The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Inlay, M. A.
Right arrow Articles by Xu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Inlay, M. A.
Right arrow Articles by Xu, Y.
Right arrowPubmed/NCBI databases
*Gene
The Journal of Immunology, 2006, 177: 1146-1151.
Copyright © 2006 by The American Association of Immunologists

Roles of the Ig {kappa} Light Chain Intronic and 3' Enhancers in Igk Somatic Hypermutation1

Matthew A. Inlay2,*, Heather H. Gao2,*, Valerie H. Odegard{dagger}, Tongxiang Lin*, David G. Schatz{dagger} and Yang Xu3,*

* Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093; and {dagger} Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Somatic hypermutation (SHM) of the rearranged Ig genes is required for the affinity maturation of Abs. SHM is almost exclusively targeted to the rearranged Ig loci, but the mechanism of this gene-specific targeting remains unclear. The Ig {kappa} L chain locus contains multiple enhancers, including the MAR/intronic (iE{kappa}) and 3' enhancers (3'E{kappa}). Previous transgenic studies indicate that both {kappa} enhancers are individually necessary for SHM of Igk. In contrast, later studies of Ag-selected V{kappa} genes in 3'E{kappa}–/– mice found no absolute requirement for 3'E{kappa} in {kappa} SHM. To address the roles of the two {kappa} enhancers in SHM in a physiological context, we analyzed SHM of the endogenous Igk in mice with a targeted deletion of either iE{kappa} or 3'E{kappa} in Peyer’s patch germinal center B cells. Our findings indicate that, although 3'E{kappa} is quantitatively important for SHM of Igk, iE{kappa} is not required for {kappa} SHM. In addition, a reduction of {kappa} mRNA levels is also detected in activated 3'E{kappa}–/– B cells. These findings suggest that iE{kappa} and 3'E{kappa} play distinct roles in regulating Igk transcription and SHM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bcells diversify the repertoire of their Ag receptor genes through multiple and distinct processes. For example, somatic joining of the V, D, and J gene segments, mediated by the RAGs, occurs during early B cell development (1). In addition, somatic hypermutation (SHM)4 of the rearranged V(D)J sequence, mediated by the activation-induced deaminase gene (AID), occurs in activated B cells in response to antigenic challenge (2). The combinatory effects of SHM and the selection for B cells expressing Ig with a higher affinity for Ag leads to affinity maturation (3). SHM is a tightly regulated process such that mutations are targeted almost exclusively to the rearranged Ig loci, with rare but significant mistargeting to oncogenes (3). However, the mechanism that targets SHM to rearranged Ig loci remains unclear.

Accumulating evidence has identified a connection between SHM and transcription (4). For example, the expression levels of transgenes strongly correlate with the levels of mutation (5, 6, 7). In addition, hypermutation of the Ig genes begins just downstream of the promoter (8, 9) and ends well before the C region (10). This ~2-kb window from the promoter for SHM is observed regardless of which J{kappa} gene segment is rearranged (8, 11). Consistent with the notion that the distance to the promoter determines the mutability of a region, the insertion of a promoter upstream of the C region in a transgenic Igk construct caused SHM within the C region at levels similar to the V region (12).

Three enhancers have been identified within the Igk locus: one within the intron between J{kappa}5 and C{kappa} (called the intronic enhancer, or iE{kappa}) (13), another 9 kb 3' of C{kappa} (the 3' enhancer, or 3'E{kappa}) (14), and a third 8 kb downstream of 3'E{kappa} (the downstream enhancer, or Ed) (15). The deletion of either iE{kappa} or 3'E{kappa} through gene targeting leads to a quantitative decrease in Igk rearrangement (16, 17), and a near-complete block in Igk rearrangement is observed with the loss of both enhancers, indicating that these two enhancers are collectively essential for Igk rearrangement (18).

However, the roles of these enhancers in regulating {kappa} SHM have been more controversial. Transgenic studies indicated that both iE{kappa} and 3'E{kappa} were necessary for the SHM of Igk transgenes (19, 20). In this context, deletion of iE{kappa} or 3'E{kappa} resulted in a dramatic reduction of SHM of the V{kappa} region within Igk transgenes to levels equivalent to that caused by PCR error (20). However, analysis of mice bearing a deletion of the endogenous 3' enhancer leads to a different conclusion. When immunized with the hapten 2-phenyl-5-oxazolone (ph-Ox), 3'E{kappa}–/– mice underwent wild-type (Wt) levels of SHM of their V{kappa} regions, demonstrating that 3'E{kappa} is not absolutely required for SHM of Igk (21). In addition, the amount of anti-ph-Ox Abs present in the sera of the immunized 3'E{kappa}–/– mice was comparable with that of Wt mice. However, in these mice, the SHM of the endogenously rearranged V{kappa}Ox1/J{kappa}5 exon was analyzed after repeated immunization with ph-Ox. Because B cells expressing an Ig{kappa} with higher avidity to the hapten are selected for survival during Ag-driven affinity maturation, it is possible that the functions of 3'E{kappa} in {kappa} SHM might be masked by such stringent selection. In this context, mice with reduced capacity for SHM might still produce B cells with hypermutated Ig{kappa}. In this regard, it was noted that the mutation frequency of the strong hot spot was potentially decreased in 3'E{kappa}-deleted loci, leading to the suggestion that the Ag-driven B cell activation and/or somatic mutation might be less efficient in 3'E{kappa}-deleted cells (21).

In addition to reduced SHM, Igk transgenes lacking 3'E{kappa} also have dramatically reduced expression (20). Because transcription is closely correlated with the efficiency of SHM, it was hypothesized that the defect in SHM of the 3'E{kappa}-deleted Igk transgene was caused by the reduced transcription. In 3'E{kappa}–/– mice, surface expression of Igk on mature resting B cells is reduced. However, Igk transcription appeared normal in 3'E{kappa}–/– B cells activated by LPS (17). Because stimulation of B cells by LPS involves many pathways but not the ones involved in signaling through the Ag receptor or CD40, it remains to be tested whether 3'E{kappa} is important for {kappa} transcription during an immune response.

iE{kappa} plays a quantitatively more important role than 3'E{kappa} in activating Igk rearrangement (18). However, in contrast to 3'E{kappa}–/– B cells, {kappa} expression in iE{kappa}–/– B cells is normal (16). Although transgenic studies have indicated a critical role of iE{kappa} in SHM of the transgenic Igk (20), the physiological roles of iE{kappa} in SHM of Igk remain to be determined. To address these issues, we used a protocol to analyze SHM of Igk in germinal center (GC) B cells during a polyclonal immune response. In this context, Peyer’s patch (PP) B cells are exposed to continuous antigenic challenge in the gut lumen, and hence undergo robust SHM (22). We also examined splenic GC B cells following immunization with the Ag Sheep RBC (SRBC), which induces a polyclonal response (23, 24, 25). Additionally, only mutations within the 5' end of the J{kappa}C{kappa} intron, which have no bearing on the specificity of the Ab and therefore will not be selected by Ag, were analyzed. Our findings indicate a quantitatively important role for 3'E{kappa}, but not iE{kappa}, in {kappa} SHM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of iE{kappa}–/– mice

iE{kappa} and associated MAR of the endogenous {kappa} allele was replaced with the LoxP-flanked PGK-Neor gene in ES cells exactly as previously described (16). The heterozygous mutant ES cells were used to generate chimeric mice that transmitted the mutant allele into germline. To excise the PGK-Neor gene from the targeted allele, the germline heterozygous mutant mice with the PGK-Neor gene inserted in the targeted allele were bred with CMV-Cre transgenic mice that express the Cre enzyme in zygotes. The resulting heterozygous mutant mice with the PGK-Neor gene deleted from the targeted allele were intercrossed to generate homozygous iE{kappa}–/– mice.

Immunizations and splenic and PP GC B cell sorting

PP were harvested from iE{kappa}–/–, 3'E{kappa}–/–, and Wt littermates. Cells were stained with anti-mouse B220 or CD19 conjugated to PE (BD Pharmingen) and anti-mouse GL7 conjugated to FITC (BD Pharmingen). Live GL7– and GL7+ B cells were sorted using a FACSVantage cell sorter (BD Biosciences). Alternatively, PP cells were stained with anti-mouse Ig{kappa} conjugated to PE (BD Pharmingen) and anti-mouse GL7 Abs, and then Ig{kappa}+GL7+ cells were sorted. 3'E{kappa}–/– and Wt littermates of 5 mo of age were immunized with 2 x 108 SRBC (catalog no. R3378; Sigma-Aldrich). At day 7, mice were given a booster of 2 x 108 SRBC. At day 14, spleens and lymph nodes were collected from these mice and purified into single-cell suspensions, which were stained with anti-mouse B220 (BD Pharmingen) Abs conjugated to PE (BD Pharmingen) and peanut agglutinin (PNA)-FITC (Vector Laboratories). Live (PI–) B220+PNAhigh GC B cells were sorted.

Analysis of SHM

Genomic DNA from sorted cells was purified, and the J{kappa}C{kappa} intron was amplified by PCR using KOD hot-start polymerase (Calbiochem) according to the manufacturer’s protocol, with the following primers: V{kappa}D.bam, 5'-ccggatccGGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3'; K1.eco, 5'-ggcgaattcGTGACAAATTTTAGAATAAGAGTCACACCTC-3'.

Sequences in lowercase were added to introduce restriction sites for cloning. The PCR conditions were 95°C for 2 min, followed by 32–35 cycles of 94°C for 20 s, 57°C for 30 s, and 68°C for 1 min, concluding with a final extension at 68°C for 10 min. Purified PCR products were cloned into pBluescript and sequenced. Clones were sequenced using the T3 primer. Sequences were aligned using ClustalX software and scored for mutations. The unpaired t test was used. A value of p < 0.05 was considered significant.

B cell stimulations and flow cytometry analysis

Single-cell suspensions of spleens from 3'E{kappa}–/– and Wt littermates were stained with microbead-conjugated anti-CD43 Abs (Miltenyi Biotec) and biotinylated anti-Ig{lambda} Abs (BD Pharmingen), followed by a secondary stain with microbead-conjugated anti-biotin Abs (Miltenyi Biotec). {kappa}+ B cells were enriched using the MACS system (Miltenyi Biotec) and were 90–95% pure. Purified {kappa}+ B cells were cultured in RPMI 1640 medium supplemented with 15% FBS, penicillin/streptomycin, L-glutamine, and 100 mM 2-ME at 106 cells/ml, and then stimulated with 10 µg/ml anti-IgM (Jackson ImmunoResearch Laboratories) and 5 µg/ml anti-CD40 (BD Pharmingen) Abs, 50 nM PMA (Calbiochem) and 1 µM ionomycin (Calbiochem), or 10 µg/ml LPS (Sigma-Aldrich). PP cells were stained with PE-conjugated anti-mouse B220, FITC-conjugated PNA, and biotin-conjugated anti-mouse Ig{kappa}. Biotinylated Ab was revealed with streptavidin-conjugated allophycocyanin.

Real-time quantitative PCR analysis

Unstimulated and stimulated {kappa}+ B cells from 3'E{kappa}–/– and Wt littermates were harvested, and their RNA was purified, converted to cDNA, and analyzed by real-time PCR as described previously (26). Primers for Cµ and C{kappa} are as described (26).

The University of California, San Diego, Animal Subjects Committee approved all experiments that involved mice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Normal Igk expression in iE{kappa}–/– B cells

To examine whether Igk expression in activated B cells requires iE{kappa}, {kappa} surface expression on B cells derived from the PP of Wt and iE{kappa}–/– littermates was analyzed by flow cytometry. The B cell activation in PP was similar between Wt and iE{kappa}–/– mice (Fig. 1A). In addition, surface Ig{kappa} levels were normal in unactivated (GL7–) as well as activated iE{kappa}–/– B cells (GL7+) when compared with those in Wt controls (Fig. 1, B and C). Because {kappa} gene rearrangement is compromised in iE{kappa}–/– B cells, as published previously (16), knockout mice exhibit a much higher proportion of Ig{kappa}-negative (Ig{lambda}-positive) cells. Thus, consistent with the conclusion from the transgenic studies (20), iE{kappa} is not required for {kappa} expression in activated B cells.


Figure 1
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1. {kappa} expression in Wt and iE{kappa}–/– GC and non-GC B cells. A, Comparison of the B cell populations in Wt (right) and iE{kappa}–/– (left) PP. Gates and percentages of non-GC (GL7–B220+) and GC (GL7+B220+) B cells are shown. B, Comparison of surface {kappa} expression between non-GC and GC B cells. B220+ PP cells are plotted for the surface {kappa} expression and intensity of GL7 staining. C, Surface {kappa} expression in non-GC (left) and GC (right) B cells in Wt (solid gray) and iE{kappa}–/– (dotted black) PP.

 
Normal {kappa} SHM in iE{kappa}–/– mice

To determine the role of iE{kappa} in {kappa} SHM, GC {kappa}+ B cells ({kappa}+B220+GL7+) were sorted from the pooled PP of three 4-mo-old Wt and iE{kappa}–/– littermates. To amplify only the rearranged Igk allele from genomic DNA purified from the sorted B cells, we used the degenerate V{kappa} primer, which binds to ~90% of V{kappa} gene segments (27), and a primer ~600 bp downstream of J{kappa}5 (Fig. 2A). PCR products were cloned into pBluescript and sequenced. Because mutations within the intronic region 3' of J{kappa}5 are not selected by Ag, we analyzed the mutation frequency in the 500-bp intronic sequence. The mutation rate was 12.6 x 10–3 mutations/bp for Wt B cells and 15.8 x 10–3 for the iE{kappa}–/– B cells (Fig. 2B and Table I). Although {kappa} SHM appeared slightly elevated in iE{kappa}–/– B cells, this was due to the lower number of sequences analyzed with zero mutations, because the mutation frequency on sequences containing at least one mutation was equivalent between iE{kappa}–/– and Wt (Table I). The mutational spectra were also similar between Wt and iE{kappa}–/– B cells (Fig. 2C), resulting in similar percentages of transitions and transversions (Table I). The mutations identified from both groups showed the hallmarks of mutations generated by SHM, because they were RGYW/WRCY hot spot targeted and biased toward transitions (Table I). Thus, {kappa} SHM is normal in iE{kappa}–/– mice.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 2. {kappa} SHM in pooled PP {kappa}+ GC B cells of three pairs of 4-mo-old Wt and iE{kappa}–/– littermates. A, PCR strategy for detecting SHM. Genomic DNA from purified GC B cells was amplified by PCR using the degenerate V{kappa} primer and the K1 primer, which anneals ~700 bp downstream of J{kappa}5. B, Pie chart of {kappa} SHM in Wt (left) and iE{kappa}–/– (right) samples. Each pie slice represents the proportion of sequences containing the number of mutations listed on the perimeter. C, Mutation spectrum of {kappa} in Wt and iE{kappa}–/– PP GC B cells. The nucleotides listed on the y-axis represent the germline nucleotide, and the nucleotide on the x-axis shows the mutation. The proportions of each type of substitution are displayed in the chart, adjusted for the percentage of each type of nucleotide in the 500-bp sequence analyzed (G, 28.2%; A, 31.6; T, 26.4%; C, 13.8%).

 

View this table:
[in this window]
[in a new window]
  		Table I. SHM of the {kappa} intronic region in Wt and iE{kappa}–/– GC B cells

 
Reduced {kappa} mRNA levels in activated 3'E{kappa}–/– B cells

Because deletion of 3'E{kappa} leads to a reduction in {kappa} surface expression in unactivated B cells (17), we examined how {kappa} expression is affected in activated 3'E{kappa}–/– B cells. Previous studies indicated that 3'E{kappa} was not necessary for {kappa} expression in B cells activated by LPS (17). However, because LPS activates B cells independently of the BCR, we analyzed {kappa} mRNA levels in Wt and 3'E{kappa}–/– B cells activated in vitro by multiple stimuli using real-time PCR. Consistent with earlier findings, there was a ~3-fold decrease in {kappa} mRNA levels in resting 3'E{kappa}–/– B cells when compared with that in resting Wt B cells (17) (Fig. 3A). In addition, similar levels of {kappa} expression were detected between 3'E{kappa}–/– and Wt B cells stimulated with LPS (Fig. 3A). However, when stimulated with anti-IgM and anti-CD40 Abs that mimic B cell activation during an immune response, a ~3-fold reduction in {kappa} mRNA levels was detected in 3'E{kappa}–/– B cells when compared with that in Wt B cells (Fig. 3A). Because the lower surface expression of IgM in 3'E{kappa}–/– B cells might affect the signaling through the BCR, we treated B cells with PMA and ionomycin, which directly activate signaling pathways downstream of the BCR independent of BCR engagement. {kappa} mRNA levels were 5-fold lower in 3'E{kappa}–/– B cells than in Wt B cells after stimulation with PMA/ionomycin (Fig. 3A). The differences in {kappa} mRNA levels also resulted in analogous differences in {kappa} surface expression in the activated B cells (Fig. 3B). To further ensure that the reduced transcription in activated 3'E{kappa}–/– B cells is not due to impaired activation, we compared the activation efficiency between Wt and 3'E{kappa}–/– B cells under each of the stimulation conditions. The percentage of activated B cells, which express the early activation marker CD69 and are actively dividing, were similar between 3'E{kappa}–/– and Wt B cells after various stimulations (Fig. 3B).


Figure 3
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 3. Analysis of {kappa} expression in stimulated Wt and 3'E{kappa}–/– B cells. A, Ratio of {kappa} mRNA levels in stimulated 3'E{kappa}–/– B cells vs those in stimulated Wt B cells. {kappa}+ B cells were purified by MACS and activated by a variety of stimuli. Untreated (UT) B cells were harvested immediately after sorting, and stimulated B cells were harvested 2 days after stimulation. Quantitative real-time PCR using primers specific for the {kappa} and µ C regions was used to analyze the gene expression of {kappa} and µ, respectively. All samples were normalized to Cµ expression. B, {kappa} surface expression profile in stimulated Wt (solid gray) and 3'E{kappa}–/– (dotted black) B cells. Only cells within the gates displayed in Fig. 3C are shown. C, Analysis of the activation efficiency in 3'E{kappa}–/– and Wt B cells by flow cytometry. Samples from each type of stimulation were stained with anti-B220, -CD69, and -{kappa} Abs. CD69 expression (y-axis) and cell size (x-axis) of live, B220+ lymphocytes are shown. The percentage of CD69+, blasting Wt (left column) and 3'E{kappa}–/– (right column) B cells are listed inside the corresponding gate. IONO, Ionomycin.

 
To examine {kappa} mRNA levels in activated B cells in vivo, we sorted {kappa}+ GC B cells from the PP of five pairs of Wt and 3'E{kappa}–/– littermates. The mRNA levels of {kappa} were ~2-fold reduced in 3'E{kappa}–/– GC B cells than in Wt GC B cells (Fig. 4A). However, when the total PP B cells were analyzed by flow cytometry, {kappa} surface expression in 3'E{kappa}–/– GC B cells was only slightly lower than Wt (Fig. 4B). This is caused in part by the reduction of surface IgM in activated GC B cells (B220+PNAhigh) compared with non-GC B cells (28, 29) (Fig. 4C), likely as a result of a reduction in Cµ mRNA levels in the B220+PNAhigh GC B cells (Fig. 4D). The lower {kappa} mRNA levels are not due to a defect in B cell activation, because the proportion of activated B cells (Fig. 4E), or specifically activated {kappa}+ B cells (Fig. 4C), is similar between the PP of 3'E{kappa}–/– and Wt mice. When compared with that in 3'E{kappa}–/– B cells stimulated by PMA and ionomycin in vitro, the reduction of {kappa} mRNA levels in 3'E{kappa}–/– GC B cells vs Wt controls is less dramatic, likely due to that the constant stimuli of PP B cells include LPS present on the bacterial flora.


Figure 4
View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 4. {kappa} expression in Wt and 3'E{kappa}–/– PP B cells. A, Analysis of {kappa} mRNA levels in {kappa}+ Wt ({square}) and 3'E{kappa}–/– ({blacksquare}) PP GC (GL7+) and non-GC (GL7–) B cells. mRNA levels were normalized to CD19 mRNA levels. {kappa} mRNA levels of all samples are shown relative to Wt non-GC sample. B, {kappa} surface expression profile of non-GC (left) and GC (right) B cells of Wt (solid gray) and 3'E{kappa}–/– (dotted black) PP. C, Comparison of {kappa} surface expression in PP B cells in 3'E{kappa}–/– and Wt mice. Total B220+ PP cells were plotted for surface {kappa} expression and intensity of PNA staining. D, Analysis of Cµ mRNA levels in B220+PNAhigh GC B cells. Samples were normalized to CD19 mRNA levels and are shown relative to Wt non-GC. E, Proportions of non-GC (B220+PNAlow) and GC (B220+PNAhigh) in Wt (left) and 3'E{kappa}–/– (right) PP. Gates and percentages of GC and non-GC B cells are shown.

 
Quantitative decrease in {kappa} SHM in 3'E{kappa}–/– GC B cells

Although 3'E{kappa} was essential for the SHM of an Igk transgene, analysis of Ag-driven SHM of Igk in 3'E{kappa}–/– mice indicated that 3'E{kappa} is not required for {kappa} SHM (20, 21). However, it remains possible that 3'E{kappa} could still have an influence on overall efficiency of {kappa} SHM. Additionally, given the correlation between SHM and transcription efficiency, the reduction in {kappa} mRNA levels we observed in 3'E{kappa}–/– GC B cells suggests the possibility of a defect in {kappa} SHM in these cells. To address this possibility, we re-examined SHM in 3'E{kappa}–/– mice by analyzing mutations within the J{kappa}C{kappa} intron in GC B cells after immunization with SRBCs. Five-month-old Wt and 3'E{kappa}–/– littermates were immunized with 2 x 108 SRBCs, and then given a booster injection at day 7. At day 14, spleen cells were purified, stained with an Ab for the B cell marker B220, and with PNA, which stains brightly for GC B cells, and GC B cells (B220+, PNAhigh) were sorted. Genomic DNA from these GC B cells was purified, and V{kappa}J{kappa}5 rearrangements were amplified by PCR, cloned, and sequenced. In Wt GC B cells, we detected an average of 3.7 mutations per 500 bp of sequence in the samples (7.3 x 10–3 mutations per base) (Table II and Fig. 5A). In contrast, only 1.3 mutations per 500 bp were detected in sequences derived from 3'E{kappa}–/– GC B cells (2.6 x 10–3 per base), an ~3-fold reduction. Furthermore, the number of unmutated sequences was ~2.5-fold greater in 3'E{kappa}–/– GC B cells (51%) compared with Wt (18%) (Fig. 5A). This did not fully account for the SHM defect, because the average number of mutations on sequences containing at least one mutation was still 2-fold reduced in 3'E{kappa}–/– GC B cells, compared with Wt (Table II). The mutation spectrum was similar between Wt and 3'E{kappa}–/– splenic GC B cells (Fig. 5B), and displayed equivalent percentages of hot-spot mutations and transitions (Table II).


View this table:
[in this window]
[in a new window]
  		Table II. SHM of the {kappa} intronic region in Wt and 3'E{kappa}–/– GC B cells

 

Figure 5
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 5. Analysis of {kappa} SHM in GC B cells of Wt and 3'E{kappa}–/– mice. A, Pie chart of {kappa} SHM in splenic GC B cells of 5-mo-old Wt and 3'E{kappa}–/– littermates after SRBC immunization. Each pie slice represents the proportion of sequences containing the number of mutations indicated on the perimeter of each slice. The data are pooled from three pairs of immunized Wt and 3'E{kappa}–/– littermates. B, Mutation spectrum of {kappa} in Wt and 3'E{kappa}–/– splenic GC B cells. The nucleotides listed on the y-axis represent the germline nucleotide, and the nucleotide on the x-axis shows the mutation. The proportions of each type of substitution are displayed in the chart, adjusted for the percentage of each type of nucleotide in the 500-bp sequence analyzed (G, 28.2%; A, 31.6%; T, 26.4%; C, 13.8%). C, Pie chart of {kappa} SHM in pooled PP GC B cells of 6-mo-old Wt and 3'E{kappa}–/– mice. Each slice represents the proportion of the sequences with the number of mutations listed on the perimeter. D, Pie chart of {kappa} SHM in pooled {kappa}+ GC B cells of four pairs of 6-mo-old Wt and 3'E{kappa}–/– mice.

 
To confirm the defect in Igk SHM observed in 3'E{kappa}–/– splenic GC B cells, we also examined SHM in GC B cells from PP of 6-mo-old unimmunized mice. GC B cells (CD19+GL7+) were purified from the PP, and the J{kappa}C{kappa} intronic region was sequenced (Fig. 5C). In Wt GC B cells, the mutation frequency of the analyzed region was 16 x 10–3 (Table II). However, in 3'E{kappa}–/– GC B cells, the mutation frequency was reduced 2.5-fold. The same proportion of sequences contained mutations in both groups, but the sequences from 3'E{kappa}–/– GC B cells had a lower mutation load than the Wt. Wt and 3'E{kappa}–/– B cells show a similar spectrum of mutations (data not shown). Additionally, the mutations analyzed from both groups are targeted to RGYW/WRCY hotspots and transition biased (Table II). The variation in the mutational frequency in the Wt GC B cells between different experiments was likely due to the different ages of mice analyzed, because older mice accumulate more mutations in Igk of GC B cells.

Although the sorted GC cells in the previous two experiments included {lambda}+ B cells, which are a significant population in 3'E{kappa}–/– mice, we would not expect that these {lambda}+ B cells contribute to the SHM data, because the rearranged Igk loci are mostly deleted through RS rearrangement in 3'E{kappa}–/– {lambda}+ B cells (17). However, to rule out the possibility that a larger proportion of {lambda}+ B cells in sorted GC B cells could lead to skewed data, we repeated the analysis of sorted {kappa}+ GC cells ({kappa}+GL7+) from the PP of 6-mo-old Wt and 3'E{kappa}–/– littermates. In Wt and 3'E{kappa}–/– GC {kappa}+ B cells, the mutation frequencies of the analyzed region were 22 x 10–3 and 10 x 10–3, respectively, a 2.2-fold difference (Fig. 5D, Table II). Therefore, 3'E{kappa} is quantitatively important for {kappa} SHM.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recruitment of the SHM machinery specifically to the rearranged Ig loci is critical for Ab maturation as well as the maintenance of genetic stability via the prevention of hypermutation in other parts of the genome. cis-elements within the Ig loci have been thought to play critical roles in this recruitment process. Transgenic studies implicated essential roles for both iE{kappa} and 3'E{kappa} in driving {kappa} SHM (20). In this study, we report the first analysis of SHM of the endogenous Igk allele in the absence of iE{kappa}. In contrast with the findings of the transgenic studies (20), we have observed no apparent defect in {kappa} SHM in iE{kappa}–/– mice. Given the very small size of transgenes compared with the endogenous 3.2-Mb Igk locus, the discrepancy is likely due to the presence of other cis-elements that play redundant roles in activating {kappa} SHM. One potential candidate is the recently discovered downstream enhancer (Ed), which was not contained in the {kappa} transgenes (15).

Previous studies of the functions of 3'E{kappa} in {kappa} SHM have reached conflicting conclusions regarding its importance. Transgenic studies indicated that 3'E{kappa} is critical for the expression and SHM of Igk in randomly integrated transgenes in the presence as well as absence of stringent Ag selection (20, 30). However, analyses of 3'E{kappa}–/– mice showed that 3'E{kappa} is not important for {kappa} transcription following LPS-induced activation (17) and also that 3'E{kappa} is not required for Ag-driven SHM (21). However, these authors noted a potentially reduced mutation frequency and proposed that 3'E{kappa} might be involved in promoting the optimal efficiency of {kappa} SHM. By analyzing SHM in the J{kappa}C{kappa} intron following immunization with the polyclonal response-inducing Ag SRBC, or in unimmunized PP, we confirm that the 3'E{kappa} is indeed not required for {kappa} SHM. However, our findings clearly demonstrate that 3'E{kappa} is quantitatively important for {kappa} SHM.

Because of the correlation between transcription and SHM, we analyzed {kappa} mRNA levels in 3'E{kappa}–/– B cells activated by a number of stimuli. Consistent with earlier observations (17), normal {kappa} mRNA levels are observed in 3'E{kappa}–/– B cells activated by LPS. However, {kappa} expression appears to be impaired in 3'E{kappa}–/– B cells when signaling pathways downstream of the BCR and CD40 are activated. We therefore conclude that 3'E{kappa}, similar to its function in developing and mature B cells, is an important mediator of {kappa} transcription in activated B cells. The surface {kappa} expression of Wt and 3'E{kappa}–/– GC B cells is similar, possibly due to reduced IgH transcription in GC B cells. Therefore, the modest defect in {kappa} transcription, but not surface expression, observed in 3'E{kappa}–/– GC B cells could explain the reduced mutation of Igk in 3'E{kappa}–/– mice.

Our findings that neither 3'E{kappa} nor iE{kappa} is critical for {kappa} SHM suggest that these two enhancers might play redundant roles in activating {kappa} SHM. A similar scenario has been observed in the regulation of Igk rearrangement by the two enhancers (18). Furthermore, mice bearing a deletion of the core region of the IgH Eµ enhancer show no defect in SHM of IgH (31). Eµ can drive a low level of SHM in transgenic constructs (32, 33, 34), but not others (35, 36), implicating a role for multiple elements in the regulation of SHM at the IgH locus.

Transcription factors binding to the enhancer elements might be involved in SHM recruitment. In this context, two accidentally introduced E2A binding sites, in addition to those already present in iE{kappa} and 3'E{kappa}, were found to increase SHM in a {kappa} transgene with no apparent impact on the transcription of the {kappa} transgene (25). As both iE{kappa} and 3'E{kappa} contain E2A binding sites, these enhancers might activate SHM via the recruitment of E2A. iE{kappa}’s two E2A binding sites have been shown to be important for Igk rearrangement (26). Therefore, similar to their redundant roles in the regulation of {kappa} rearrangement, the intronic and 3' enhancers might play redundant roles in activating {kappa} SHM (18). Targeted replacement of J{kappa} with a rearranged V{kappa}J{kappa} driven by a constitutive promoter in the context of an enhancerless Igk allele might reveal the redundant roles of the two Igk enhancers in targeting SHM to Igk loci.


   Acknowledgments
 
We thank Drs. Mark Schlissel and Fred Alt for the 3'E{kappa}–/– mice, and Dr. Fred Alt for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 work was supported by National Institutes of Health Grant AI44838 (to Y.X.). D.G.S. is a Howard Hughes Medical Institute investigator. Back

2 M.A.I. and H.H.G. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Yang Xu, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322. E-mail address: yangxu{at}ucsd.edu Back

4 Abbreviations used in this paper: SHM, somatic hypermutation; iE{kappa}, {kappa} intronic enhancer; 3'E{kappa}, {kappa} 3' enhancer; {kappa}, Ig {kappa} L chain; Igk, Ig {kappa} L chain locus; ph-Ox, 2-phenyl-5-oxazolone; Wt, wild type; GC, germinal center; PP, Peyer’s patch; PNA, peanut agglutinin; SRBC, sheep RBC; iono, inomycin. Back

Received for publication January 28, 2005. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bassing, C. H., W. Swat, F. W. Alt. 2002. The mechanism and regulation of chromosomal V(D)J recombination. Cell 109: S45-S55. [Medline]
  2. Honjo, T., K. Kinoshita, M. Muramatsu. 2002. Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu. Rev. Immunol. 20: 165-196. [Medline]
  3. Wagner, S. D., M. S. Neuberger. 1996. Somatic hypermutation of immunoglobulin genes. Annu. Rev. Immunol. 14: 441-457. [Medline]
  4. Storb, U., A. Peters, E. Klotz, N. Kim, H. M. Shen, K. Kage, B. Rogerson, T. E. Martin. 1998. Somatic hypermutation of immunoglobulin genes is linked to transcription. Curr. Top. Microbiol. Immunol. 229: 11-19. [Medline]
  5. Goyenechea, B., N. Klix, J. Yelamos, G. T. Williams, A. Riddell, M. S. Neuberger, C. Milstein. 1997. Cells strongly expressing Ig{kappa} transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers. EMBO J. 16: 3987-3994. [Medline]
  6. Fukita, Y., H. Jacobs, K. Rajewsky. 1998. Somatic hypermutation in the heavy chain locus correlates with transcription. Immunity 9: 105-114. [Medline]
  7. Bachl, J., C. Carlson, V. Gray-Schopfer, M. Dessing, C. Olsson. 2001. Increased transcription levels induce higher mutation rates in a hypermutating cell line. J. Immunol. 166: 5051-5057. [Abstract/Free Full Text]
  8. Lebecque, S. G., P. J. Gearhart. 1990. Boundaries of somatic mutation in rearranged immunoglobulin genes: 5' boundary is near the promoter, and 3' boundary is approximately 1 kb from V(D)J gene. J. Exp. Med. 172: 1717-1727. [Abstract/Free Full Text]
  9. Rada, C., A. Gonzalez-Fernandez, J. M. Jarvis, C. Milstein. 1994. The 5' boundary of somatic hypermutation in a V {kappa} gene is in the leader intron. Eur. J. Immunol. 24: 1453-1457. [Medline]
  10. Hackett, J., Jr, B. J. Rogerson, R. L. O’Brien, U. Storb. 1990. Analysis of somatic mutations in {kappa} transgenes. J. Exp. Med. 172: 131-137. [Abstract/Free Full Text]
  11. Weber, J. S., J. Berry, T. Manser, J. L. Claflin. 1991. Position of the rearranged V{kappa} and its 5' flanking sequences determines the location of somatic mutations in the J{kappa} locus. J. Immunol. 146: 3652-3655. [Abstract]
  12. Peters, A., U. Storb. 1996. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4: 57-65. [Medline]
  13. Queen, C., D. Baltimore. 1983. Immunoglobulin gene transcription is activated by downstream sequence elements. Cell 33: 741-748. [Medline]
  14. Meyer, K. B., M. S. Neuberger. 1989. The immunoglobulin kappa locus contains a second, stronger B-cell-specific enhancer which is located downstream of the constant region. EMBO J. 8: 1959-1964. [Medline]
  15. Liu, Z.-M., J. B. George-Raizen, S. Li, K. C. Meyers, M. Y. Chang, W. T. Garrard. 2002. Chromatin structural analyses of the mouse Ig{kappa} gene locus reveal new hypersensitive sites specifying a transcriptional silencer and enhancer. J. Biol. Chem. 277: 32640-32649. [Abstract/Free Full Text]
  16. Xu, Y., L. Davidson, F. W. Alt, D. Baltimore. 1996. Deletion of the Ig {kappa} light chain intronic enhancer/matrix attachment region impairs but does not abolish V{kappa} J{kappa} rearrangement. Immunity 4: 377-385. [Medline]
  17. Gorman, J. R., N. vanderStoep, R. Monroe, M. Cogne, L. Davidson, F. W. Alt. 1996. The Ig {kappa} 3' enhancer influences the ratio of Ig {kappa} versus Ig {lambda} B lymphocytes. Immunity 5: 241-252. [Medline]
  18. Inlay, M., F. W. Alt, D. Baltimore, Y. Xu. 2002. Essential roles of the {kappa} light chain intronic enhancer and 3' enhancer in {kappa} rearrangement and demethylation. Nat. Immunol. 3: 463-468. [Medline]
  19. Sharpe, M. J., M. Neuberger, R. Pannell, M. A. Surani, C. Milstein. 1990. Lack of somatic mutation in a {kappa} light chain transgene. Eur. J. Immunol. 20: 1379-1385. [Medline]
  20. Betz, A. G., C. Milstein, A. Gonzalez-Fernandez, R. Pannell, T. Larson, M. S. Neuberger. 1994. Elements regulating somatic hypermutation of an immunoglobulin {kappa} gene: critical role for the intron enhancer/matrix attachment region. Cell 77: 239-248. [Medline]
  21. van der Stoep, N., J. R. Gorman, F. W. Alt. 1998. Reevaluation of 3'E{kappa} function in stage- and lineage-specific rearrangement and somatic hypermutation. Immunity 8: 743-750. [Medline]
  22. Gonzalez-Fernandez, A., D. Gilmore, C. Milstein. 1994. Age-related decrease in the proportion of germinal center B cells from mouse Peyer’s patches is accompanied by an accumulation of somatic mutations in their immunoglobulin genes. Eur. J. Immunol. 24: 2918-2921. [Medline]
  23. Rogerson, B. J.. 1995. Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members. Int. Immunol. 7: 1225-1235. [Abstract/Free Full Text]
  24. Kim, N., G. Bozek, J. C. Lo, U. Storb. 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190: 21-30. [Abstract/Free Full Text]
  25. Michael, N., H. M. Shen, S. Longerich, N. Kim, A. Longacre, U. Storb. 2003. The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription. Immunity 19: 235-242. [Medline]
  26. Inlay, M. A., H. Tian, T. Lin, Y. Xu. 2004. Important roles for E protein binding sites within the immunoglobulin {kappa} chain intronic enhancer in activating V{kappa} J{kappa} rearrangement. J. Exp. Med. 200: 1205-1211. [Abstract/Free Full Text]
  27. Schlissel, M. S., D. Baltimore. 1989. Activation of immunoglobulin {kappa} gene rearrangement correlates with induction of germline {kappa} gene-transcription. Cell 58: 1001-1007. [Medline]
  28. Butcher, E. C., R. A. Reichert, R. L. Coffman, C. Nottenburg, I. L. Weissman. 1982. Surface phenotype and migratory capability of Peyer’s patch germinal center cells. Adv. Exp. Med. Biol. 149: 765-772. [Medline]
  29. Butcher, E. C., R. V. Rouse, R. L. Coffman, C. N. Nottenburg, R. R. Hardy, I. L. Weissman. 1982. Surface phenotype of Peyer’s patch germinal center cells: implications for the role of germinal centers in B cell differentiation. J. Immunol. 129: 2698-2707. [Abstract]
  30. Sharpe, M. J., C. Milstein, J. M. Jarvis, M. S. Neuberger. 1991. Somatic hypermutation of immunoglobulin {kappa} may depend on sequences 3' of C{kappa} and occurs on passenger transgenes. EMBO J. 10: 2139-2145. [Medline]
  31. Perlot, T., F. W. Alt, C. H. Bassing, H. Suh, E. Pinaud. 2005. Elucidation of IgH intronic enhancer functions via germ-line deletion. Proc. Natl. Acad. Sci. USA 102: 14362-14367. [Abstract/Free Full Text]
  32. Azuma, T., N. Motoyama, L. E. Fields, D. Y. Loh. 1993. Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer. Int. Immunol. 5: 121-130. [Abstract/Free Full Text]
  33. Sohn, J., R. M. Gerstein, C. L. Hsieh, M. Lemer, E. Selsing. 1993. Somatic hypermutation of an immunoglobulin mu heavy chain transgene. J. Exp. Med. 177: 493-504. [Abstract/Free Full Text]
  34. Klotz, E. L., U. Storb. 1996. Somatic hypermutation of a {lambda}2 transgene under the control of the {lambda} enhancer or the heavy chain intron enhancer. J. Immunol. 157: 4458-4463. [Abstract]
  35. Hackett, J., Jr, C. Stebbins, B. Rogerson, M. M. Davis, U. Storb. 1992. Analysis of a T cell receptor gene as a target of the somatic hypermutation mechanism. J. Exp. Med. 176: 225-231. [Abstract/Free Full Text]
  36. Hengstschlager, M., M. Williams, N. Maizels. 1994. A {lambda}1 transgene under the control of a heavy chain promoter and enhancer does not undergo somatic hypermutation. Eur. J. Immunol. 24: 1649-1656. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. Xiang and W. T. Garrard
The Downstream Transcriptional Enhancer, Ed, Positively Regulates Mouse Ig{kappa} Gene Expression and Somatic Hypermutation
J. Immunol., May 15, 2008; 180(10): 6725 - 6732.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Kothapalli, D. D. Norton, and S. D. Fugmann
Cutting Edge: A cis-Acting DNA Element Targets AID-Mediated Sequence Diversification to the Chicken Ig Light Chain Gene Locus
J. Immunol., February 15, 2008; 180(4): 2019 - 2023.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. Y. Yang, S. D. Fugmann, and D. G. Schatz
Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences
J. Exp. Med., December 25, 2006; 203(13): 2919 - 2928.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Inlay, M. A.
Right arrow Articles by Xu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Inlay, M. A.
Right arrow Articles by Xu, Y.
Right arrowPubmed/NCBI databases
*Gene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS