) 
3-08 hv 3.3+ 1b (16.7) 0 (0.0) 0b (0) 0 (0.0) 
3-09 DP-31 0 (0) 1 (2.4) 4 (7) 11 (3.1) 
3-11 DP-35 2b (33.3) 8 (19.5) 1b (1.8) 6 (1.7) 
3-13 DP-48 0 (0) 1 (2.4) 1 (1.8) 3 (0.9) 
3-15 DP-38 0 (0) 2 (4.9) 1 (1.8) 11 (3.1) 
3-23 DP-47 0 (0) 9 (22.0) 2 (3.5) 45 (12.9) 
3-30 DP-49 0 (0) 2 (4.9) 2 (3.5) 21 (6.0) 
3-33 DP-50 0 (0) 3 (7.3) 2 (3.5) 10 (2.9) 
3-53 DP-42 0 (0) 1 (2.4) 0 (0) 10 (2.9) 
3-64 DP-61 1b (16.7) 0 (0.0) 0b (0) 2 (0.6) 
3-72 DP-29 0 (0) 1 (2.4) 0 (0) 1 (0.3) 
3-74 DP-53 0 (0) 2 (4.9) 2 (3.5) 4 (1.1) 
      
Total  41 57 350 
GeneSLENormals
Nonproductive No. (% of total)Productive No. (% of total)Nonproductive No. (% of total)Productive No. (% of total)
2-05 DP-76 0 (0) 1 (2.4) 1 (1.8) 5 (1.4) 
2-70 DP-28 1 (16.7) 2 (4.9) 2 (3.5) 2 (0.6) 
1-24 DP-5 0 (0) 1 (2.4) 0 (0) 4 (1.1) 
4-31.1 DP-65 0 (0) 1 (2.4) 0 (0) 0 (0.0) 
3-30.3 DP-46 0 (0) 2 (4.9) 1 (1.8) 22 (6.3) 
4-34 DP-63 1 (16.7) 2 (4.9) 4 (7) 13 (3.7) 
3-07 DP-54 0 (0) 2 (4.9) 3 (5.3) 20 (5.7) 
3-08 hv 3.3+ 1b (16.7) 0 (0.0) 0b (0) 0 (0.0) 
3-09 DP-31 0 (0) 1 (2.4) 4 (7) 11 (3.1) 
3-11 DP-35 2b (33.3) 8 (19.5) 1b (1.8) 6 (1.7) 
3-13 DP-48 0 (0) 1 (2.4) 1 (1.8) 3 (0.9) 
3-15 DP-38 0 (0) 2 (4.9) 1 (1.8) 11 (3.1) 
3-23 DP-47 0 (0) 9 (22.0) 2 (3.5) 45 (12.9) 
3-30 DP-49 0 (0) 2 (4.9) 2 (3.5) 21 (6.0) 
3-33 DP-50 0 (0) 3 (7.3) 2 (3.5) 10 (2.9) 
3-53 DP-42 0 (0) 1 (2.4) 0 (0) 10 (2.9) 
3-64 DP-61 1b (16.7) 0 (0.0) 0b (0) 2 (0.6) 
3-72 DP-29 0 (0) 1 (2.4) 0 (0) 1 (0.3) 
3-74 DP-53 0 (0) 2 (4.9) 2 (3.5) 4 (1.1) 
      
Total  41 57 350 
a

There were no significant differences in the frequency of gene usage between the nonproductive repertoire of the SLE patient compared to that of normals.

b

, p < 0.05, significant differences between the frequency nonproductive repertoire of the SLE patient and normals (p < 0.002 for VH 3-08, p < 0.001 for VH 3-11, and p < 0.002 for VH3-64, χ2 test); †, significant difference between the frequency found in the productive repertoire of the SLE patient and in normals (p < 0.001, χ2 test).

We thank Dr. Qin-Chang Cheng for help in determining appropriate statistical analyses.

1

This work was supported by National Institutes of Health Grant AI 31229 and Deutsche Forschungsgemeinschaft Grants Do 491/2-1 and 4-1 (to T.D.).

3

Abbreviations used in this paper: SLE, lupus erythematosus; BCR, B cell Ag receptor.

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