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Signals That Initiate Somatic Hypermutation of B Cells In Vitro¹

Sigridur Bergthorsdottir^*, Aoife Gallagher^2,*, Sandra Jainandunsing^*, Debra Cockayne, James Sutton^*, Tomas Leanderson and David Gray^2,3,*

^* Department of Immunology, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom; Neurobiology Unit, Roche Bioscience, Palo Alto, CA 94304; and Immunology Unit, University of Lund, Lund, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Somatic hypermutation is initiated as B lymphocytes proliferate in germinal centers. The signals that switch on the mutation process are unknown. We have derived an in vitro system to define signals that will initiate mutation in normal, naive splenic B cells. We find that three signals are required to allow detection of somatic mutation in vitro; these are anti-Ig, anti-CD40, and anti-CD38. If any one of these is omitted, mutation remains off. We show that CD40 is obligatory in vivo, as CD40 knockout mice exhibit no Ag-driven mutation. In contrast, CD38 is not, as CD38 knockout mice mutate normally. We believe that, in vitro, CD38, in combination with other stimuli, drives extensive cell division, allowing the detection of mutated sequences. However, in germinal centers in vivo, proliferative activity is instigated by a different molecule. This is the first demonstration of the initiation of hypermutation in vitro with normal splenic B cells using defined stimuli.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Affinity maturation of the Ab response occurs largely as a result of selection of the Ig V gene products that have undergone a process of somatic hypermutation. The vast majority of late primary and secondary response Abs exhibit changes from the germline V sequence (1, 2), and most memory B cells carry somatically mutated B cell receptors (BCR)⁴ (3). In generating the repertoire for affinity selection to act upon, somatic hypermutation forms an integral part of the process of memory B cell development, and the two processes seem to be intimately linked, both occurring in the same anatomical site. Germinal centers (GC) are necessary for the generation of memory B cells (4) and are sites in which B cell hypermutation is switched on (5). Under normal, physiological circumstances GC appear to be the only sites of mutation (6), although, if pushed to the extreme by repeated immunization with enormous amounts of Ag, mutations can be detected in lymphotoxin-^-/- mice that lack GC (7).

B cell memory development, GC reaction, and somatic hypermutation occur only during responses to T-dependent Ags (generally proteins), suggesting that signals from T cells are necessary at some point in all of these processes. The exact molecular identity of the signals driving these processes is still unknown. We do not know what initiates the GC reaction, what drives GC B cell proliferation, or what causes differentiation within the GC (e.g., the transition of centroblast to centrocyte). CD40 is implicated in the first two functions as CD40 knockout mice develop no GC (8, 9); however, its role in GC induction may be an indirect one as rudimentary GC can be regenerated in these mice simply by injection of CD40-Ig (10). However, CD40 signals do seem to be crucial during the final stage of memory differentiation in GC; the final rescue of mutated GC B cells from apoptosis (11) and entry into the memory pool (12). Signals via the BCR are clearly also crucial, as without recognition and uptake of Ag the B cell would be unable to elicit T cell help. Whether the BCR signals generated by small protein Ags have any role to play in driving the cell through cell cycle is not known. Ag uptake and subsequent processing for presentation is important at two stages of the B cell response: the initiation and for the selection of mutants in the GC for survival in the memory pool (13). So we know that the processes involved in memory generation require signals via surface immunoglobulin and signals derived from T cells; indeed, we have shown that somatic mutation could be maintained (although not initiated) by culturing B cells with anti-Ig and helper T cells (14). This has since been supported by similar findings using human B cell lines (15, 16) and tonsillar B cells (17). The exact identity of these T cell-derived signals is still mysterious.

In this study we have set out to find the signals that initiate somatic mutation of B cells in vitro. To do this we have stimulated B cells from a transgenic mouse carrying a VOx1 gene with all of the upstream elements required to target somatic mutation to the V gene (18). As this V is rearranged to a rat C the transgene is readily identified, avoiding the problem of unequivocal identification of the germline equivalent of a mutated V that is often difficult in normal mice due to close similarities within V gene families. B cells from these mice were stimulated and maintained in culture under a number of conditions. These included BCR cross-linking in combination with the various effector molecules of T cell help (i.e., CD40 ligand and cytokines). B cells stimulated using anti-Ig together with anti-CD40, and supernatants from Th2 clones did not accumulate somatic mutations in the VOx1 transgene. However, the replacement of cytokines with an Ab to CD38 led to the detection of somatic mutation in vitro.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice and immunizations

ELK mice, carrying a transgene incorporating VOx1 and upstream regulatory elements (18), were provided by Dr. Michael Neuberger (LMB, Cambridge, U.K.). CD38 knockout mice (B6 x 129 F₂) (19) were provided by Dr. Maureen Howard (Anergen, Redwood City, CA) and CD40 knockout mice (8), originally made by Dr. Hitoshi Kikutani (Osaka, Japan) have been maintained in our laboratory for some time and backcrossed to C57BL/6 for seven generations. Wild-type mice were C57BL/6 strain. All mice were bred and maintained under standard laboratory conditions in the animal facilities of Imperial College School of Medicine, Hammersmith Hospital and later at the Ashworth Laboratories, University of Edinburgh. Mice were used at 8–12 wk of age. Mice were immunized via the i.p. route with 100 µg of alum-precipitated phenyloxazolone-chicken serum albumin together with 10⁹ killed Bordetella pertussis and then boosted 3 wk later with 100 µg of soluble phenyloxazolone-chicken serum albumin.

B cell stimulations

In early experiments B cells were prepared from spleens by the depletion of T cells using IgM Abs against Thy1, CD8, and CD4 and lysis with mouse-absorbed young rabbit complement (C-Six Diagnostics, Mequon, WI). Residual T cells were depleted using Dynabeads (Dynal, Oslo, Norway) coated with IgG Abs to the same surface markers (Abs and procedure are described in Ref. 20). In more recent experiments B cells have been prepared by negative selection using anti-CD43 MACS microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according the manufacturer’s instructions and described in Skok et al. (21). Using both methods, B cell preps were routinely >95% pure with <1% contamination with CD3⁺ T cells. One million B cells/well were plated out in 24-well tissue culture plates (Costar, Corning, NY) in IMDM supplemented with 5% FCS, 2 mM L-glutamine (Life Technologies, Paisley, Scotland), 50 mM 2-ME, and penicillin-streptomycin (50 mg/ml). The cultures were maintained at 37°C in a humidified 5% CO₂ atmosphere. The following stimuli were added singly or in combination: LPS (Salmonella typhosa 0901) at 5 µg/ml (Difco, Detroit, MI), anti-CD38 supernatant at 1:200 (NIMR-5, Ref. 22 , provided by Dr. Michael Parkhouse; IAH, Pirbright, Surrey, U.K.), anti-CD40 (FGK-45, Ref. 23) at 10 µg/ml, and anti- light chain (187.1) at 10 µg/ml. Supernatants from alloreactive Th2 clone (20) produced as described previously were used at a 1:10 dilution. The cells were harvested after 7 days.

Analysis of cell division

The number of cell divisions undergone in culture was measured using FACS analysis (FACSCaliber running CellQuest; Becton Dickinson, Mountain View, CA) at appropriate times of cells loaded with the dye CFSE (Molecular Probes, Eugene, OR) at a concentration of 10 nM for 10 min at 37°C as previously described (24).

PCR amplifications and sequencing

Total RNA was prepared using RNAzol B (AMS Biotechnology, Oxon. U.K.), then cDNA was made using a Promega Reverse Transcription kit (Promega, Madison, WI) containing all the reagents for synthesis of single-stranded cDNA (both according to manufacturer’s instructions). The VOx1 transgene (600 bp) was amplified from the cDNA using the following primers: EK16, 5'-GCCGGAATTCCCAGAGGACAAATTGTTC-3' (VKOx1); and RtCk, 5'-GCCCGGATCCGACGGGTGAGGA-3' (ratC); and the following protocol: 95°C for 5 min and then 30 cycles of 95°C for 1 min, 53°C for 1 min, 72°C for 1 min, and then a 72°C extension for 10 min. The PCR product was purified using a QIAquick PCR purification kit (Qiagen, Chatsworth, CA) and ligated into P-Gem-T easy vector (Promega) before transforming competent JM109 bacteria. Colonies were picked and minipreps made (QIAprep spin plasmid miniprep kit; Qiagen). Colonies containing the correct insert were selected by digestion with ApaI and PstI. Sequencing was performed by MWG Biotech (Ebersberg, Germany).

For analysis of the J-C intron flanking the 3' border of V_HJ558 genes, the method used was essentially that described by Jolly et al. (25). Briefly, genomic DNA was isolated using a QIAprep tissue kit (Qiagen). The J_H4 intron was amplified using a primer specific for framework region 3 of J_H558 family of V_H genes (5'-GGAATTCGCCTGACATCTGAGGACTCTGC-3') and the 3' end of the intronic enhancer (5'-GACTAGTCCTCTCCAGTTTCGGCTGAA-3') using a two-stage protocol: 10 cycles of 94°C for 15 s, 64°C-55°C (reducing 1°C per cycle) for 15 s, 72°C for 4 min, and then 25 cycles of 94°C for 15 s, 63°C for 15 s, and 72°C for 4 min. The J_H4 rearrangement gives a product of 1200 bp; this was excised, extracted from the gel using QexII (Qiagen), cloned into pBluescript at the EcoRV site, and sequenced using a primer specific for J_H4 (5'-TATGCTATGGACTACTGG-3'). The polymerase error rate was calculated by sequencing 20 clones derived after RT-PCR of message from a hybridoma expressing germline VOx1. This error rate was 1:1200.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Experimental system

All in vitro experiments were conducted with B cells from a transgenic mouse (ELK) expressing VOx1 linked to a rat C. The transgenic construct contains both the 3' enhancer and the intron enhancer that together facilitate targeting of somatic mutation to the transgene (18). The gene product is expressed at the cell surface and is secreted into the serum; however, as far as this experiment is concerned, it is used as a passenger transgene in that we do not analyze Ag-driven responses. It has the advantage that we can readily identify the mutated transgene by amplifying with a PCR primer for the rat C together with a VOx1-specific primer. This is important as the unequivocal identification of mutated V genes in mice is complicated by the close homology within families and the possibility that not all the members of that family are yet cloned. Thus, splenic B cells were purified (>98%) and placed in culture under a variety of conditions for 7 days. The culture medium and stimuli were replenished after 4 days (see Materials and Methods). After harvesting cells, RNA was prepared and the VOx1 transgene was amplified by RT-PCR, cloned, and sequenced.

Initiation of somatic mutation in vitro

The stimuli used in the cultures were designed to mimic signals delivered to B cells during T-dependent responses, i.e., antigen (anti-Ig), CD40 ligand (anti-CD40), and cytokines (delivered as a mixture of Th2 cytokines contained in a supernatant from an activated Th2 clone). We also tested an Ab to CD38 that has strong mitogenic properties on mouse B cells (22). As previous studies had indicated that mutation did not occur during culture of B cells following LPS stimulation (26), we used LPS as a negative control. As demonstrated in Fig. 1A, the B cells cultured with LPS for 7 days exhibited no mutation of the VOx1 transgene above the Taq error rate (1:1200). Somatic hypermutation was found under only one culture condition, containing three stimuli together: anti-Ig, anti-CD40, and anti-CD38. Fig. 1B shows a significant number of point mutations scattered through the VOx1 transgene from B cells cultured in this way. Approximately half (9 of 20) of the sequences analyzed contained mutations (see Table I), and most had two or more mutations per sequence. Both Table I and Fig. 2 indicate that the difference in mutation frequency between LPS (1:1140) and the triple stimulus (1:290) is statistically significant. Table I and Fig. 2 also show that mutation can only be initiated if all three stimuli are present; omission of any one results in loss of significant mutation. A very striking result is the observation that the addition of Th2 cytokines to the anti-Ig, anti-CD40, anti-CD38 culture inhibits the accumulation of somatic mutations, we think because they drive differentiation of B cells to plasma cells. In Table II we show data from four independent experiments that confirm that the triple stimulus of anti-Ig, anti-CD40, anti-CD38 reproducibly induces mutation in vitro. This culture condition is the only one tested in which the difference in mutation frequency compared with the LPS control, which could not occur due to chance, according to Fisher’s exact test. The frequency of mutations observed with dual stimuli was greater than that in the LPS controls but did not reach statistical significance. For stimulation conditions involving anti-CD40 this increase was consistent in four independent experiments; for anti-Ig plus anti-CD38 the increase was seen in only two of the three experiments. This could indicate that CD40 signals are sufficient on their own to switch on mutation that is detectable only when other stimuli are present to enhance cellular proliferation.

View larger version (89K): [in this window] [in a new window]   FIGURE 1. Mutation initiated in vitro. Sequences of the VOx1 transgene from B cells cultured with LPS (A) and anti-, anti-CD40, and anti-CD38 (B). The top line shows the germline sequence; only changes from the germline are indicated. Note that similar results were obtained in three other experiments.

View this table: [in this window] [in a new window]   Table I. Frequency of somatic mutations in the VOx1transgenic in splenic B cells stimulated in vitro

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Mutation frequency in B cell cultures. The frequency of mutation obtained in the VOx1 transgene following 7-day culture of B cells with combinations of anti- (anti-Ig), anti-CD40, and anti-CD38. Only the combination of all three stimuli was statistically, significantly different from the LPS control (using Fisher’s exact test). This result is representative of three other experiments

We have analyzed the mutations incorporated into the VOx1 transgene in B cells stimulated with anti-Ig, anti-CD40, anti-CD38 for hallmarks of somatic mutation. The point mutations detected are scattered throughout the V region, with more than half in the framework regions; however, there does seem to be an accumulation in complementarity-determining region 2 (Fig. 1B). This pattern of mutation in sequences from triple stimulation cultures was similar in four independent experiments (80 sequences). It may be that the complementarity-determining region targeting of mutations observed previously is related to antigenic selection, of which there is none in this system. As expected, for this reason, the ratio of the number of replacement mutations compared with the number silent mutations is little different from the predicted value (data not shown). Table III shows that there is an over-representation of transitions compared with transversions in the set of mutations from the triple stimulation culture. A preference for transitions is an intrinsic feature of the hypermutation machinery. Transitions should make up only 33% of the mutations; however, in this data set 56% of the mutations are transitions. We have to keep in mind that up to 25% of the mutations we see could be taq-induced (our taq error rate = 1:1200) and these would also be biased toward transitions. If we take away 25% of the observed transitions, the proportion of such mutations is, at 43%, still some 10% greater than the expected frequency. Some changes are particularly prevalent, such as CT, AG, and GT; these have been noted previously in a study of mutation in a cell line (16). Mutation in the intrinsic mutational hotspots is not observed in the sequences illustrated here (Fig. 1); thus we see no mutation of the serine 26, 31, or 77 codons (28, 29). Moreover, these mutations were not observed in the larger data set (Table II) above the frequency expected by chance.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Proliferation of B cells in culture. Flow cytometric analysis of CFSE labeling of B cells cultured for 7 days with various stimuli (A–E, as indicated). These data are representative of four such experiments.

CD38 might play one of two roles in the initiation of somatic mutation in vitro. First, it could provide a specific signal to start mutation or second, it could drive proliferative expansion of mutated cells to a level within the cultures at which they are readily detectable. We analyzed the proliferative activity of B cells stimulated in various ways by labeling the starting population with CFSE and counting the number of cell divisions (by halving of CFSE fluorescence) at the end of 7 days. Fig. 3C shows that anti-CD38 and anti-Ig cause the majority of cells to enter cell division and proceed to the maximum number of six divisions. This confirms what we already know (Table I and Fig. 2), that CD38 is not an "on switch" for somatic mutation, at least, not in the absence of CD40 signals, as even with maximal cell division no mutation is seen. Anti-CD38, in combination with anti-CD40 and anti-Ig, increases the number of cells proceeding through the maximum six divisions (compare Fig. 3, D and E) and as such may well be working to drive expansion of mutants to a detectable level. It should also be noted that, in the anti-CD38 stimulations, the majority of cells enter the cell cycle (98.7 vs 93.9%; Fig. 3, D and E), and so no subpopulation of B cells is left at the proliferative starting gate as happens with LPS or anti-CD40 (see Fig. 3, B and D). We have found no other stimulation conditions that have such a broad and powerful mitogenic/proliferative effect. Although we cannot rule out absolutely the possibility that some of the mutations we see in our cultures derive from the expansion of pre-existing memory cells, the proliferation data suggest otherwise. Anti-CD38 plus anti-Ig seem to push most cells into cycle (Fig. 3C), but mutation is not significantly enhanced. We also have in excess of 40 ELK transgene sequences from splenic B cells, which exhibit no evidence of mutation despite the presence of ongoing ‘irrelevant’ immune responses (e.g., splenic GCs) in the mice.

In relation to the cell proliferation it is clear that none of the sequences obtained from these cultures are clonally related, i.e., no sequential accumulation of mutation during culture could be detected. This is not unexpected given the cell input number and the relatively small sample size. We are currently trying to extend the useful life of the cultures in the hope of achieving a larger clone size and observing such clonal development. It might then also be possible to introduce selection into the system.

Somatic mutation in CD40 and CD38 knockout mice

To see whether CD40 and CD38 play a role in hypermutation in vivo we immunized CD40- and CD38-deficient mice (19) and analyzed the accumulation of mutations in their V regions. To do this we sequenced the J-C intron flanking the 3' border of V_HJ558 genes in B cells from immunized mice, as described by Jolly et al. (25). The data are summarized in Table IV. As expected, in the light of the absence of GC in CD40^-/- mice (8, 9), no somatic mutation was found. This may not be surprising, however, it is the first formal demonstration of impaired somatic mutation in these mice. The role of CD40 in the mutation process is obligatory in vitro and in vivo. The obvious interpretation is that CD40 directly initiates the mutation process by inducing expression of one of the components of the "mutator complex"; however, it might act indirectly following induction of other costimulatory receptors on the B cell surface. The latter possibility is indicated by the report that an EBV-negative Burkitt’s lymphoma (BL2) mutates following anti-Ig stimulation and interaction with T cells, independently of CD40 (34).

Concluding remarks

Our data suggest that resting B cells require nothing other than Ag and CD40 ligand as a donor of T cell help to initiate the hypermutation process. We found that CD38 signaling was required in our in vitro model before mutations were evident, but its role was to enhance proliferation rather than to act as a specific on-switch: This pro-proliferative role for CD38 was dispensable in vivo. We propose that, in addition to CD40, another, still unidentified, molecular signal drives proliferation of centroblasts in GC.

	Acknowledgments

 
We thank Dr. Dominic van Essen for help with the statistical analysis and Drs. John Pound and Andy Knight for critical comments.

	Footnotes

 
¹ This work was supported by grants from the Wellcome Trust and the Medical Research Council (U.K.). T.L. was supported by grants from the Swedish Medical Research Council and the Swedish Cancer Society.

² Current address: Institute of Cell, Animal and Population Biology, University of Edinburgh, Ashworth Laboratories, King’s Buildings, West Mains Road, Edinburgh EH9 3JT, U.K.

³ Address correspondence and reprint requests to Dr. David Gray, Institute of Cell, Animal and Population Biology, University of Edinburgh, Ashworth Laboratories, King’s Buildings, West Mains Road, Edinburgh EH9 3JT, U.K.

⁴ Abbreviations used in this paper: BCR, B cell receptor(s); GC, germinal center(s).

Received for publication June 23, 2000. Accepted for publication November 20, 2000.

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