Myeloma

Myeloma isotype-switch variants in the murine 5T myeloma model: evidence that myeloma IgM and IgA expressing subclones can originate from the IgG expressing tumour

Abstract

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cμ region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

Introduction

Multiple myeloma (MM) is a B cell malignancy characterized by a monoclonal plasma cell population in the bone marrow, secreting high levels of immunoglobulins, mainly of the IgG or IgA isotype. It is generally accepted that the malignant cells originate from a mature, antigen selected B cell that has passed through the germinal centre because of the presence of somatic mutations. The hypermutation process has changed the MM-Ig genes in a way that favours antigen selection in most cases.1,2,3 The variable part of the MM-Ig gene is a good tumour-specific marker due to the presence of unique mutations in the CDR regions as well as the hypervariable CDR3 region. PCR assays that are based on amplifying these variable Ig sequences can therefore be used to trace cells that belong to the myeloma clone. Besides plasma cells also the presence of mature B cells, belonging to the MM clone, has been demonstrated in the peripheral blood.4,5,6,7 Intriguingly, cells that expressed the same variable region as the myeloma cells but linked to a different isotype were also found in the peripheral blood and bone marrow.8,9,10 Such cells are called isotype-switch variants of the malignant clone. Especially the presence of IgM isotype-switch variants tempted us to speculate about the existence of a possible pre-switch MM precursor cell.8,11 However, numerous efforts to isolate and expand these pre-switch precursor cells to analyse their clonogenic behaviour and karyotype have been unsuccessful.12 In order to gather more information on the biological significance of these isotype-switch variants we chose to use a mouse model for multiple myeloma. The 5T series of MM, which developed spontaneously from ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble those of the corresponding human disease and can be used as in vivo experimental models.13,14,15 The 5T series of MM have since been propagated by intravenous transfer of bone marrow myeloma cells in young syngeneic animals and have a nearly exclusive homing to the bone marrow. Two in vivo 5T lines (the 5T2MMvivo and the 5T33MMvivo) have further been characterized in previous studies.16,17 An in vitro bone marrow stroma-independent variant of the 5T33MM cells, the 5T33MMvitro cell line, has also been obtained. This offers us an experimental system allowing studies quite impossible to pursue in humans. We first demonstrated that isotype-switch variants are present in these two 5TMMvivo lines but not in the 5T33MMvitro line. Furthermore, we studied the appearance and disappearance of isotype-switch variants respectively in vivo and in vitro. Therefore, the 5T33MMvitro cell line was injected into young recipients, tumour cells isolated from these mice were cultured in vitro during several months. The data generated from this mouse model indicate that the MM-related IgM and IgA isotype-switch variants originate from the IgG myeloma clone which argues against the existence of a pre-switched pre-malignant myeloma precursor cell from which the cells, expressing the dominant isotype, develop.

Materials and methods

Animals

C57BL/KalwRij mice were obtained from Harlan CPB (Horst, The Netherlands). Male mice were 6–10 weeks old when used. They were housed under conventional conditions and had free access to water and food. They were sacrificed by CO2 asphyxiation. The care and use of mice were in accordance with the guidelines of the Vrije Universiteit Brussel.

Cell lines

The 5T2MM and 5T33MMvivo cell lines originated from aged C57BL/KalwRij mice which spontaneously developed MM.13,14 Both the 5T2MM and the 5T33MM express an IgG-κ isotype. The cells have since been expanded into young syngeneic animals by intravenous transfer of the diseased bone marrow. Progression of MM in diseased animals was followed up by electrophoretic quantification of serum paraprotein.16 Animals were killed when a paraprotein concentration of 10 mg/ml was reached. MM cells were purified from the bone marrow and the percentage of tumour cells was determined by FACS analysis as described elsewhere.17,18

The 5T33MMvitro cell line has been obtained by Radl's group13,14 who developed the 5TMM experimental mouse model. This cell line resulted from cultured 5T33MMvivo bone marrow cells and grows indefinitely without the need of bone marrow stroma. Cells were cultured and maintained in DMEM (Life Technologies, Merelbeke, Belgium) supplement with 10% bovine serum (Fetal Clone I; Hyclone, Logan, UT, USA), penicillin–streptomycin, glutamine and MEM (Life Technologies). They were harvested by simple resuspension. Subclones of this 5T33MMvitro cell line were obtained by seeding 0.3 cells per well in a 96-well plate.

Detection of Ig isotype-switch variants

Total RNA was extracted from tumour cells (either from bone marrow cell suspensions, perivertebral solid tumours or cultures) by a guanidine isothiocyanate/acid-phenol modified method with TRIzol reagent (Life Technologies) and reversed transcribed using an oligo d(T) primer and the SuperScript Preamplification System (Life Technologies). The Ig heavy chain sequence expressed by the 5T2MM and 5T33MM tumour cells has already been published.19 Tumour-specific primers and probes were designed in the CDR1 and the CDR3 region: 5T2MM-CDR1 sense primer: 5′ CAC-AAC-TGC-TTG-GAA-TGC-AGT-G 3′; 5T2MM-CDR3 probe: 5′ CCG-CCT-GGT-TTG-CTT-ACT-GG 3′; 5T33MM-CDR1 sense primer: 5′ CAC-TAA-TTA-CTT-GAT-AGA-GTG-G 3′ and 5T33MM-CDR3 probe: 5′ GCA-GTT-ACC-ATA-AGC-CTC-TC 3′. 5T2MM and 5T33MM myeloma-related sequences were amplified using the specific CDR1 primers together with different Ig constant region-specific primers: Cμ: 5′ GCT-CTC-GCA-GGA-GAC-GAG-GGG-GA 3′; Cγ: 5′ GCG-AAT-TCC-CTT-GAC-CAG-GCA-TCC 3′ and Cα: 5′ CTC-GGA-TCC-TCA-CAT-TCA-TCG-TGC-C 3′. The amplification reaction was performed on 1 μl of first strand cDNA, corresponding to roughly 100000 cells, in a 50 μl reaction volume containing 1 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTP, 10 pmol of each primer and 0.625 U of Taq polymerase (Life Technologies). Normal C57Bl/KalwRij bone marrow cDNA was used as negative control and water was used as control for contamination.

Each PCR cycle consisted of 94°C heat denaturation for 30 s and primer annealing at 55°C for 30 s, followed by primer extension at 72°C for 1 min. The first cycle was preceded by a 2-min denaturation step at 94°C and the last elongation step was prolonged to 10 min to ensure full-length products. Forty cycles were performed in a Perkin Elmer GeneAmp PCR system (Perkin Elmer, Zaventem, Belgium). The PCR amplified DNA fragments were analysed by standard agarose gel electrophoresis, blotted on to Hybond N-plus membranes (Blotting-Nylon 66 Membrane type B, positive, pore size 0.45 μm; Fluka, Switzerland) and hybridised to 5T2MM and 5T33MM-specific CDR3 oligomers that were end-labelled with 32P-γATP. Hybridisation was performed overnight in 3 × SSC at 42°C. Membranes were washed at 50°C in 2 × SSC, 0.1% sodium dodecyl sulphate (SDS). With this myeloma-specific PCR assay it is possible to detect at least one tumour cell in 100000 normal cells.20 Positive PCR samples were cloned into the pCRII vector (Invitrogen, Groningen, The Netherlands), bacterial colonies were screened by hybridisation with the CDR3 probe. Plasmids from positive colonies were sequenced using the autoload solid phase sequencing kit and the ALFexpress (Pharmacia, Roosendaal, The Netherlands).

FISH analysis

Painting probes for chromosomes 12 and 15 (CAMBIO, Villers-Poterie, Belgium) were used in FISH analysis to determine the number of these chromosomes present in the 5T33MMvitro cell line. Slides were treated using standard techniques also used for human cells.21 In short, the slides were treated with Rnase, followed by Pepsine (10%), post-fixated in formaldehyde 37%, denatured for 3 min at 73–74°C in 70% formamide/2 × SSC, followed by dehydration. The directly labelled probes were denatured at 75°C for 5 min in 70% formamide/2 × SSC, preannealed (30 min) and hybridised overnight at 37°C in a moist chamber. Washing and detection were done according to the manufacturer's recommendations. The FISH data were analysed and collected on a Leitz DMRB fluorescence microscope equipped with a triple band pass filter and a cooled black and white CCD camera run by Smart capture software (Vysis, Stuttgart, Germany).

Results

Detection of isotype-switch variants in 5T2MM and 5T33MM bone marrow samples

Bone marrow samples from tumour bearing mice were tested for the presence of isotype-switch variants by a 5T2MM or 5T33MM specific PCR assay (Table 1). The MM-IgG sequences were detected as a strong CDR3 hybridisation signal on the CDR1/Cγ PCR products already after 2 h of exposure in both the bone marrow cell suspensions from 5T2MM as well as 5T33MM bearing mice. Only weak to moderate signals were observed after 1–3 days of exposure when CDR1/Cμ and CDR1/Cα PCR products were hybridised with the CDR3 probes. The specificity of the signals was confirmed by cloning the PCR products and sequencing the bacterial colonies that hybridised to the CDR3 probe. In all cases the sequences were of tumour origin and of the isotype determined by the isotype-specific antisense PCR primer (Figure 1). The 5T33MM-VDJ sequence differed slightly (only three nucleotides) from the published sequence19 (Figure 1).

Table 1 Isotype-switch variants in 5T2MMvivo and 5T33MMvivo bone marrow
Figure 1
figure1

Comparison between the nucleotide sequence of 5T33MM VH gene published by Zhu et al19 and the amplified VH gene from 5T2MM (a) and 5T33MM (b) bone marrow cells and/or 5T33MM in vitro culture cells. Amplification was performed with a 5T2MM or 5T33MM-CDR1-specific primer and the different constant region primers. Primers are depicted in italics. Dashes (-) represent identical nucleotides. Deviating nucleotides which result in amino acid replacements are underlined. CDR and CH1 indications are according to Kabat et al.25 The sequences of the CH1 regions amplified with the different isotype constant-region specific primers were identical to the CH1 regions as reported by Kabat et al.25

Isotype-switch variants can be induced in vivo

The in vitro variant from the 5T33MM line was also tested for the presence of isotype-switch variants. Amplification with the CDR1 primer and the different constant region primers only revealed a PCR product when the Cγ primer was used. Even the addition of 10 × more cDNA in the PCR reaction or increasing the cycle number until 60 did not change this result. Also after hybridisation with the CDR3 probe no signals were observed in the CDR1/Cμ or the CDR1/Cα PCR products (Figure 2). The CDR1/Cγ PCR product of the 5T33MMvitro line was cloned and sequenced (Figure 1). The VDJ-region was identical between the 5T33MMvitro and 5T33MMvivo cells which proves that the 5T33MMvitro cell line was indeed derived from the original 5T33MMvivo bone marrow cells.18 Moreover, the hypermutation mechanism had stopped in these cells since no accumulation of further mutations in the VDJ-region of the heavy chain Ig gene occurred during the long period of in vitro culture.

Figure 2
figure2

T33MM-CDR3 specificity of isotype-specific PCR products from 5T33MM tumour cells in different locations. RT-PCR products generated with either 5T33MM-CDR1/Cμ, 5T33MM-CDR1/Cγ or 5T33MM-CDR1/Cα primer combinations were blotted and hybridised with the 32P-labelled 5T33MM-CDR3 oligomer. Lane 1, femur BM of 5T33MMvivo; lane 2, solid tumour of 5T33MMvt-vv; lane 3, femur BM of 5T33MMvt-vv; lane 4, 5T33MMvitro; and lane 5, Femur BM of control C57BL/KalwRij mice. The CDR1/Cμ and the CDR1/Cα blots were exposed for 18 h while the CDR1/Cγ blot was exposed for only 2 h. The femur BM of the 5T33MMvt-vv mice was less infiltrated with tumour cells than the femur bone marrow of the 5T33MMvivo mice (10% and 86%, respectively). The actin RT-PCR products of these samples show good quality RNA; lane 6, water control.

5T33MMvitro cells were injected in syngeneic mice (n = 3). When disease developed, RNA was extracted from isolated bone marrow cells and a perivertebral solid tumour that had developed in two out of these three mice. The same PCRs were performed as above and high expression of the 5T33MM-IgG sequence was found as expected. However, also a clear specific signal was observed in the perivertebral tumour after hybridisation of the CDR1/Cα and CDR1/Cμ products (Figure 2).

Such switch-variants were not detected in the bone marrow of 5T33MMvitro bearing mice. This difference between 5T33MMvivo and 5T33MMvitro bearing mice may be explained by the fact that the tumour infiltration in the femurs in diseased mice was always much less in the case of the 5T33MMvitro line (+/−10% and >80%, respectively).

After cloning and sequencing, the 5T33MM/Cγ sequence was again identical to the original 5T33MM VDJ sequence. The CDR1/Cμ sequence revealed the true 5T33MM VDJ region linked to the Cμ constant region. Similar results were obtained for the CDR1/Cα sequence.

To exclude the slightest possibility that these Cμ and Cα expressing cells in the tumour originated from Cμ and Cα expressing cells already present in the in vitro 5T33MM line, but under the detection limit of the PCR assay, we subcloned the 5T33MMvitro line. Subclones were generated and tested for the presence of isotype-switch variants. Only 5T33MM-Cγ expressing clones were found in 11 subclones tested. Three randomly chosen subclones (wells 1, 2 and 11) were injected i.v. and bone marrow cells from the femur and perivertebral tumour masses were isolated from diseased mice (n = 8). Again, isotype-switch variants were found in the solid tumour only but not in the bone marrow (Table 2).

Table 2 Isotype-switch variants originate from Cγ expressing 5T33MM cells

5T33MM isotype-switch variants disappear in vitro

Cells from the 5T33MM perivertebral tumours were isolated and again cultured. These cells are called 5T33MMvt-vv-vt hereafter. RNA was extracted from the cells at different time points, starting from 0 days until 4 months, and analysed for the presence of isotype-switch variants (Table 3). Already after 2 months the Cμ expressing cells could no longer be detected while the Cα expressing cells seem first to increase during culture and then also disappear. After 4 months no isotype-switch variants could be detected.

Table 3 The appearance of isotype-switch variants changes during culture

Isotype-switch variants are not PCR artefacts

RT-PCR artefacts due to in vitro recombination do occur.22 5T33MM-Cμ sequences could be generated in the PCR reaction by priming of an incomplete non-clonotypic mu cDNA, that terminates in the JH sequences, to the 5T33-VDJ sequence. This phenomenon could occur when abundant non-clonotypic mu or alpha transcripts are present next to the tumour cells that exclusively express clonotypic gamma transcripts. To exclude this possible explanation for the appearance of isotype-switch variants we mixed the 5T33MMvitro cell line, in which no isotype-switch variants were found, with normal spleen cells of C57BL/KalwRij mice or with bone marrow cells from 5T2MM bearing mice. Ratios were 100/0, 75/25, 50/50, 25/75 and 0/100. RNA was extracted, first strand cDNA was synthesised and 5T33MM-specific PCRs were performed as described in Materials and methods. Results are shown in Figure 3. Hybridisation signals with the 5T33MM-specific CDR3 oligo were only observed on the 5T33MMCDR1/Cγ PCR products and not on the 5T33MMCDR1/Cα or Cμ products despite the fact that non-clonotypic mu and alpha sequences were present in the spleen and 5T2MM-specific isotype-variants in the bone marrow. A control hybridisation on 5T33MMvivo bone marrow cells, in which isotype-switch variants are detected, is shown on the right.

Figure 3
figure3

Isotype-switch variants are not RT-PCR artefacts. 5T33MMvitro cells were mixed with normal spleen cells or bone marrow cells from 5T2MM bearing mice. RT-PCR and CDR3 hybridisation was performed as in Figure 2. Hybridisation signals are only observed when the 5T33CDR1/Cγ primer combination is used. No 5T33-Cμ or 5T33-Cα is detected. The 5T33MMvivo bone marrow is used as positive control for the detection of isotype-switch variants.

FISH analysis reveals a normal number of chromosome 12

Multiple myeloma cells often have a complex karyotype with many numeric changes. To exclude the possibility that more than two copies of chromosome 12, harbouring the Ig heavy chain locus, are present in 5T33MM, FISH analysis with a painting probe for chromosome 12 was performed. Also a painting probe for chromosome 15, harbouring the c-myc gene, was used to see whether major changes had occurred in this chromosome. No numeric or structural changes of chromosome 12 and 15 were found. Therefore, complex recombinations between several Ig loci to explain the presence of these isotype-switch variants could be excluded.

Discussion

Ig sequence analysis showed the presence of α and μ Ig isotype-switch variants next to the major MM IgG sequence in the 5T2MMvivo and the 5T33MMvivo cells similar to human MM.8,9,10 This emphasises again that the 5TMM mouse model is very close to the human situation. Several hypotheses have been put forward to explain the presence of isotype-switch variants,11 including the possibility for the presence of a clonogenic pre-switched precursor myeloma cell that could give rise to IgG and IgA expressing cells by downstream switching. In the cultured 5T33MMvitro cells no evidence for the presence of isotype-switch variants was found. However, when these 5T33MMvitro cells were injected in vivo, isotype-switch variants were again present in the tumour masses. However, they only could be found in a large tumour mass such as the solid tumour that emerged from the vertebrae but not in the bone marrow itself, emphasising the low frequency of these isotype-switch variants.

In vitro recombination during the RT-PCR reaction as explanation for the presence of isotype-witch variants in general, was ruled out by a control experiment mixing the 5T33MMvitro line with normal spleen cells or bone marrow cells from 5T2MM bearing mice.

The emergence of IgM and IgA expressing cells indicates that IgG expressing myeloma cells can switch to IgM and to IgA. The possibility, that there were small numbers of IgM and IgA expressing cells already present in the 5T33MMvitro cell line under the detection level of the PCR assay, that could explain the emergence of isotype-switch variants in vivo, could be ruled out by using IgG expressing subclones derived from the 5T33MMvitro line for injection. Again, in the cases where a solid tumour emerged, isotype-switch variants could be found. Also, the number of chromosome 12 was normal in the 5T33MMvitro cell line, ruling out complex recombinations between several chromosomes 12. Switching from IgG to IgA can occur by a recombination event between one of the γ switch regions and the downstream located α switch regions on the same chromosome. This sequential productive IgH switching has already been described for mouse plasmacytoma lines, occurring at frequencies of 0.01–1% among all cells.23,24 However, switching from IgG to IgM can only be explained by recombination events between sister chromatids (trans-switching) as in a normal switch events from IgM to IgG the Cμ locus is deleted from the CIS chromosome. Switch recombinations in trans, ie between S regions from active and inactive IgH loci have been described occasionally.24 The fact that these switch-variants can only be found in the in vivo situation and not in the in vitro line indicates that the local microenvironment might play an important role in inducing these switch events. It is known that switching is directed towards particular isotypes by lymphokines such as IL4, IFN-γ and TGF-β that are produced by T cells (reviewed in Ref. 24). After transfer of the 5T33MMvitro line into mice, isotype-switch variants appear and remain present even when these cells were isolated and cultured again. The μ+ cells disappear after a few months of culture while the α expressing cells exist for a longer period of time. A possible explanation for this is the lack of microenvironmental signals necessary for more switch events in the major IgG clone. Since the total number of IgG cells is so much higher than the number of IgM and IgA cells it is not surprising that over time the IgG clone takes over completely.

From these data it is clear that in these models, closely resembling human MM both μ and α isotype-switch variants can originate from the major IgG MM clone. These switch events may be considered as a rather normal phenomenon which become detectable in MM due to the high number of clonally identical tumour cells. These data argue against the existence, at least in this mouse model, of a pre-switched myeloma precursor cell as we postulated before.8,11 In this respect, the 5T33MMvitro cells can acquire the same characteristics as the 5T33MMvivo cells upon injection in vivo.

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Bakkus, M., Asosingh, K., Vanderkerken, K. et al. Myeloma isotype-switch variants in the murine 5T myeloma model: evidence that myeloma IgM and IgA expressing subclones can originate from the IgG expressing tumour. Leukemia 15, 1127–1132 (2001). https://doi.org/10.1038/sj.leu.2402164

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Keywords

  • multiple myeloma
  • class switch
  • immunoglobulin
  • precursor myeloma
  • trans-switching

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