Department of Internal Medicine and Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas TX 75235, USA
Correspondence to: P. E. Lipsky
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: B lymphocytes, Ig, selection, somatic hypermutation, VDJ rearrangement
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Engagement of CD40, a type I transmembrane protein, is critical in many steps of B cell activation. CD40 ligation induces proliferation of immature and mature B cell subsets, differentiation and Ig production, isotype switching, rescue from apoptosis, phenotypic differentiation of B cells into germinal center cells, and skewing of the maturation of germinal center cells into memory cells rather than plasma cells (46).
Most research has concentrated on the effect of CD40 stimulation on mature B cells. However, CD40 could play a role in some aspects of B cell maturation as it is expressed very early in human B cell ontogeny immediately after the expression of CD10 and CD19 (7). Moreover, ligation of CD40 on pre-B cells and immature B cells induces growth as well as CD23 expression in the presence of IL-3 (8,9).
Although the expression of CD40 precedes rearrangement of the Ig genes (7), engagement of CD40 is not thought to play a role in the molecular generation of the Ig repertoire as this appears to be grossly normal in mice that have been engineered to lack expression of CD40 or CD154 (1013). CD40 ligation may, however, play a role in influencing the expressed Ig repertoire as signaling through this molecule could play a role in the positive and negative selection of B cell precursors after they express a functional Ig receptor. In addition, CD40 ligation plays a critical role in the transition of naive B cells to memory B cells in response to antigenic stimulation. Thus, alterations of the Ig repertoire that might occur as a result of T cell-dependent antigenic stimulation, including that resulting from the induction of somatic hypermutation and subsequent selection, are largely precluded in X-HIgM patients (3). The overall impact of CD40 ligation in shaping the preimmune Ig repertoire and its subsequent influence on the immune repertoire have not been delineated.
The opportunity to analyze the Ig repertoire expressed by B cells of a child with X-HIgM and compare it with that of B cells from normal donors provided the means to deduce the impact of CD40 ligation on the distribution of expressed Ig gene products. To achieve this, a single-cell PCR technique was utilized (14) to analyze VH rearrangements from genomic DNA of individual CD19+/IgM+ B cells isolated from the peripheral blood.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Single-cell PCR
Genomic DNA of individual CD19+/IgM+ B cells was analyzed for rearranged Ig genes using a single-cell PCR technique that permits the amplification of both productive and non-productive VH rearrangements (14). An initial primer extension preamplification step employing random 15mers and 60 rounds of amplification with Taq polymerase supplied by Promega (Madison, WI) was used to produce sufficient DNA for multiple subsequent specific amplifications with nested VH family-specific primers (14). Aliquots (10 µl) of the final PCR products were then separated by electrophoresis using a 1.5% agarose gel and analyzed for products of the anticipated size.
Sequence analysis
PCR products were cut from the gel and purified by gel electrophoresis through 2% SeaKem LE agarose from FMC Bioproducts (Rockland, ME) using GeneCapsule obtained from Geno Technology (St Louis, MO) or GenElute from Supelco (Bellefonte, PA) and directly sequenced with an automated DNA sequencer (ABI Prism 377; Perkin-Elmer, Norwalk, CT) using the ABI Prism dye termination cycle sequencing kit from Perkin-Elmer and the 5' primer used for the second round of amplification. The DNA sequences obtained were analyzed using GeneWorks software, release 2.45, by IntelliGenetics (Mountain View, CA) and Sequencher supplied by Gene Codes Corp. (Ann Arbor, MI) and the V BASE Sequence Directory (17). A D segment was identified when it shared at least seven consecutive nucleotides with a known D segment or eight nucleotides of sequence identity interrupted by no more than one nucleotide of substitution (18). Whenever a sequence was found that contained stop codons within FR1 to FR4 or an out of frame rearrangement, it was considered to be non-productive, because it could not encode a protein product. All sequences determined in this study have been submitted to the GeneBank, EMBL and DDBJ Nucleotide Sequence Database under accession nos AF077410AF077525.
Control VHDJH repertoire from normal donors
The VH repertoire from normal donors reported previously (14,19; accession nos X87006X87089 and Z80363Z80770) was used as the control. Since no significant differences were found concerning the distribution of VH, D or JH gene segments or families in the different donors; the data were combined.
Estimation of the Taq polymerase error
To delineate whether the error rate found in the VH sequences of the X-HIgM patient were indeed introduced as a result of the method or might result from a CD154-independent pathway of somatic mutation as recently suggested (20), a known Ig sequence of 238 bp was repetitively subjected to the preamplification step and subsequent nested amplifications. Two sequence errors were detected in 81 resulting copies (19,261 nucleotides) and, thus, few if any of the nucleotide changes encountered in this analysis can be ascribed to amplification errors.
Statistical methods
To determine significant differences in distributions in productive or non-productive rearrangements, the 2 test was used. P
0.05 was assumed to be significant. VH pseudogenes were omitted in the comparison of the productive and the non-productive repertoires. Overall statistical significance between observed and expected frequencies was calculated employing the
2 goodness-of-fit statistic. If significant, each of the single degree of freedom
2 was examined for significant contribution to the total. The P value for the significance of these single degree of freedom
2 was than adjusted for the accumulation of errors related to multiple testing according to the Bonferroni method (21). In addition, the MannWhitney test was utilized to analyze CDR3 length, number of N nucleotides and JH exonuclease activity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
VH repertoire in an X-HIgM patient
The distribution of VH families in the productive as well as the non-productive repertoire of the X-HIgM patient was different from the distribution of genes in each family in the genome (Table 1). In the non-productive repertoire, the VH3 family was over-represented and the VH4 family was under-represented. In the productive repertoire, the VH3 family was also found most often, followed by the VH4 and VH1 families. However, the VH3 family was not over-represented in the productive repertoire and the VH4 family was found at almost the same frequency as the VH3 family, even though it has only half the number of family members. Furthermore, when the non-productive and the productive repertoires were compared, there was a significant increase in the VH4 family, and a significant decrease in the VH3 and the VH6 family in the productive repertoire. This was in contrast to normal adults, in which the VH3 family was found significantly more frequently than the other families and significantly more often in the productive than the non-productive repertoire. In the normal productive repertoire, as opposed to that found in the X-HIgM patient, the VH4 family was less than half as frequent as the VH3 family and was significantly diminished in the productive compared to the non-productive repertoires.
|
|
|
|
Composition of the CDR3 in an X-HIgM patient
The distribution of the CDR3 length in the X-HIgM patient was not statistically different than that found in normal individuals (Fig. 2). Of note, there was a significant decrease in the number of sequences with a CDR3 >72 nucleotides in the productive rearrangements of both normals and the X-HIgM patient (0/90 and 3/421) compared to the respective non-productive repertoires (4/24 and 8/70, P
0.0001). In contrast to the X-HIgM patient, in normal donors there was an additional shift in the productive rearrangements towards shorter CDR3s. Thus, in the normal productive rearrangements, significantly more CDR3s of 2536 nucleotides were observed than were found in non-productive rearrangements (144/421, 34.2% versus 10/70, 14.3%; P
0.0009) and significantly less were found with a CDR3 length of >61 nucleotides (Fig. 2
). This tendency toward shorter CDR3 lengths in the productive rearrangements of normals was not seen in the productive X-HIgM repertoire.
|
Somatic hypermutation in an X-HIgM patient
Of the 90 productive and 24 non-productive rearrangements analyzed, 17 and seven sequences respectively contained one to three mutations. The total number of mutations in the productive rearrangements was 25 in a total of 23,725 bp. In the non-productive repertoire there were 14 nucleotide substitutions in 6206 bp. The overall mutational frequency of productive VH genes was half of that for non-productive rearrangements (1.1x103 and 2.3x103/bp, P 0.02). When the sequences that contained mutations were analyzed separately, the mutational frequencies were comparable between productive and non-productive rearrangements with a frequency of 5.7x103 mutations/bp for productive and a frequency of 7.8x103 mutations/bp for non-productive rearrangements (25/4385 and 14/1802 bp, respectively). In normal adults (22,23) the frequency of mutations in the productive rearrangements was 21.2x103 (2303 mutations/108,559 bp) and in the non-productive 19.9x103 (357 mutations /17,909 bp), representing a 10- to 20-fold decrease in the overall mutational frequency in the X-HIgM patient. Comparing the frequency of mutations within mutated sequences between the X-HIgM patient and normal adults, the difference was ~5-fold. Thus, in normal adults the number of mutations per mutated sequence was 2303 mutations/72,969 bp (31.6x103) in the productive repertoire and 357 mutations/9498 bp (37.6x103) in the non-productive repertoire.
All mutations in the CDR1 or 2 resulted in replacement mutations (five in the productive and three in the non-productive rearrangements), whereas mutations in framework regions led either to replacement or silent mutations (13 replacement and six silent mutations in the productive and six replacement and five silent mutations in the non-productive repertoire).
Of note, mutations of individual nucleotides occurred more equally in the non-productive repertoire, whereas in the productive repertoire mutations of purines dominated (A 36.0%, G 32.0%) and T mutations occurred infrequently (Table 4). Moreover, despite the low number of mutated nucleotides, there was a slight increase in the frequency of transitions in the productive (52.0%) compared to the non-productive repertoires (42.8%), similar to that previously noted in the normal VH repertoire (53.3 and 46.4% respectively).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, the distribution of VH families in the non-productive repertoire of the X-HIgM patient and normal donors was not significantly different. This result implies that the VHDJH rearrangement process is intact in this X-HIgM patient and, moreover, suggests that VHDJH rearrangements are not affected by age, as the X-HIgM patient was 4 years old, whereas the normal donors were 26, 32 and 45 years old.
In contrast to the non-productive repertoire, when the productive repertoires were analyzed, a significant increase in the frequency of the VH4 family and a significant decrease in the VH3 family was noted in the X-HIgM patient. From our previous analysis of normals (19), >40% of IgM+ B cells contained mutated genes and, therefore, were memory B cells (25). When unmutated and mutated VH rearrangements were compared in normal donors, the over-representation of the VH3 family appeared to be related to preferential expansion of VH3-expressing B cells in the memory population (19). Thus, some of the differences found in the X-HIgM and normal donors were likely to have resulted from the absence of memory B cells in the X-HIgM patient (3). This conclusion is consistent with recent data showing that the IgD CD27+ memory B cell population is absent from X-HIgM patients (26). In this regard, the frequency of the VH3 family in the X-HIgM patient was not significantly different than that found in the unmutated VHDJH rearrangements from normal donors (38/90 versus 120/251, P>0.36), suggesting that CD154-dependent antigen-stimulated development of memory B cells is likely to play a role in the differences found in the frequency of the VH3 family in normals and the X-HIgM patient.
The conclusion that the bias in the VH3 family members relates to the development of memory B cells, however, does not appear to apply to VH3-23/DP-47, since it was found at the same frequency in the productive repertoire of the X-HIgM patient as in that of normals. In both the normals and the X-HIgM patient, VH3-23/DP-47 was over-represented in the productive compared to the non-productive repertoire. It has been shown that VH3-23/DP-47 is overexpressed very early in B cell ontogeny (27,28), and its over-representation was noted in both unmutated and mutated rearrangements of IgM+ B cells (19). Moreover, in normal donors no preferential D or JH segment was associated with the over-represented VH3-23/DP-47 genes, and, therefore, it was assumed that overexpression resulted from positive selection based on the VH gene segment per se and not the antigen-binding CDR3 (19). The data from the X-HIgM patient further support this conclusion, since no similarities in the CDR3 composition of the over-represented VH3-23/DP-47-encoded Ig genes were found. Together, these results suggest that over-representation of VH3-23/DP-47 during early B cell development reflects positive selection into the preimmune repertoire based on the specific VH gene sequence and is independent of CD40CD154 interactions. Since selection is independent of the CDR3, it may reflect the activity of an endogenous material with B cell superantigen-like activity, as recently proposed (29,30).
Over-representation of the VH4 family in the productive repertoire of the X-HIgM patient was also noted, even when compared to the unmutated B cell repertoire of normal donors (36/90, 40.0% versus 60/251, 23.9%; P 0.004). The VH4 family is subjected to negative selection in normal donors, especially in IgM+/CD5 B cells (19), presumably to avoid autoimmunity, since members of this family (31,32) encode many autoantibodies. The increased frequency of VH4-expressing B cells and the absence of negative selection in the X-HIgM B cells suggests that CD154 might be involved in this process. It should be noted that the expansion of VH4-expressing B cells occurs at the expense of VH3- and VH6-expressing cells, each of which is present less frequently than was noted in the non-productive repertoire. Of note, the lack of expansion of the VH3-expressing cells in the productive repertoire of X-HIgM appears to reflect the absence of memory B cells. Similarly, in the normal productive repertoire, the VH6 family appears to be highly mutated (95.6 ± 3.4%, mean ± SD homology to the germline sequence). Therefore, the apparent diminution in both the VH3 and the VH6 families from the productive repertoire of the X-HIgM patient may reflect a compensatory decrease secondary to the increase in the VH4 family in the absence of the generation of memory B cells.
As already demonstrated for the normal B cell repertoire, the usage of VH genes is quite restricted (19,28). This was also the case for the repertoire of the X-HIgM patient. Thus, 60% of the productive rearrangements utilized one of seven VH gene segments and all seven were used more frequently than expected by random chance. Recombinational bias appears to be responsible only for the over-representation of one gene segment, VH3-11/DP-35, as it was found more frequently than expected in the non-productive repertoire. Although it has been suggested that VH3-11/DP-35 is expressed at a high frequency in pre-B and immature B cells but rarely in the mature B cell population (28), we have found that it appeared at the expected frequency in both the non-productive and productive repertoires in normal donors, and was subjected to neither positive nor negative selection (Table 2). Whether this difference is related to the techniques used for analysis, single-cell PCR versus in situ hybridization, is unknown. The pattern of expression of VH3-11/DP-35 in the X-HIgM was similar to that of normals, but its amplitude of expression was greater.
An additional VH gene segment, VH4-31/DP-65, that was negatively selected in normal donors, was found to be over-represented in the productive repertoire of the X-HIgM patient, suggesting that this VH segment may be positively selected in the X-HIgM but not in normals. It is possible that this reflects expansion secondary to T-independent mechanisms perhaps in response to microbial pathogens, as the X-HIgM child was subjected to multiple infections. The single most common VH gene segment in the X-HIgM patient was VH4-59/DP-71. Whereas its over-representation in normals seems to be related to preferential recombination, as evidenced by its increased frequency in the non-productive repertoire, this was not the case in the X-HIgM patient. Rather the distribution of this particular VH4 gene in the non-productive rearrangements of the X-HIgM patient was within the expected range of random usage but, similar to the VH3-23/DP-47 gene, VH4-59/DP-71 was expanded into the productive repertoire by a mechanism that must not involve CD40 stimulation. No indication for clonal expansion was found for B cells utilizing this VH4 family member, since the CDR3s of the over-represented VH4-59/DP-71-containing VH rearrangements were different (data not shown). The step of B cell differentiation at which this VH gene segment is positively selected is unclear, but expansion of this VH4 family member could result from T-independent antigenic stimulation perhaps in response to microbial infection, as mentioned for VH4-31/DP-65. Although the effect of T-independent stimulation might also occur in normal donors, it could be of a smaller magnitude and, therefore, not apparent in the presence of T cell-dependent B cell stimulation.
From this analysis is appears that CD40CD154 interaction affects the expressed peripheral VH repertoire in several ways. Certain aspects of negative selection appear to require CD154, as indicated by the loss of the normal negative selection of the VH4 family. In addition, positive selection especially into the memory (mutated) population requires CD40 signaling and, thus, the VH3 family is not expanded in the X-HIgM patient. Finally, in the absence of T-dependent positive selection of memory B cells, CD154-independent positive selection of specific genes, such as VH4-59/DP-71 and VH4-31/DP-65, can be seen that might be related to T-independent antigen stimulation.
The composition of the CDR3 appeared to be comparable to that previously reported for normal donors. We used the same criteria as previously employed for peripheral blood CD19+ (14) or IgM+ CD5+ and CD5 B cells (19) to allow comparison even though a more complete mapping of the D segments has recently been reported (33). Even with the more recent criteria, D segment utilization in the X-HIgM patient is similar to normal donors (data not shown). Furthermore, selection did not appear to effect the distribution of the D or the JH gene segments when the productive and the non-productive repertoires of the X-HIgM patient were compared. These data imply that there is no major impact of CD40CD154 interactions in biasing the CDR3 composition of the expressed repertoire. As previously described for normal donors (20), the productive repertoire in the X-HIgM patient is biased towards shorter CDR3 (Fig. 2), indicating that this bias is not dependent on CD40CD154 interaction. However, the increase in sequences with a CDR3 length of 812 amino acids in the productive repertoire of normals is not observed in the X-HIgM repertoire. This finding is consistent with the observation that shorter CDR3s are characteristic of memory B cells with mutated Ig genes (34) and, therefore, reflects CD40CD154-mediated positive selection of memory B cells.
Whether X-HIgM patients are capable of somatic hypermutation is still controversial. Two recent studies that addressed this question came to different conclusions. In one report, Chu et al. (16) analyzed cDNA libraries obtained from peripheral blood of three X-HIgM patients and noted that 7/102 VH6 IgM and 1/6 VH6 IgG rearrangements contained two to nine mutations per sequence for a mutational frequency of 1.4x102. In a second report, Razanajaona et al. (35) noted that the frequency of mutations obtained from cDNA libraries of two X-HIgM patients was much lower (1x103/nucleotide) and was most likely the result of Taq error, whereas a third patient with a partially functional CD154 exhibited a mutational frequency of 1.6x102. This suggested that some functional activity of CD154 was necessary to induce somatic hypermutations.
In the patient studied here, CD154 contained two mutations that completely prevented its expression (15). Despite this, a low level of mutations was noted (1.1x103/nucleotide in productive and 2.3x103/nucleotide in non-productive rearrangements). It is possible that this level of mutations might relate to Taq polymerase error, although several observations made this unlikely. First, if the mutations were the result of errors introduced in the amplification steps, they should be similar in productive and non-productive rearrangements. Since clear differences in the mutational patterns in non-productive and productive rearrangements were noted, it is more likely that these base changes had been introduced in vivo by a CD154-independent process and, thereafter, had been subjected to selection. Moreover, A to G and T to C substitution have been reported to be the most frequent Taq polymerase misincorporation events (36), whereas each nucleotide was found to be mutated with a comparable frequency in non-productive rearrangements, as has been noted previously in normal donors (23). Finally, direct measurement of the error rate of the amplification procedures used in this technique indicated a greater fidelity than anticipated, presumably because the PCR products are directly sequenced without cloning. In this regard, when a known PCR product was repetitively subjected to primer extension preamplification and nested amplification two errors were found in 81 resultant copies (19,261 nucleotides). Therefore, the errors introduced by the technique appear to be negligible. This makes it much more likely that the low level of mutation noted in the X-HIgM patient reflects the activity of a mutational mechanism that is independent of CD154. In this regard, analysis of the induction of Ig mutations in cell lines in vitro has shown that CD40 ligation is neither sufficient nor necessary to activate the mutational machinery (37,38).
The 10-fold higher mutational frequency in the productive repertoire of X-HIgM (1.4x102 versus 1.1x103) reported by Chu et al. (20) could relate to the limitation of the analysis to the single member VH6 family. Although this approach has the advantage of excluding the possibility that mutations found in the VH genes are actually polymorphisms since this gene had been demonstrated to be non-polymorphic in humans (39,40), it may bias the analysis by focusing on a frequently mutated VH gene (41). Thus, in several studies it has been shown that in the peripheral VH repertoire of children or adults, VH6 is frequently hypermutated (14,19,24,4144). Moreover, VH6 may be more mutated in the productive repertoire than other VH gene families. Thus, the productive VH6 genes in the peripheral repertoire of human B cells (14,19) were found to be less homologous to their germline counterparts (95.6 ± 3.4%, mean ± SD homology; n = 8) than all other VH genes in the same B cell population (97.9 ± 2.9%, mean ± SD homology; n = 413; P 0.03). This suggests that VH6 is uniquely targeted by the mutational machinery, that unmutated VH6 genes are eliminated from the productive repertoire or that mutated VH6 are positively selected, resulting in a population of expressed VH6 rearrangements that is enriched in mutations. Therefore, analysis of VH6-encoded Ig could yield a higher mutational frequency compared to that found for all other VH genes. Independent of this, the data reported here indicate that a low level of mutation can be induced in the absence of a functional CD154. Whether these mutations are generated by the same mechanisms as in normal donors cannot be answered, but recent reports have indicated that CD40 signals are not necessary for the initiation of proliferation of B cells (45) or Ig production (4,46) and that somatic hypermutation and selection can be found even in the absence of germinal centers (47).
![]() |
Acknowledgments |
---|
![]() |
Abbreviations |
---|
CDR complementarity-determining region |
X-HIgM X-linked hyper-IgM syndrome |
![]() |
Notes |
---|
Transmitting editor: A. Fischer
Received 12 August 1999, accepted 8 February 2000.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|