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The Journal of Immunology, 2004, 173: 7368-7376.
Copyright © 2004 by The American Association of Immunologists

Genetic Dissection of Lupus Pathogenesis: Sle3/5 Impacts IgH CDR3 Sequences, Somatic Mutations, and Receptor Editing1

Masatoshi Wakui*, Jinho Kim*, Edward J. Butfiloski*, Laurence Morel*,{dagger} and Eric S. Sobel2,*,{dagger}

* Department of Medicine and Division of Rheumatology and Clinical Immunology, and {dagger} Center for Mammalian Genetics and Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sle3/5 is a lupus susceptibility locus identified on mouse chromosome 7 of the New Zealand Black/New Zealand White (NZB/NZW)-derived NZM2410 strain. Based on previous observations, this locus appears to contribute to lupus pathogenesis through its impact on diversification of immune responses. To understand how Sle3/5 affects somatic diversification of humoral responses, we analyzed IgH rearrangements preferentially encoding hapten-reactive IgG1 repertoires after immunization and assessed peripheral IgH VDJ recombination activities in C57BL/6 (B6) mice congenic for Sle3/5 (B6.Sle3/5). In addition to altered somatic VH mutation profiles, sequences from B6.Sle3/5 mice exhibited atypical IgH CDR3 structures characteristic of autoreactive B cells and consistent with peripheral B cells bearing putatively edited receptors. Significant expression of Rag genes and circular VHD gene excision products were detected in splenic mature B cells of B6.Sle3/5 but not B6 mice, showing that peripheral IgH rearrangements occurred beyond allelic exclusion. Taken together, on the nonautoimmune background, Sle3/5 affected VHDJH junctional diversity and VH mutational diversity and led to recombinational activation of allelically excluded IgH genes in the periphery. Such impact on somatic IgH diversification may contribute to the development of autoreactive B cell repertoires. This is the first report to present evidence for significant association of a lupus susceptibility locus, which has been mapped to a chromosomal region in which no Ig genes have been identified, with somatic IgH sequence diversity and peripheral H chain receptor editing or revision without relying upon Ig transgene strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Th progress in genome-wide approaches has allowed several groups to identify the positions of lupus-susceptibility loci in humans and mice (1). Our group has identified three major genomic intervals linked to glomerulonephritis in the lupus prone New Zealand Black (NZB)3/New Zealand White (NZW)-derived NZM2410 mouse strain (2). To dissect the pathogenesis of lupus genetically and functionally, each of the lupus susceptibility intervals was subsequently transferred individually onto the nonautoimmune C57BL/6 (B6) genome using the strategy of speed congenics (3, 4). Two of the most critical regions are Sle1 on chromosome 1 and Sle3/5 on chromosome 7. B6 mice congenic for Sle1 (B6.Sle1, previously called B6.NZMc1) spontaneously developed high titers of IgG specific to H2A/H2B/dsDNA subnucleosomes, autoreactive T cells responding to histone epitopes, and an increase in expression of the early cell activation marker CD69 but no renal disease (5, 6). B6 mice congenic for Sle3/5 (B6.Sle3/5, previously called B6.NZMc7) developed autoantibodies to a variety of nuclear Ags with a low titer and penetrance, an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells that were relatively resistant to activation-induced apoptosis, and a low but significant incidence of glomerulonephritis (5, 7). The subsequent linkage study revealed that the Sle3/5 genetic interval consisted of two subintervals, Sle5 on centromeric chromosome 7 and Sle3 telomeric to Sle5 (8). Sle5 was linked to anti-dsDNA IgG production while Sle3 was linked to the development of glomerulonephritis as well as anti-ssDNA IgM and anti-thyroglobulin IgG production. The bicongenic strain B6.Sle1.Sle3/5 strain showed a much more robust phenotype, as characterized by high titers and a broad spectrum of antinuclear Abs along with severe glomerulonephritis (9, 10). Although the etiology and mechanisms of lupus nephritis still remain under investigation, cross-reactivity of anti-dsDNA Ab with renal tissue components has been reported, suggesting its potential role in the pathogenesis of nephritis (11). Consistent with it, B6.Sle1.Sle3/5 mice produced anti-glomerular basement membrane Ab (9, 10). Based on these observations, Sle3/5 appears to play a major role in lupus pathogenesis through its impact on diversification of immune responses.

To understand better how Sle3/5 affects humoral immune responses, we examined IgH rearrangements preferentially encoding (4-hydroxy-3-nitrophenyl) acetyl (NP) hapten-reactive IgG1 repertoires in B6 and B6.Sle3/5 mice after immunization. This is an extremely well-characterized immune response on the B6 background (12, 13, 14). In addition, Rag gene expression and circular gene excision products derived from somatic recombination of VH and D genes were examined in spleens from unimmunized B6 and B6.Sle3/5 mice. The results showed that Sle3/5 affected VHDJH junctional diversity as well as VH mutational diversity and led to recombinational activation of allelically excluded IgH genes resulting in peripheral receptor editing or revision.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and immunization

B6 mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal colony. The B6.Sle3/5 congenic mouse strain and its development has been described (3, 7). The preparation of NP hapten-conjugated SRBC (NP-SRBC) followed a published protocol (15). NP-SRBC were washed and resuspended in PBS at a final concentration of 5%. B6 and B6.Sle3/5 mice at 10 wk of age were immunized by i.p. injection with 0.1 ml of this thymus-dependent Ag (TD-Ag).

Analysis of VH186.2-D-JH-C{gamma}1 gene rearrangements

Spleens were harvested from mice at 14 days postimmunization. Because it has been well documented that the somatic mutation frequencies peak around day 14 after immunization with NP-conjugated TD-Ags (12, 13), we used this time point for our studies. Total RNA was extracted from the spleens using the TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and subjected to cDNA synthesis by the SuperScript II RNase H– reverse transcriptase (Invitrogen Life Technologies) using oligo(dT) primers. Control reactions were in the absence of the reverse transcriptase. VH186.2-D-JH-C{gamma}1 rearrangement transcripts, which preferentially encode NP-reactive IgG1 repertoires, were amplified by nested PCR (14). For the first 35 cycles, 20-µl reactions contained 1 µl of cDNA, 0.04 U/µl Taq polymerase (Fisher Scientific, Pittsburgh, PA) with 1x reaction buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 0.8 µM external VH186.2 sense primer (5'-CTCTTCTTGGCAGCAACAGC-3'), and 0.8 µM external C{gamma}1 antisense primer (5'-GCTGCTCAGAGTGTAGAGGTC-3'). For the second 35 cycles, 1 µl of product from the first round PCR, 0.8 µM internal VH186.2 sense primer (5'-GTGTCCACTCCCAGGTCCAAC-3'), and 0.8 µM internal C{gamma}1 antisense primer (5'-GTTCCAGGTCACTGTCACTG-3') were used in identical conditions. Each set of PCR cycles began with a 94°C incubation for 5 min followed by 35 cycles of 94°C for 15 s, 50°C for 45 s, and 72°C for 90 s and then ended with a 72°C incubation for 5 min. The nested PCR products were cloned using the TOPO TA Cloning for Sequencing (Invitrogen Life Technologies). The obtained clones were randomly picked up and sequenced using the ABI 373A or 377 automated DNA sequencer (Applied Biosystems, Foster City, CA). The sequence profiles were analyzed using the National Center for Biotechnology Information BLAST program (National Center for Biotechnology Information, Bethesda, MD), DNASIS software (Hitachi Software Engineering America, San Bruno, CA), and some published references to D gene sequences (16, 17, 18).

Cell fractionation

Fractionation of spleen cells was performed using magnetic cell sorting or T cell-depletion with mAbs and complement. The magnetic cell sorting was conducted with anti-FITC microbeads (Miltenyi Biotec, Auburn, CA) after staining of spleen cells with FITC-conjugated primary mAb or treatment with biotin-conjugated primary mAb followed by staining with streptavidin-conjugated FITC (Jackson ImmunoResearch Laboratories, West Grove, PA) under optimal conditions. In this procedure, CD19 (mAb clone 1D3) or B220 (mAb clone RA3-6B2) was used as a B cell marker, and combinations of CD4 (mAb clone H129.19), CD8 (mAb clone 53-6.7), and CD90.2 (mAb clone 30-H12) were used as T cell markers. A cell surface marker 493 (mAb clone 493) was used as a splenic immature/transitional B cell marker (19). IgM (mAb clone R6-60.2) and IgD (mAb clone 11-26c.2a) were also used as B cell subset markers. All primary mAb reagents were purchased from BD Pharmingen (San Diego, CA). The T cell-depletion with mAbs and complement was performed as previously described (20). Briefly, a mixture of anti-CD4 (clone 172-4), anti-CD8 (clone 31M), and anti-CD90.2 (clone MmT1) was used followed by lysis with Low-Tox-M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada). The purity or the depletion efficacy after the cell fractionation was evaluated by flow cytometric analysis using the BD FACSCalibur system (BD Biosciences, San Jose, CA). The purity was over 90% in the B cell fractionation, IgM-positive selection, and IgD-positive selection whereas it was above 70% in the 493-positive selection (data not shown). The depletion efficacy was over 95% in the non-B cell fractionation, IgM-negative selection, IgD-negative selection, and 493-negative selection (data not shown).

Detection of Rag gene expression

Total RNA was prepared from bone marrow and fractionated or nonfractionated spleen cells of unimmunized mice and subjected to cDNA synthesis as described above. As internal controls, {beta}-actin gene transcripts were amplified by PCR with a range from 28 to 38 cycles. The set of PCR cycles for Rag-1 and Rag-2 began with 94°C incubation for 2 min followed by a range from 30 to 45 cycles of 94°C for 30s, 60°C for 30 s, and 72°C for 1 min. Each PCR product was electrophoresed on a 2% agarose gel and analyzed using the FOTO/Analyst Archiver systems (Fotodyne, Hartland, WI). The primer sequences were as follows: {beta}-actin (sense), 5'-ATGGATGACGATATCGCT-3', (antisense) 5'-ATGAGGTAGTCTGTCAGGT-3'; Rag-1 (sense), 5'-CCAAGCTGCAGACATTCTAGCACTC-3', (antisense), 5'-CTGGATCCGGAAAATCCTGGCAATG-3'; and Rag-2 (sense), 5'-CACATCCACAAGCAGGAAGTACAC-3', (antisense), 5'-GGTTCAGGGACATCTCCTACTAAG-3'.

Detection of circular VHD gene excision products

One million cells were suspended in 200 µl of the lysis buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1.5 mM MgCl2, 0.5% Tween 20, and 1.5 mg/ml proteinase K. This suspension was incubated at 56°C for 60 min and 95°C for 10 min. The obtained cell lysates were directly subjected to seminested PCR for detection of circular VHD gene excision products. The first round was done using P5 (conserved 3' of VHJ558 family: 5'-TGAGGAGGAGGTAATAAATGGACA-3') and P71 (conserved 5' of DFL16.1/DSP2.2: 5'-TCAAAGCACAATGCCTGGCTTGGG-3') primers (21, 22). The second round was performed using P6 (conserved 3' of VHJ558 and more proximal to the coding sequence than P5: 5'-TCCCAGTGCAGCTTCCTGCTCCT-3') and P71 primers (21). The first round began with a 94°C incubation for 2 min followed by 15 cycles of 94°C for 30s, 62°C for 30 s, and 72°C for 1 min. The second round began with a 94°C incubation for 2 min followed by 35 cycles of 94°C for 30s, 65°C for 30 s, and 72°C for 45 s. As an internal control, {gamma}-actin gene was amplified by PCR using sense (5'-ATGTTTGAAACCTTCAATAC-3') and antisense (5'-GGAAGGAAGGCTGGAAGAGT-3') primers. The set of PCR cycles for {gamma}-actin began with a 94°C incubation for 2 min followed by 35 cycles of 94°C for 30s, 50°C for 30 s, and 72°C for 1 min. Each PCR product was evaluated by electrophoresis as described earlier. In some experiments, the PCR products were subjected to sequencing to verify that they were derived from circular VHJ558-DFL16.1/DSP2.2 gene excision.

Statistical analysis

Statistical analysis was done using the two-sample t test with or without Welch’s correction or the {chi}2 test for independence. All values for p < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Somatic mutations of VH186.2 genes

To investigate whether Sle3/5 affects somatic VH mutational diversity and VHDJH junctional diversity, we attempted to collect VH186.2-D-JH-C{gamma}1 rearrangements, which were derived from splenic B cells with IgH gene rearrangements preferentially encoding NP-reactive repertoires undergoing isotype-switching to IgG1. However, some VHJ558 family gene sequences other than VH186.2 were also obtained. These were present both in B6 and B6.Sle3/5 mice at a similar frequency (10% vs 12%). This finding has also been reported by others using the same nested PCR strategy (14), and these sequences were excluded from further analysis. Through sequencing of plasmid DNAs that were randomly selected after TA cloning, 20 VH186.2-D-JH-C{gamma}1 rearrangements were obtained from each immunized mouse. In total, 60 VH186.2-D-JH-C{gamma}1 rearrangements from three B6 mice and 60 from three B6.Sle3/5 mice were collected and subjected to further analysis. With respect to Ig sequence profiles, there was little intrastrain variability in either strain, except for the molecular tree analysis, as described below. Although the frequencies of replacement or total mutations of VH186.2 genes were not significantly different between B6 and B6.Sle3/5 mice, B6.Sle3/5 mice exhibited higher frequencies of silent mutations (B6: 5.6 ± 2.78 vs B6.Sle3/5: 7.2 ± 3.29 bases, p < 0.005) (Fig. 1, A–C). Ten or more silent mutations were more frequently observed in the B6.Sle3/5 rearrangements (B6: 7% vs B6.Sle3/5: 30%, p < 0.005). The distribution of replacement to silent mutation (R/S) ratio was also different (p < 0.001) (Fig. 1D). High R/S ratio (>3) was frequently observed in B6 mice (B6: 37% vs B6.Sle3/5: 8%). In contrast, low R/S ratio (<2) was frequently observed in B6.Sle3/5 mice (B6: 20% vs B6.Sle3/5: 45%). Some rearrangements shared CDR3 sequences, showing the existence of genealogical relationship explained by intraclonally mutational developments. The molecular tree analysis, which was based on the identification of CDR3 mutations, revealed the existence of genealogies exhibiting evolutionarily branched patterns in B6 and B6.Sle3/5 mice (Fig. 2). In the B6 group, one mouse presented three genealogies but the other two mice did not. In the B6.Sle3/5 group, two or three genealogies were obtained from each of the three mice. Despite the lack of significant differences in replacement and total mutations between the two strains, intraclonal sequence diversity appeared more frequently in B6.Sle3/5 mice. The contribution of replacement mutations to such intraclonal evolution of B cell repertoires tended to be smaller in B6.Sle3/5 mice, but the difference was not statistically significant. Collectively, altered somatic mutation profiles were observed in sequences from B6.Sle3/5 mice in response to immunization, as characterized by increased silent mutations or a low R/S ratio without significant changes in replacement or total mutation frequencies.



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  		FIGURE 1. Somatic mutation profiles of VH186.2 genes. A, The mean frequency of replacement mutations was 15.1 ± 6.92 bases in B6 mice and 14.2 ± 6.38 bases in B6.Sle3/5 mice (NS). B, The mean frequency of silent mutations was 5.6 ± 2.78 bases in B6 mice and 7.2 ± 3.29 bases in B6.Sle3/5 mice (p < 0.005). Ten or more silent mutations were observed in 7% of the B6 rearrangements but in 30% of the B6.Sle3/5 rearrangements (p < 0.005). C, The mean frequency of total mutations was 20.7 ± 9.03 bases in B6 mice and 21.4 ± 9.38 bases in B6.Sle3/5 mice (N.S.). D, High replacement to silent mutation (R/S) ratio (>3) was observed in 37% of the B6 rearrangements but in 8% of the B6.Sle3/5 rearrangements (p < 0.001). Low R/S ratio (<2) was observed in 20% of the B6 rearrangements but in 45% of the B6.Sle3/5 rearrangements (p < 0.001).

 


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  		FIGURE 2. Molecular tree analysis of VH186.2-D-JH-C{gamma}1 rearrangement sequences. For individual groups of rearrangements with shared CDR3 sequences, genealogical trees were constructed from patterns of shared and unique mutations by assuming that shared mutations represent single events and not independent parallel mutations and by assuming the minimum number of mutational events. Individual cloned sequences ({circ}) and putative progenitors (•) bearing the germline (GL) VH186.2 segment and the shared CDR3 are represented. Hypothetical intermediates (broken circles) are also represented. Numbers near branches indicate the number of replacement (R) or silent (S) mutations to the next node. Three and eight genealogies exhibiting evolutionarily branched patterns were observed in sequence data from B6 mice (A) and from B6.Sle3/5 mice (B), respectively. In the B6 group, all three genealogies (Tree A1-A3) originated from one individual. In the B6.Sle3/5 group, in contrast, the eight genealogies originated from all three individuals (Tree B1-B3, B4, and B5) and B6-B8, respectively. The percentages of replacement mutations among total mutations involved in individual genealogies in B6 and in B6.Sle3/5 mice were 70–100% (mean, 80 ± 13.9%) and 46–78% (mean, 66 ± 9.3%), respectively (NS).

 
IgH CDR3 profiles

IgH CDR3s are derived from VHDJH junctional diversity before somatic VH mutational diversification. To investigate whether Sle3/5 affected VHDJH junctional diversity, we analyzed CDR3 profiles of VH186.2-D-JH-C{gamma}1 rearrangements collected from B6 and B6.Sle3/5 mice using the Kabat definition (23). By this definition, CDR3 begins with position 95 and ends with position 102. Position 92 is the invariant FR3 cysteine and position 103 is the invariant FR4 tryptophan. The mean CDR3 length was greater in B6.Sle3/5 mice (B6: 10.2 ± 2.37 vs B6.Sle3/5: 11.4 ± 2.96 aa, p < 0.02) (Fig. 3A). Long CDR3s (14 aa or more) were more frequently observed in B6.Sle3/5 mice (B6: 5% vs B6.Sle3/5: 22%, p < 0.01). The mean number of arginine residues in CDR3 was greater in B6.Sle3/5 mice (B6: 1.0 ± 0.59 vs B6.Sle3/5: 1.4 ± 0.96, p < 0.002) (Fig. 3B). CDR3s bearing three or more arginine residues were found only in B6.Sle3/5 mice (12%, p < 0.01). The mean number of tyrosine residues in CDR3 was larger in B6.Sle3/5 mice (B6: 2.1 ± 1.13 vs B6.Sle3/5: 3.1 ± 2.04, p < 0.002) (Fig. 3C). CDR3s bearing five or more tyrosine residues was more frequently observed in B6.Sle3/5 mice (B6: 2% vs B6.Sle3/5: 27%, p < 0.001). The mean number of aromatic residues (histidine, phenylalanine, tryptophan, and tyrosine) in CDR3 was slightly greater in B6.Sle3/5 mice (B6: 3.1 ± 1.54 vs B6.Sle3/5: 3.8 ± 1.93, p < 0.05) (Fig. 3D). CDR3s bearing six or more aromatic residues were frequently observed in B6.Sle3/5 mice (B6: 3% vs B6.Sle3/5: 18%, p < 0.01).



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  		FIGURE 3. IgH CDR3 amino acid profiles. A, The mean CDR3 length was 10.2 ± 2.37 aa in B6 mice and 11.4 ± 2.96 aa in B6.Sle3/5 mice (p < 0.02). Long CDR3s (14 aa or more) were observed in 5% of the B6 rearrangements but in 22% of the B6.Sle3/5 rearrangements (p < 0.01). B, The mean number of arginine residues was 1.0 ± 0.59 in B6 mice and 1.4 ± 0.96 in B6.Sle3/5 mice (p < 0.002). CDR3s bearing three or more arginine residues were found in 12% of the B6.Sle3/5 rearrangements but not in any of the B6 rearrangements (p < 0.01). C, The mean number of tyrosine residues was 2.1 ± 1.13 in B6 mice and 3.1 ± 2.04 in B6.Sle3/5 mice (p < 0.002). CDR3s bearing five or more tyrosine residues was observed in 2% of the B6 rearrangements but in 27% of the B6.Sle3/5 rearrangements (p < 0.001). D, The mean number of aromatic residues was 3.1 ± 1.54 in B6 mice and 3.8 ± 1.93 in B6.Sle3/5 mice (p < 0.05). CDR3s bearing six or more aromatic residues were observed in 3% of the B6 rearrangements but in 18% of the B6.Sle3/5 rearrangements (p < 0.01).

 
D-D fusion, which results from an unusual recombination of two or more different D genes at a single rearrangement, frequently occurred in B6.Sle3/5 mice (B6: 3% vs B6.Sle3/5: 32%, p < 0.001) (Fig. 4A and Table I). DQ52, the most downstream D gene was used in 15% of the B6 rearrangements but not in any B6.Sle3/5 rearrangement (p < 0.002) (Fig. 4A). DFL16, the most upstream D gene family was used in 35% of B6 rearrangements but in 48% of B6.Sle3/5 rearrangements (p < 0.05) (Fig. 4A). Except for DST4, D genes have three reading frames (RFs), RF1, RF2, and RF3. RF3 of most D genes contains arginine (24). RF3 was more frequently used in the B6.Sle3/5 mice (B6: 7% vs B6.Sle3/5: 22%, p < 0.02) (Fig. 4B). JH3 and JH4 are located more downstream than JH1 and JH2. The rearrangements using JH3 or JH4 were more frequent in the B6.Sle3/5 mice (B6: 30% vs B6.Sle3/5: 48%, p < 0.05) (Fig. 4C).



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  		FIGURE 4. Features of D and JH gene usage. A, D-D fusion was observed in 3% of VH186.2-D-JH-C{gamma}1 rearrangements from B6 but in 32% of those from B6.Sle3/5 mice (p < 0.001). DQ52 gene was used in 15% of the B6 rearrangements but not in any B6.Sle3/5 rearrangement (p < 0.002). DFL16 gene family was used in 35% of the B6 rearrangements but in 48% of the B6.Sle3/5 rearrangements (p < 0.05). B, RF3 of D genes was used in 7% of the B6 rearrangements but in 22% of the B6.Sle3/5 rearrangements (p < 0.02). C, The rearrangements using JH3 or JH4 were more frequent in the B6.Sle3/5 mice (B6: 30% vs B6.Sle3/5: 48%, p < 0.05).

 

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  		Table I. D-D fusion in B6 and B6.Sle3/5 mice

 
Taken together, atypical CDR3s were more frequently observed in B6.Sle3/5 mice, as characterized by a greater length, more frequent presence of an arginine/tyrosine/aromatic residue-rich structure, more frequent usage of RF3 for D genes, skewed D and JH usage, and much more frequent D-D fusion.

Correlation between VH 186.2 mutations and CDR3 profiles in B6.Sle3/5 mice

In B6.Sle3/5 mice, the mean CDR3 lengths of the rearrangements bearing 10 or more silent mutations of VH186.2 genes and those bearing <10 silent mutations were 12.8 ± 3.17 aa and 10.8 ± 2.65 aa, respectively (p < 0.02) (Fig. 5A). Long CDR3s (14 aa or more) were observed in 39% of the rearrangements with 10 or more silent mutations but in 14% of those with <10 silent mutations (p < 0.05). The mean numbers of arginine residues in CDR3 of the rearrangements bearing ≥10 silent mutations and those bearing <10 silent mutations were 2.1 ± 1.09 and 1.1 ± 0.71, respectively (p < 0.002) (Fig. 5B). CDR3s bearing three or more arginine residues were found in 33% of the rearrangements with 10 or more silent mutations but in 2% of those with <10 silent mutations (p < 0.001). The difference in the number of tyrosine residues or aromatic residues in CDR3 was not statistically significant between the rearrangements bearing 10 or more silent mutations and those bearing <10 silent mutations (data not shown). Collectively, in B6.Sle3/5 mice, the higher incidence of silent mutations of VH186.2 correlated with greater lengths and arginine-rich structures of CDR3s. A similar correlation was also observed between R/S ratio of VH186.2 and CDR3 profiles in B6.Sle3/5 mice. The mean CDR3 lengths of the rearrangements with low R/S ratio (<2) and those with ≥2 or more R/S ratio was 12.3 ± 2.56 aa and 10.6 ± 3.14 aa, respectively (p < 0.05) (Fig. 5C). Although long CDR3s (14 aa or more) appeared more frequently in the rearrangements with low R/S ratio, the difference was not statistically significant. The mean numbers of arginine residues in CDR3 of the rearrangements with low R/S ratio and those with a ≥2 R/S ratio were 1.9 ± 1.07 and 1.0 ± 0.65, respectively (p < 0.001) (Fig. 5D). CDR3s bearing three or more arginine residues were found in 26% of the rearrangements with low R/S ratio but not in any of those with a ≥2 R/S ratio (p < 0.005). The differences in the number of tyrosine residues or aromatic amino acid residues in CDR3 was not statistically significant between the rearrangements with low R/S ratio and those with a ≥2 R/S ratio (data not shown).



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  		FIGURE 5. Correlation between VH186.2 mutations and CDR3 profiles in B6.Sle3/5 mice. A, The mean CDR3 lengths of rearrangements bearing 10 or more silent mutations (S mut) and those bearing <10 silent mutations were 12.8 ± 3.17 aa and 10.8 ± 2.65 aa, respectively (p < 0.02). Long CDR3s (14 aa or more) were observed in 39% of the rearrangements with 10 or more silent mutations but in 14% of those with <10 silent mutations (p < 0.05). B, The mean numbers of arginine residues in CDR3 of the rearrangements bearing 10 or more silent mutations and those bearing <10 silent mutations were 2.1 ± 1.09 and 1.1 ± 0.71, respectively (p < 0.002). CDR3s bearing three or more arginine residues were found in 33% of the rearrangements with 10 or more silent mutations but in 2% of those with <10 silent mutations (p < 0.001). C, The mean CDR3 lengths of the rearrangements with low R/S ratio (<2) and those with 2 or more R/S ratio were 12.3 ± 2.56 aa and 10.6 ± 3.14 aa, respectively (p < 0.05). D, The mean numbers of arginine residues in CDR3 of the rearrangements with low R/S ratio (<2) and those with 2 or more R/S ratio were 1.9 ± 1.07 and 1.0 ± 0.65, respectively (p < 0.001). CDR3s bearing three or more arginine residues were found in 26% of the rearrangements with low R/S ratio (<2) but not in any of those with a ≥2 R/S ratio (p < 0.005).

 
Rag gene expression by splenic mature B cells of B6.Sle3/5 mice

A unique subset of human peripheral B cells has been recently reported to express both surrogate and conventional L chains on cell surfaces and to have significant levels of Rag gene expression, suggesting the existence of edited BCRs in the periphery (25). IgH CDR3s of such B cells were characterized by a greater length, tyrosine-rich structure, skewed JH usage, and frequent D-D fusion and resembled the atypical CDR3s observed in B6.Sle3/5 mice as well as in human or mouse autoimmune diseases (26, 27, 28, 29, 30). This similarity led us to examine the peripheral Rag gene expression in B6 and B6.Sle3/5 mice. By RT-PCR, significant expression of Rag-1 and Rag-2 genes by splenic B cells of unimmunized B6.Sle3/5 mice was observed, whereas little or no expression was seen in unimmunized B6 mice (Fig. 6, lanes 4, 5, 10, and 11). Rag-1 expression in splenic B cells was positive for all seven B6.Sle3/5 mice, whereas only two of the seven B6 mice showed very weak positivity. In addition, splenic B cells from all seven B6.Sle3/5 mice were positive for Rag-2 expression, a feature not seen in any of the seven spleen samples from the B6 controls. A cell surface marker 493 is expressed on pro-B, pre-B, and immature/transitional B cells but not on mature B cells (19). To determine whether this finding reflected re-expression by mature B cells or continued expression by B cells recently migrated from the bone marrow, we conducted RT-PCR assays on cells fractionated by 493. The Rag gene expression was detected in a splenic 493-negative population but not in a 493-positive population, showing that peripheral Rag gene expression was by mature B cells but not immature/transitional B cells or earlier developmental B stage (Fig. 6, lanes 6, 7, 12, and 13). These observations are similar to a recent report showing an increase in peripheral mature B cells expressing Rag genes in patients with lupus, suggesting a potential role of peripheral secondary Ig rearrangements in lupus pathogenesis (31).



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  		FIGURE 6. Expression of Rag-1 and Rag-2 genes in splenic mature B cells of B6.Sle3/5 mice. Each lane shows the RT-PCR product from fractionated or unfractionated cells. Rag-1 and Rag-2 gene transcripts were observed in both B6 and B6.Sle3/5 bone marrow (BM) as positive controls (lanes 2, 3, 8, and 9). The RT-PCR study detected significant expression of Rag-1 and Rag-2 genes by splenic (SP) B cells of unimmunized B6.Sle3/5 mice, whereas little or no expression was observed in unimmunized B6 mice (lanes 4, 5, 10, and 11). The Rag gene expression was detected in splenic 493-negative (–) cells but not in splenic 493-positive (+) cells, showing peripheral Rag gene expression by mature B cells but not immature/transitional B cells or earlier developmental B stage (lanes 6, 7, 12, and 13). Representative results from experiments using seven mice in each strain are shown.

 
Circular VHD gene excision products in splenic mature B cells of B6.Sle3/5 mice

As a result of somatic recombination, VHDJH rearrangements and circular gene excision products are generated. VHDJH rearrangements persist within the chromosome and are propagated during cell proliferation, encoding BCRs and/or secreted Igs in the progeny. In contrast, circular gene excision products exist extrachromosomally and are not propagated. Their existence reflects active recombination (32). To determine whether the peripheral Rag gene expression in B6.Sle3/5 mice was functionally active, we used seminested PCR to examine the circular gene excision products derived from the somatic recombination of VH and D genes (Fig. 7A). The P6P71 PCR products obtained after the second PCR round were detected in the spleens of all five unimmunized B6.Sle3/5 mice but were not detected in any of the spleens from the five unimmunized B6 mice (Fig. 7B, lanes 4 and 5). The sequence analysis showed that the P6P71 products had recombination signal sequence (RSS) joints, verifying that they were derived from circular VHJ558-DFL16.1/DSP2.2 gene excision products (Fig. 7C). To ascertain that the recombinational events occurred in a mature B cell population as suggested by the Rag gene expression profiles, we conducted seminested PCR assays on cells fractionated by cell surface IgM, IgD, or 493. The gene excision products were observed in spleen IgM-positive, IgD-positive, and 493-negative cell populations but not IgM-negative, IgD-negative, or 493-positive cells, showing that secondary IgH recombination occurred in mature B cells of B6.Sle3/5 mice (Fig. 7B, lanes 6–11). VH replacements use the cryptic RSS within the VH coding region of VHDJH rearrangement and result in recombination of an upstream VH gene and a VHDJH rearrangement at the same allele. In contrast, secondary recombination of a VH gene and a DJH rearrangement can take place only at the other allele but not the allele once used for making of BCRs because all unrearranged D genes at the same allele have been deleted at the primary recombination. Taken together, these observations show that peripheral IgH rearrangements took place in splenic mature B cells of B6.Sle3/5 mice using allelically excluded genes.



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  		FIGURE 7. Circular VH-D gene excision products in splenic mature B cells of B6.Sle3/5 mice. A, The strategy for detection of circular VHJ558-DFL16.1/DSP2.2 gene excision products is shown. B, The P6P71 PCR products were detected in both B6 bone marrow (lane 2) and B6.Sle3/5 bone marrow (lane 3) as positive controls. The products were also detected in B6.Sle3/5 splenic B cells (lane 5) but not B6 splenic B cells (lane 4). The products were observed in B6.Sle3/5 splenic IgM-positive (+) cells (lane 6), IgD-positive (+) cells (lane 8), and 493-negative (–) cells (lane 11) but not B6.Sle3/5 splenic IgM-negative (–) cells (lane 7), IgD-negative (–) cells (lane 9), or 493-positive (+) cells (lane 10). Lane 1 shows 100 bp DNA ladder. Representative results from experiments using five mice in each strain are shown. C, Representative data from sequence analysis of the P6P71 PCR products are shown. This sequence contains the RSS joint, which consists of VHJ558-derived nonamer/spacer/heptamer and DFL16.1-derived heptamer/spacer/nonamer, showing that the P6P71 product is derived from circular VHJ558-DFL16.1 gene excision.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Normally, mature B cells with the appropriate Ig receptor, having once encountering TD-Ags and cognate CD4+ T cell help, enter the germinal center (GC) reaction, proliferate highly, and undergo somatic Ig mutation and isotype switching. These so-called GC B cells experience affinity maturation by mutational replacements of amino acids and are selected by interaction with follicular dendritic cells (FDCs) via Ag-binding affinity (33). Selected GC B cells receive signals for survival and differentiate into memory B cells or plasma cells. During the GC reaction, B cells with higher R/S ratio accumulate under selective pressure (33). It was therefore of interest to observe that B cells from B6.Sle3/5 mice exhibited higher frequencies of VH silent mutations and a lower R/S ratio, compared with those from B6 mice. These findings suggest that the altered VH mutational diversity in B6.Sle3/5 mice may result from inappropriate selection for low affinity repertoires due to impaired selective pressures. Altered selection was also suggested by the molecular tree analysis, which showed that intraclonal sequence diversity appeared more frequently in B6.Sle3/5 mice. It was also of interest to observe that in sequences from B6.Sle3/5 mice, a higher incidence of VH silent mutations and a lower R/S ratio correlated with IgH CDR3s having a greater length and an arginine-rich structure. Such CDR3 structures are thought to provide binding capacities for a number of different self-Ags or a critical binding site for dsDNA (26, 27, 34). Long CDR3s might interfere with normal pairing and interaction between H chain and L chain, affecting the selection process based on BCR signals (25). The correlation between altered VH mutation profiles and atypical CDR3s raises a possibility that the physical nature of atypical CDR3s may interfere with affinity-based selection.

Alternatively, somatic mutations might inappropriately occur outside of GCs in B6.Sle3/5 mice as demonstrated recently in MRL/lpr lupus-prone mice (35). In this case, in the absence of a functional follicular dendritic cells network, dendritic cells or other populations may play a role in the selection of somatically mutated B cells (35, 36). Such cells may lead to an alteration in selective pressures. Taken together, the alteration in VH mutational diversity in B6.Sle3/5 mice may reflect an inappropriate selection process for affinity maturation, which possibly ties to polyclonal reactivity of B cell repertoires (5). Preliminary data regarding the affinity maturation of the anti-NP response support this concept. Inhibition ELISA have shown a decreased average affinity (avidity) of serum IgG1 anti-NP from B6.Sle3/5 when compared with B6 (M. Wakui and E. S. Sobel, unpublished observations). This suggests that Sle3/5 reduced the rate and the final degree of affinity maturation of NP-reactive IgG1-positive B cells and is consistent with our hypothesis of altered selection.

It is of considerable interest that the mutation frequencies in our study are higher than those reported by other groups (12, 13, 14). A number of studies have used NP-conjugated carrier proteins, such as chicken gammaglobulin or keyhole limpet hemocyanin, with adjuvant for immunization. In contrast, we immunized mice with NP-SRBC in the absence of adjuvant. SRBC is a strong TD-Ag even without use of adjuvant. This was used to good effect in a comprehensive flow cytometry-based study of the phenotype of GC B cells (37). Compared with a single protein carrier, SRBC likely produces a larger variety of foreign peptides, which potentially activate a more diverse T cell repertoire. As reported in a recent study using a chronic graft-versus-host model of autoimmunity, a diverse T cell repertoire enhances B cell activation (38). Indeed, the somatic mutation frequencies in the chronic graft-versus-host model have been reported to be higher than those seen during secondary immune responses to foreign Ags or to those seen in the MRL/lpr mouse model (39). Likewise, help from a diverse repertoire of activated, SRBC-reactive T cells may contribute to the higher mutation frequency rate seen in our study.

Ig sequences from B6.Sle3/5 mice also exhibited an increase in atypical IgH CDR3s, showing that Sle3/5 affected VHDJH junctional diversity. Atypical CDR3 sequences have been reported to be a hallmark of autoreactive B cell repertoires, and have been proposed to have structural features that predispose to binding to self-Ags (24, 25, 26, 27, 28, 29, 30, 34, 40). In the mouse models, such observations have been made using well-known lupus-prone strains such as MRL/lpr and NZB/NZW, both of which have unique IgH haplotypes, which could potentially alter somatic Ig sequence diversity. Sle3/5 has been mapped to chromosome 7 of the NZB/NZW-derived NZM2410 strain in a location where no Ig genes have been identified. This suggests that atypical CDR3s relevant to autoreactive repertoires are likely generated independently of the IgH haplotype. Although there is yet no direct evidence, we suggest two possible mechanisms that could explain the unusual CDR3s in B6.Sle3/5 mice. One possibility is that Sle3/5 may contribute to altered regulation of somatic VDJ recombination events. Consistent with this hypothesis was our finding that in B6.Sle3/5 mice, peripheral IgH rearrangements took place in mature splenic B cells using allelically excluded genes. Another possibility is that Sle3/5 impairs negative selection for B cells with unusual CDR3s in the bone marrow and/or in the periphery. In this case, the primary frequencies of atypical V-D-J junctions by somatic recombination may or may not be different between B6 and B6.Sle3/5 strains. To clarify this point, the preimmune repertoire should be examined and compared using VDJ sequences from the bone marrow and the periphery.

Frequent D-D fusion as well as skewed D and JH usage suggests that the secondary DJH rearrangements take place more frequently in B6.Sle3/5 mice than in B6 mice. Klonowski et al. (30, 41) have reported similar observations in MRL mice. Although D-D fusion was frequently observed in both MRL newborn livers and adult pre-B cells, the skewed D and JH usage observed in the MRL newborn livers was not present in adult pre-B cells, suggesting that the IgH rearrangement process persists later during B cell life in lupus prone mice. Based on these results, they proposed a model of peripheral IgH receptor revision in autoimmune mice (41). A number of studies on in vivo secondary Ig rearrangements in mouse models have been reported, suggesting that receptor editing or revision plays a role in maintenance of self-tolerance in nonautoimmune mice or contrarily in development of autoreactive repertoires in autoimmune mice (39, 42, 43, 44). In addition, a break in allelic exclusion has been observed in lupus prone mice (39, 45). These studies have exclusively used Ig transgene strategies, which make it easy to determine whether secondary recombination occurs in B cells. However, it is possible that Ig transgenes artificially interfere with regulation of the somatic recombination process (46). Secondary IgL rearrangements in peripheral mature B cells have also been shown in nonautoimmune mice immunized without relying upon Ig transgene strategies (47, 48, 49). To our knowledge, however, this present report is the first to provide evidence for peripheral H chain receptor editing or revision without Ig transgene strategies and to show its association with a lupus susceptibility locus. As this was happening spontaneously only in B6.Sle3/5 mice, it supports the model of peripheral receptor revision contributing to autoimmunity (41).

How Rag genes are re-expressed by peripheral mature B cells remains unclear. Stimulation with LPS plus IL-4 or with anti-CD40 Ab plus either IL-4 or IL-7 can induce Rag expression by mature B cells in vitro (50). Treatment with anti-CD40 Ab up-regulates IL-7R on mature B cells (50). In B6.Sle3/5 mice, signals from chronically activated CD4+ T cells may be responsible for Rag gene re-expression by mature B cells along with some cytokines. Somatic Ig recombination requires not only Rag expression but also Ig gene accessibility. IL-7 can enhance the accessibility of 5' distal VH gene families, such as VHJ558 gene family, via histone acetylation in mature B cells re-expressing IL-7R under CD40 stimulation, leading to recombinational activation of allelically excluded Ig genes (51). Collectively, CD40 and IL-7 signals might play a critical role in peripheral IgH rearrangements occurring beyond allelic exclusion in B6.Sle3/5 mice. Efforts to clarify this point are underway.

There are two possibilities as to why mature B cells bearing atypical IgH CDR3s appear in the periphery of B6.Sle3/5 mice. One possible explanation is that atypical CDR3s have already been generated in the primary B cell repertoire of the bone marrow before migration to the periphery. This process would likely be T cell-independent. Another possibility is that the production of atypical CDR3s is completed in the periphery. In this case, in concordance with the model proposed by Klonowski and Monestier (41), secondary DJH rearrangements may completely or incompletely occur in both the bone marrow and the periphery followed by recombination of VH and DJH at the mature B cell stage. Although the peripheral IgH rearrangements might depend on an increase in activated CD4+ T cells in B6.Sle3/5 mice as described above, it is also plausible that the frequent appearance of unique CDR3 structures may reflect functional expression of Sle3/5 by non-T cells.

Our ultimate goal is to identify genes functionally contributing to lupus pathogenesis through the Sle3/5 phenotype. The positional candidate approach is a growing strategy for identification of susceptibility genes in polygenic autoimmune diseases (1, 52, 53). Indeed, an allelic variant of Cr2 with a functional consequence has been successfully identified as one of the molecules encoded by the Sle1 locus following the development of subinterval congenics with smaller intervals (54). Because the genomic interval represented by Sle3/5 contains a large number of genes related to immune response as well as many uncharacterized genes, it is still difficult to apply the positional candidate approach simply to the Sle3/5 locus. Further functional characterization of Sle3/5 along with finer mapping of mouse chromosome 7 should continue to complement each other. Our laboratory has concentrated on mixed bone marrow chimera models to elucidate the functional patterns of lupus susceptibility loci (20, 55, 56). Previous observations from these studies demonstrated that the elevated CD4:CD8 ratio was not an intrinsic property of the T cell lineage and presented evidence for functional expression of Sle3/5 by non-T cells of bone marrow origin (56). The impact of Sle3/5 on somatic IgH diversification revealed in the present study also supports the functional expression of Sle3/5 by non-T cells as described above. Mixed chimera studies are underway to determine whether the findings obtained here are an intrinsic or extrinsic property of the B cell lineage.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the National Institutes of Health Grants AI43454 and AI39824. Back

2 Address correspondence and reprint requests to Dr. Eric S. Sobel, Department of Medicine, Box 100221, J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610-0221. E-mail address: sobeles{at}medicine.ufl.edu Back

3 Abbreviations used in this paper: NZB, New Zealand Black; NZW, New Zealand White; NP, (4-hydroxy-3-nitrophenyl)-acetyl; TD-Ag, thymus-dependent Ag; R/S, replacement to silent mutation; RF, reading frame; RSS, recombination signal sequence; GC, germinal center. Back

Received for publication June 1, 2004. Accepted for publication October 4, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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