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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 198-207
Light Chain Shifting: Identification of a Human Plasma Cell Line
Actively Undergoing Light Chain Replacement
By
Hirofumi Tachibana,
Hirotaka Haruta, and
Koji Yamada
From the Department of Food Science and Technology, Faculty of
Agriculture, Kyushu University, Fukuoka, Japan.
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ABSTRACT |
We identified an antibody-secreting human B-cell line (HTD8), which
actively replaces the production of the original  light chain with a
new chain (light chain shifting) at a high rate. Loss of the
original rearranged light chain occurs by significantly reducing
the amount of transcript expressed. Expression of the new chain,
which replaces the original chain, occurs by rearranging new VJ
segments on a previously excluded allele. V gene usage of these new
rearrangements are biased toward V 4, V6, and V10 families, which are known to be the least
frequently used. In striking contrast to the plasma cell phenotype,
recombination activating genes, RAG-1 and RAG-2, were expressed in the
HTD8 cells and were shown to be necessary, but insufficient for
inducing expression of the new chain. These results suggest that
human plasma cells have the potential to actively undergo light chain replacement.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALLELIC EXCLUSION is a fundamental
principle in B-cell development and is the basis for clonal selection,
which limits one cell to produce a single antibody with a certain
specificity. Ig gene rearrangements occur in a highly ordered fashion
during B-cell development1 and is initiated by DNA
rearrangement at the heavy-chain locus.2 In developing B
cells, the expression of a complete Ig heavy-chain protein is
accompanied by a drastic change in the targeting of V(D)J recombinase
activity. Recombinase activity changes from being predominantly active
at the heavy-chain locus in pro-B cells to being exclusively restricted
to the light chain loci in pre-B cells. This switch in locus-specific
recombinase activity results in allelic exclusion at the Ig heavy-chain
locus.3 The resulting pre-B cells then can rearrange their
light chain genes, although some pro-B cells have been shown to be able
to rearrange their light chain genes before their heavy-chain
genes.4 Expression of the light chain genes and assembly
with the heavy chains leads to the formation of complete Ig molecules
that are expressed on the cell surface. To ensure antibody
monospecificity, it is generally believed that further light chain
rearrangement is shut off as soon as a functional light chain gene
rearrangement is generated, resulting in expression of the complete IgM
on the cell surface of immature B cells.2,5,6 However, the
mechanism for allelic exclusion at the light chain locus has not been
clearly elucidated.3 Continuous rearrangement of the light
chain genes has been reported in some cell surface Ig positive
(sIg+) B-cell lines7-10 and in
mature B cells in vivo,11,12 but not in Ig-secreting plasma
cells. We have shown that long-term culture of a human B-cell hybridoma
line with concanavalin A (Con A) induce at a high frequency production
of various novel light chains, which replaced the original light
chain.13,14 This differential light chain expression led to
an alteration of the original antigen binding ability. We call this
process light chain shifting.14 To investigate the
mechanism of how this alteration occurs in antibody-secreting cells, we
established a plasma cell clone (termed HTD8). Clone HTD8 had undergone
secondary rearrangement after losing its ability to express the
originally rearranged chain at a high frequency. To explain how the
loss of original light chain expression occurs, the receptor editing
model has been proposed. It has been demonstrated that the V genes
coding for an autoreactive light chain can be replaced with V genes
coding for a functional nonautoreactive light chain in mice expressing a transgenic sIg with antiself specificity.15,16 In this
model, rearrangement of the new light chain occurs on the
originally rearranged allele, which results in the deletion of the
original light chain. In contrast, we see in our plasma cells that loss of the original chain production not only precedes expression of the
new light chain, but is also independent of the new light chain
gene rearrangement.
Components of the recombinational machinery necessary for Ig
rearrangement include the recombination activating genes, 1 and 2 (RAG-1 and RAG-2).17 Mature B cells, in which RAG-1 and
RAG-2 expression is downregulated,18 have been shown to
undergo no further Ig gene rearrangement. However, recently RAG-1 and
RAG-2 proteins have been shown to be reexpressed in mature B cells that have been stimulated in vitro with lipopolysaccharide and interleukin-4 (IL-4) and also in germinal center B cells in immunized mice. This
indicates that the control of RAGs expression has yet to be fully
elucidated.19-22 In striking contrast to antibody producing cells in the plasma phenotype stage, the RAG genes are expressed in our
established plasma cell line. B cells leaving the bone marrow have been
hypothesized to have fixed sIg that can only be altered by somatic
hypermutation. We describe a possible mechanism by which expression of
the original light chain is replaced with a new light chain in cells at
the plasma stage of B-cell development.
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MATERIALS AND METHODS |
Cells.
The human hybridoma HB4C5, which secretes antibody specific to human
histone H2B, was generated by fusing a B lymphocyte with the IgM
secreting human plasma line NAT-30.23 Con A-resistant clones derived from HB4C5 cells were isolated as previously
described.24 These cells were cultured in ERDF
medium (Kyokuto Pharmacy, Tokyo, Japan) containing 5%
fetal calf serum (Whittaker Bioproducts, Walkersville,
MD). The human mature B-cell line, Raji, and the human
T-cell line, Molt-4, were cultured as recommended by American Type
Culture Collection (Manassas, VA). To identify HTD8 cells, Con A-resistant variants were seeded in 96-well culture plates, and
the supernatant of each well 3 weeks after subcloning was examined for
the expression of light chains.
Cell stimulation.
Cells were cultured in a 5% CO2 atmosphere at 37°C in
the presence of 50 ng/mL of phorbol 12-myristrate 13-acetate
(PMA).25 The cells and the culture supernatants were then
obtained and assayed for the expression of light chain and RAG
proteins. PMA was purchased from Sigma (St Louis, MO).
Flow cytometric analysis.
Cultured cells were washed in phosphate-buffered saline (PBS) and
stained on ice with the appropriate monoclonal antibodies for 30 minutes in 100 µL of PBS and the relative fluorescence intensity was
detected by flow cytometry (FACS Calibur; Becton Dickinson, Mountain
View, CA). Antibodies used were mouse anti-CD20 (Southern
Biotechnology, Birmingham, AL), mouse anti-CD38
(Immunotech, Cedex, France), anti-mouse IgG1 conjugated
to fluorescein isothiocyanate (FITC) (Southern Biotechnology), and goat
anti-human Ig heavy and light chains conjugated to FITC
(Biosource, Camarillo, CA). Irreverent FITC-conjugated
mouse or goat IgG was used as a negative control.
Isolation of sIg cells.
To isolate sIg cells, cells were incubated in a
culture dish (10 cm in diameter; Becton Dickinson) coated with 100 µg/mL of anti-human IgM antibody (Biosource). The cell population
left unattached to the dish was harvested, and these cells were
cultured in ERDF medium containing 5% fetal calf serum.
Western blot analysis.
Cell extracts were prepared by one cycle of freeze-thaw lysis (1 × 107 cells) in PBS containing 1 mmol/L phenylmethyl
sulfonyl fluoride (PMSF) as described.26 The resulting
lysate was spun for 5 minutes at 4°C, and supernatant was retained
as whole-cell extract. Samples from supernatant cell extract were
boiled for 5 minutes in sample buffer, electrophoresed on sodium
dodecyl sulfate (SDS)-polyacrylamide gels (10%) and transferred to a
nitrocellulose membrane.27 The blotted RAG proteins were
detected by immunoblotting with 1 µg/mL of anti-human RAG-1 or RAG-2
antibodies (Pharmingen, San Diego, CA). Immunoreactive
proteins were detected by incubation with horseradish peroxidase
(HRP)-conjugated goat anti-mouse IgG (Sigma) at 1:2,000 dilution for 1 hour at room temperature; immobilized HRP was visualized using an
enhanced chemiluminescence assay (Amersham, Buckinghamshire,
UK). To detect the human light chain, the blotted membrane was incubated in a 1:500 dilution of HRP-conjugated goat anti-human light chain (Biosource) for 1 hour. The membranes were
washed in PBS containing 0.05% Tween 20 and developed with 1.6 mmol/L
4-chloro-1-naphthol, 0.01% H2O2 in PBS with
20% methanol.
Nucleotide sequence analysis.
Total RNA prepared using the TRIzol reagent (GIBCO-BRL, Gaithersburg,
MD) was subjected to cDNA synthesis and the resultant cDNA served as a
template for polymerase chain reaction (PCR) amplification using
specific primers. cDNA was synthesized from total RNA using a kit
(Amersham) according to the instructions provided by the manufacturer.
PCR was done in 50-µL reaction volumes containing 10 mmol/L Tris-HCl
(pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 20 µg/mL
gelatin, 1 µmol/L of each primer, 0.2 mmol/L of each
deoxyribonucleotide triphosphate, and 1 U of Taq
polymerase (Takara, Osaka, Japan). A single PCR cycle consisted of an
incubation period of 0.7 minutes at 94°C, 1 minute at 54°C, and
1 minute at 72°C. The PCR primers used were as follows. The
variable regions of the light chain genes28:
P-LLF, 5 -ACTAGAATTCATG(AG)-
CCTG(CG)(AT)C(CT)CCTCTC(CT)T(CT)CT(CG)(AT)(CT)CC-3 and
P-LLR, 5-ACTAGCGGCCGCCTATGAACATTC(CT)G(CT)AGGGGC-3;
EBNA-1 gene: EBNA1F, 5-CCGAAATTTGAGAACATTGC-3
and EBNA1R, 5-TCACTCCTGCCCTTCCTCAC-3; LMP-1 gene: LMP1F,
5-ACTAAGCTTATGGAACGCGACCTTGAGAG-3 and LMP1R, 5-ACTAGCGGCCGCTTAGTCATAGTAGCTTAGC-3 and
glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH): P-G3PDF,
5-CATCACCATCTTCCAGGAGC-3 and P-G3PDR,
5-GGATGATGTTCTGGAGAGCC-3. The nucleotide sequences of V
region genes were determined after subcloning into pGEM-T vector
(Promega, Madison, WI), using the DyeDeoxy terminator kit (Perkin Elmer-Cetus, Norwalk, CT) on the DNA sequencer
ABI 310 (Perkin Elmer-Cetus) according to the manufacturer's
instruction.
DNA analysis.
Genomic DNA was prepared as previously described.29 A total
of 1 ng of genomic DNA was used for the PCR assay. The PCR was used to
detect the VC5-to-J coding joint for the original light chain
C5 or the VJ coding joints for the new light chains (D8-1,
D8-2, D8-3, and D8-4). The following primers: VC5P (C5-CDR1 region-specific) 5-AACAGCTCCAACATTGGGAA-3
(sense), V4P (V4-CDR1 region-specific)
5-CTCTGAGCAGTGGGCACAGC-3 (sense), V6P
(V6-CDR1 region-specific)
5-AGTTGCAGCATTTTCAGCAAC-3 (sense), and JP (conserved
3 of J) 5-TCAGTTTAGTCCCTCCGCC-3 (antisense)
were used. PCR reactions were performed as described above for reverse
transcriptase (RT)-PCR. Aliquots were withdrawn at cycles 20 and 25 for
separate analysis to ensure that amplification was a linear range.
Reaction products were run on 0.8% agarose gels, followed by Southern
blot hybridization. The PCR products were transferred to nylon transfer
membranes (Hybond-N+, Amersham) and hybridized with
32P-radiolabeled probes. cDNA fragments coding for each light chain, which were used for analyzing the variable region
sequence, was digested from their respective vectors with restriction
enzymes. The resulting fragment each containing the specific V gene was labeled using Redi-Prime (Amersham) and
 32P-deoxycytidine triphosphate (Amersham) for use as
probes for the detection of the corresponding VJ coding joints.
To check for possible Taq polymerase errors, all PCR products were
sequenced and compared with the previously defined sequences.
Northern blots.
Total RNA, 10 µg per lane, was subjected to electrophoresis through a
1% agarose gel and transferred onto nylon membranes and hybridized
with the 32P-radiolabeled probes. The membrane was
air-dried and autoradiographed on x-ray film. For original light chain, µ heavy chain, and  -actin transcripts, the appropriate PCR
fragments (VC5-J, µNAT-30, and -actin) were used as
probe.14
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RESULTS |
Isolation of a plasma clone expressing differential light chains.
To obtain glycosylation variants of Ig, we previously isolated
glycosylation mutants of Ig-secreting cells that were resistant to the
cytotoxic effect of Con A from the human hybridoma line, HB4C5. HB4C5
cells secrete IgM reactive to the human histone, H2B.13,30
The expressed light chain is relatively large (32 kD), the size of which is due to a carbohydrate chain
linked to its variable region.31 Initially, some of the
variant clones were able to secrete light chains of different
apparent molecular sizes. The various antibodies secreted from these
clonal mutants showed either a loss of reactivity or varied
cross-reactivity with different antigens.13 When we
subcloned these Con A-resistant variant cells, we isolated a
single-cell variant clone (HTD8). As with the parental HB4C5 line, HTD8
initially secreted the original light chain species. However, as
the clone was allowed to expand, other various light chain species
were detected. At week 3, a 28-kD species and at week 4 an even smaller
species were found (Fig 1A). To determine
whether a single-cell clone can secrete more than one light chain,
we further subcloned the expanded HTD8 cells using the limiting
dilution method in 96-well culture plates and allowed the cloned cells
to undergo only 12 cell divisions. Almost all of the subclones (22 of
33 randomly chosen clones) secreted again mainly the 32-kD species and
a detectable amount of the 28-kD chain. Secretion of the various
new light chains, which are determined by their difference in size
from the original, was observed in five wells (Fig 1B, lanes 6, 7, 14, 25, and 29). A complete lack of light chain expression, or at least
reduced production to undetectable amounts, was shown in six wells (Fig 1B, lanes 1, 8, 9, 11, 16, and 20). Another six randomly chosen wells
from the 96-well culture plate were cultured for a further 4 weeks, and
as in Fig 1A, week 4, almost all of these subclones again demonstrated
that the 32-kD light chain species was the dominant species and the
28-kD chain was the minor species produced (Fig 1C). From Fig 1B, four
of the five novel light chain producers were isolated and named D8-1,
D8-2, D8-3, and D8-4. In addition, a clone, which did not secrete any
light chains (D8-11), was also isolated. The original chain was
not detected in the cytoplasmic fractions of any of these novel chain-producing clones (data not shown). Although these four clones
were cultured for over 6 months, they secreted the novel chain
stably, and the original 32-kD chain was not detected at all. These
results indicate that HTD8 subpopulations obtained by expansion are
able to secrete either the original or a new light chain or none at
all.
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| Fig 1.
Immunoblot analysis of the expression of differential light chains by HTD8 cells. The pattern of light chain secretion
was assayed by immunoblotting. (A) The parental HB4C5 cells are shown
to secrete only the 32-kD light chain. During the continuous
culture of HTD8 cells, culture supernatants taken from 0, 2, 3, and 4 weeks after the initial cloning were assessed using an anti-human light chain antibody. (B) The HTD8 cells were further cloned by the
limiting dilution method in 96-well culture plates, the culture
supernatants from 33 randomly chosen wells were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a
nitrocellulose membrane, and the light chains were detected with an
anti-human light chain antibody/HRP conjugate. (C) Cloned HTD8
cells were cultured in 96-well plates for 4 more weeks, and six
randomly selected wells were assayed for chain production.
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V gene usage in the new light chain gene expression.
From the subclones secreting only the novel chain (D8-1, D8-2,
D8-3, and D8-4), cDNA was synthesized and analyzed for V gene
sequencing (Fig 2). The nucleotide
sequences of V regions of the light chains produced by the four
subclones are different from the original. The germ-line V gene for the
original light chain (C5) has been shown to belong to the
V1 family,31 as grouped by Chuchana et
al.32 The germ-line V families used in the light chain
genes from the four subclones were shown to be V4,
V6, and V10. Strikingly, these
V gene families are the least frequently
used.33 In addition to the four genes, another seven new
light chains sequenced so far also used the V genes from
these seldom used V gene families (data not shown).
These results indicate that the usage of V genes in HTD8
cells is biased towards these minor families. Although D8-2 and D8-3
cells express genes encoded by the same germ-line V gene
(HSIGLV36), they apparently arose from independent recombination
events, as judged by having distinct V-J joining sequences. This was
confirmed by genomic DNA analysis using V family-specific probes (see Fig 3). We
found no significant differences in the growth rates of these new light
chain-expressing subclones, and doubling times of all subclones were
comparable to the original cells (data not shown). Therefore, we
conclude that the bias in V gene usage is not the result
of a differential growth rate.
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| Fig 2.
Nucleotide sequences of variable regions of new light
chains expressed in HTD8 subclones. Nucleotide sequences of the
variable regions of the light chains expressed in HTD8 subclones (D8-1,
D8-2, D8-3, and D8-4) are shown in a 5 to 3 direction.
VLD8-1, VLD8-2, VLD8-3, and VLD8-4 represents the light chain genes
expressed by the subclones of HTD8. Sequences are compared with the
corresponding germ-line V (HUMIGL8V, HSIGLV36, and V1-20) and
J2/3 sequences. Designations in brackets indicate their
corresponding V gene family. Dashes sequence identity with the
germline genes. Dots indicate where gaps have been introduced to
maximize homology.
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| Fig 3.
Presence of the original V to J coding joint and
the new V to J rearrangement and quantification of the original
chain transcript. PCRs were performed with primers that amplify the
indicated VJ coding joint formation in the genomic DNA from HB4C5,
HTD8, and its subclones. (A) A diagram of the PCR primers used to
detect the VJ coding joint. The direction of primers used are indicated
with arrowheads. (B) The PCR-amplified products were run on agarose
gels and assayed for hybridization to a VC5, V4, and V6
specific probes by Southern blotting. Bands corresponding to VJ coding
joint for the original light chain C5, V4 joined to J, and
V6 jointed to J are shown (C) Expression of the original light chain and the µ heavy-chain mRNAs were examined by Northern
blotting. Cells used as a source of mRNA are indicated above each
lane.
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Rearrangement of the new chain on a previously excluded allele
and reduction of the original chain transcript are found in the
HTD8 subclones.
To determine the genetic event responsible for expression of a
differential light chain in Ig-secreting cells, we analyzed light chain gene rearrangement in HB4C5 cells, HTD8 cells, and the four
subclones secreting only the novel chain (D8-1, D8-2, D8-3, and
D8-4). The four clones were resubcloned three times by limiting
dilution before use. If rearrangement for the expression of a new light chain occurs on the previously rearranged locus, the original
VC5 to J coding joint should be replaced by the novel rearrangement and thus be deleted from the chromosome. If
rearrangement for the new light chain occurs on a previously excluded
allele, both the original and the new VJ
coding joint should be detected. PCR was performed on the genomic DNA from each cell line using primers appropriate to the CDR1 of C5 and
the J regions to detect the original
VC5-to-J coding joint formation. The
combination of these primers provides the ability to detect VJ
rearrangement necessary to produce C5 in the Ig loci. Genomic DNA
was subjected to PCR and the reaction products were analyzed by
Southern blot hybridization. A diagram of the PCR assay strategy is
shown in Fig 3A. In DNA from all of the subclones examined, the VJ
coding joint for C5 was detected (Fig 3B). Furthermore, authenticity
of all of the PCR products was confirmed by determining the partial
sequence of these products (data not shown). The presence of the VJ
coding joint for C5 suggest that the original rearranged locus is
retained in the chromosome thus implying that the rearrangement for a
new light chain occurs on another allele. To corroborate this view, we
examined for the presence of novel VJ
coding joint formation specific for the new light chain using primers
specific to the V4 and V6 families (Fig
3B). PCR products hybridized with V4 and
V6-specific probes were detected slightly in the DNA
from HTD8 cells, suggesting that the new VJ rearrangements occurred
during the cell population expansion. This result coincides with the
fact that not only the original light chain, but also a smaller amount
of the new light chains, was found in the supernatant of the HTD8 bulk
cell population (Fig 1A). Because only rearranged light chain genes
can be detected by this assay, each subclone is shown to contain both
the originally rearranged allele plus another rearranged gene on a
previously excluded Ig allele. These results indicate that the new
chain expression in HTD8 cells results from rearrangement on a
previously excluded allele. The subclones also retain the original
rearranged gene, although the original protein is not expressed. To
address the loss of production of the original light chain in these
subclones, we examined for the expression of the original light chain
transcript by Northern blot analysis (Fig 3C). The original chain
transcript was seen in all subclones, however the level was much lower
when compared with HTD8 cells and the parental HB4C5 cells. In
contrast, no difference was seen in the expression level of the µ heavy-chain transcript. The significant reduction of the level of mRNA
may be a reason why the original chain protein is not detected in the HTD8 subclones, although we cannot rule out other possibilities.
RAG genes expression is necessary, but insufficient for inducing
rearrangement of the new light chain gene in HTD8 cells.
RAG-1 and RAG-2 gene products have been shown to be essential for
initiating Ig gene rearrangement, and RAG expression is terminated
after the formation of sIg, which may be a factor in maintaining
allelic exclusion.18 To test for a relationship between the
expression of the new light chain in the HTD8 cells and RAG
expression, we examined for RAG expression in HTD8 cells (Fig 4A). We found that both proteins were
expressed in the HTD8 cells. To test whether the level of RAG
expression correlates with the ongoing new light chain expression
in HTD8 cells, we treated HTD8 cells with PMA, which has been shown to
decrease the RAG expression (Fig 4A).25 The level of RAG
expression was significantly reduced as compared with the control. To
assess the effect of the reduction of the RAG expression on the new
light chain expression, HTD8 cells were cultured in 96-well culture plates in the presence of PMA for 3 weeks, and expression of the new
light chain from eight randomly chosen wells was compared (Fig 4B).
Although the new light chain was detected in some of the
PMA-treated clones, other clones producing only the 32-kD original light chain were also found. When the PMA-treated clones expressing
only the 32-kD light chain were further maintained in PMA-free medium
for 2 weeks, only the 32-kD light chain continued to be expressed
(Fig 4C). The failure of these cells' ability to express the new light chain when treated with PMA parallels the absence of RAG
expression, suggesting that the inhibition of the new light chain
induction is a direct effect of the termination of RAG expression.
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| Fig 4.
Correlation between RAG proteins expression and new light
chain expression. (A) Effect of PMA that has been shown to moderate RAG
expression was examined. HTD8 cells were treated with (+) or without
( ) 50 ng/mL PMA. Cells were lysed in SDS lysis buffer; 50 µg/mL
protein from each sample was fractionated by 10% SDS-PAGE, and RAG-1
and RAG-2 proteins were detected by immunoblotting. (B) Lambda light
chain expression pattern in HTD8 cells treated with PMA. The pattern of
light chain secretion from HTD8 cells cultured with (+) or
without () PMA in a 96-well culture plate for 4 weeks and eight
wells were randomly chosen, and the culture supernatants were subjected
to SDS-PAGE, transferred to a nitrocellulose membrane, and light
chains were detected by immunoblotting. (C) The 32-kD original chain producing subclones generated from the PMA treatment was cloned
via the limiting dilution method, further cultured for 2 weeks in a
96-well plate, and eight randomly chosen wells were assayed for light chain production by immunoblotting. (D) The expression of RAG-1
and RAG-2 proteins in the cells indicated above each lane was assessed
by immunoblotting.
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It has been shown that there is a strong correlation between the RAG
expression levels and Ig gene rearrangement.19 Therefore, we assessed the RAG proteins level in HTD8 and the parental HB4C5 cells
(Fig 4D). The expression level for the RAG-1 and RAG-2 produced in HTD8
cells did not significantly differ from HB4C5 cells. In addition, the
RAG-1 and RAG-2 were expressed in the HTD8 subclones that produce only
the new light chain or no light chain (Fig 4D). The continuous
expression of RAG in the subclones indicates that new rearrangement and
the following expression of new light chains does not terminate RAG
expression in HTD8 cells. We previously showed that enhancement of RAG
expression in HB4C5 cells treated with dibutyryl cyclic adenosine
monophosphate (dibutyryl cAMP) did not induce light chain
shifting.14 Although the subclones of HTD8 cells (D8-1,
D8-2, D8-3, and D8-4) have been maintained for over 1 month in cell
culture medium, other new VJ rearrangements did not occur (Fig 3B).
These results suggest that constitutive and enhanced expression of the
RAG genes is insufficient to rearrange new light chain genes.
With regard to RAG expression in human mature B cells, evidence has
recently been presented, which shows that expression of the
Epstein-Barr virus nuclear antigen 1 (EBNA-1) protein was sufficient to
induce both RAG genes.34 Conversely, expression of
Epstein-Barr virus latent membrane protein (LMP)-1 downregulates RAG
genes expression.22 To confirm these findings, we performed RT-PCR to detect EBNA-1 and LMP-1 RNAs (Fig
5). Human Burkitt's lymphoma line, Raji, which has been shown to lack
RAG expression, expressed LMP-1 as shown previously.22 HTD8
and HB4C5 cells did not express LMP-1, but the EBNA-1 transcript was
detected in both cell lines. Both genes were not detected in the human T-cell line, Molt-4. These results validate findings stating that the
expression of EBNA-1 and LMP-1 are associated with RAG genes expression
in human mature B cells.
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| Fig 5.
Detection of mRNA for EBNA-1 and LMP-1. The expression of
EBNA-1, LMP-1, and G3PDH mRNA was examined by RT-PCR. Lane 1, Raji
cells; lane 2, HB4C5 cells; lane 3, HTD8 cells; and lane 4, Molt-4
cells. The PCR-amplified products were run on agarose gels and assayed
for hybridization to specific internal probes (EBNA-1 probe:
5-TGACGGAGATGAAGGAGGTG-3; LMP-1 probe:
5-TTGTGCTGTTCATCTTTGGC-3) by Southern blotting.
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Loss of sIg expression occurs at a high frequency in HTD8 cells.
We have previously shown that HB4C5 cells display characteristics
common to B cells in the plasma cell state with regard to Ig secretion
and the expression pattern of surface antigens.14 HTD8
cells and all of the subclones, except D8-11 cells, secrete IgM. To
elucidate the difference between HTD8 and the parental HB4C5 cells, we
examined for the expression of the antigens CD20 and CD38. The
expression pattern of these antigens in human plasma cells was
shown to be CD20CD38+.35
Both of our cell lines are also
CD20CD38+, indicating that the HTD8
cells are in the same plasma state as wild-type plasma cells
(Fig 6A). As determined by surface staining with anti-human Ig heavy and light chain antibodies, HB4C5 cells, as
well as all subclones of HTD8, except for the subclone D8-11, did not
contain a notable sIg subpopulation (Fig 6B). An
sIg subpopulation was observed, albeit barely, in
the HTD8 bulk population and clearly increased ( 30%) after a
further 4 weeks of cell growth (Fig 6B). This result turned out to be
the most significant difference seen between HB4C5 and HTD8 cells,
because after 4 weeks of culture, the HB4C5 sIg
subpopulation did not increase. This is consistent with previous results showing that Con A stimulation is able to induce the appearance of an sIg subpopulation from the light chain
shifting-inducible cell line, HB4C5.14 To examine the
process for the loss of sIg, we isolated the sIg
subpopulation from the HTD8 bulk population using anti-human µ light
chain antibody (Fig 6C). The sIg+ population clearly
increased from this isolated sIg HTD8 population
after 4 days in culture. This demonstrates that in HTD8 cells,
inactivation of the original light chain, which manifest as the
sIg state, actively occurs before expression of a
new light chain. We also found a few chain
clones (see Fig 1B). The subclone, D8-11, does not contain an sIg+ phenotype, indicating that the D8-11 line was probably
isolated from the sIg subpopulation. We examined the
expression of a new light chain gene in this subclone by RT-PCR
using V gene-specific primers followed by DNA sequence
analysis. It appears that the D8-11 clone expresses a transcript from
the new light chain gene, but the gene contains a frame-shift
mutation at the VJ junction. This suggests that the D8-11 subclone
cannot produce a new light chain protein due to a nonproductive
rearrangement (data not shown). Therefore, subclones that fail to
produce viable rearrangements may also be included in the
sIg population. These results taken together suggest
that the process of sIg+ to sIg
expression is initiated when the cell loses the ability to produce the
original light chain and is sustained when a nonproductive new
rearrangement occurs, which renders the cells incompetent to express a
new chain.
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| Fig 6.
Flow cytometric analysis of CD20, CD38, and surface Ig
expression. HB4C5 cells, HTD8 bulk population, and the new light chain
expressing HTD8 subclones (D8-1, D8-2, D8-3, D8-4, and D8-11) were
stained with FITC-conjugated anti-human CD20, CD38, or Ig antibodies
and analyzed by flow cytometry (shaded histogram). The negative control
was established using an irrelevant FITC-conjugated antibody of the
same isotype (clear histogram). The fluorescence intensity is shown on
the x-axis and the cell number on the y-axis. HB4C5 and HTD8 cells were
examined for CD20 and CD38 expression (A). Presence of sIg was assessed
for bulk population HB4C5 and HTD8 cells, the same cells after a 4-week
culture, as well as for the HTD8 subclones (B). After isolating
sIg HTD8 cells, these cells were examined for sIg
expression at day 0, 1, and 4 of culture using FITC-conjugated
anti-human µ antibody (C). Numbers in the upper left corners indicate
the percentage of cells that are stained negative.
|
|
| |
DISCUSSION |
In this study, we showed a novel process for regulating the light
chain expression in human plasma cells. Expression of a complete
functional surface Ig complex is thought to signal the overall
inactivation of Ig rearrangement.6,36 This process has been
shown to be completed during B-cell development, and cells at the
plasma state are thought to have lost the ability to undergo Ig
rearrangement.37 The striking features of the plasma clone,
HTD8, shown here are: (1) production of a new light chain resulting
from a rearrangement on another previously excluded allele, and (2)
active loss of expression of the previously rearranged light chain
gene. Although heavy-chain allelic exclusion has been confirmed by the
presence of feedback inhibition culminating with the formation of
membrane-bound µ chains,38,39 the mechanism of light
chain allelic exclusion remains unclear. There are a number of factors
that have been shown to be necessary for Ig light chain gene
rearrangement.40,41 Components of the recombinational machinery necessary for Ig rearrangement include the RAG-1 and RAG-2
genes. The RAG genes are expressed precisely when B cells rearrange the
Ig heavy and light chain loci during B-cell differentiation, but not in
plasma cells.17 The mechanisms that underlie the regulation
of RAG gene expression are poorly understood. Although for the most
part, expression of both of these genes is not seen in either normal or
malignant sIg+ B cells, some exceptions to the inverse
correlation between sIg expression and RAG transcription have been
found.42 Bone marrow immature B cells expressing a
transgenic sIg with anti-self specificity increased RAG transcript
expression when the cells were cross-linked with autoantigens, which
led to a secondary rearrangement.15,17 Ma et
al36 showed that sIg+ B-cell lines established
from Eµ-N-myc transgenic mice expressed relatively high
levels of the RAG genes. We show that both HTD8 cells and the parental
HB4C5 cells continuously express these RAG genes. Taken together with
our results, we assume that RAG expression and sIg expression or Ig
secretion are not exclusive, and that a developmental stage where the
two are coexpressed may exist.
With regard to RAG expression in human mature B cells, it has been
postulated that loss of sIg expression not only interrupts the signal
required to terminate RAG expression, but in fact, triggers the
upregulation of RAG expression.42 In contrast, because our
HB4C5 and HTD8 cells express the RAG genes continuously without
suffering a loss of expression of the original chain, we can
exclude the upregulation mechanism as an explanation for new
rearrangement. The RAG genes are also expressed in HB4C5 cells and the
parental NAT-30 cells, both of which are in the plasma cell
stage.14 RAG expression in both lines likely reflects a unique feature of the NAT-30 cells. NAT-30 cells are a subclone of the
Epstein-Barr virus-bearing B-cell line Namalwa. It has been shown that
expression of EBNA-1 and LMP-1 proteins was associated with RAG genes
expression in human mature B cells.22,34 The expression
pattern of both genes in HTD8 and HB4C5 cells coincided with the
findings. EBNA-1 is a multifunctional protein that controls transcription of additional EBV genes and is itself regulated in a
complex manner.43 Thus, it is possible that EBNA-1 , or an
as yet undetected viral product(s), could alter RAG expression.
Ig rearrangement activity and expression of both RAG genes is known to
be strongly linked in vivo and in various cell lines,16,25 suggesting a possible link between ongoing RAG expression and the
secondary rearrangement in our plasma cells. In fact, we found when the
RAG proteins were eliminated by treatment with PMA, the cells could not
produce a new light chain, suggesting the necessity of the RAG
genes for new gene rearrangements. However, the uninterrupted maintenance of a certain level of RAG expression may be insufficient to
induce expression of a new light chain, as shown by parental HB4C5
cells, which secrete only the original light chain and HTD8 cells,
which can secrete a new or the original light chain. We did not
detect any difference in the levels of RAG proteins between these two
cell lines, which suggests that new chain expression in HTD8 cells
is not caused by a greater amount of expression of the RAG genes. In
addition, we have found that the increased level of RAG expression
caused by dibutyryl cAMP treatment did not generate any new light chain
in HB4C5 cells. However, when HB4C5 cells were treated with Con A for a
long period of time, secondary gene rearrangement occurred on a
previously excluded allele of the Ig locus leading to the
production of a new light chain.14 This indicates that
the potential for recombinase activity is present in the HB4C5 cells,
and that other regulatory factors are needed for the activation of gene
rearrangement.
A critical difference between the HTD8 cells and the parental HB4C5
cells is the expression pattern of sIg. HTD8 cells were able to
generate subclones lacking the production of the original light
chain at a high frequency on cell proliferation, consequently leading
to a subpopulation of sIg cells. Especially
noteworthy is the fact that none of the HTD8 subclones produced both
the original and new light chains together, although subclones that
were light chain negative were found. These results indicate that loss
of the original light chain precedes expression of a new light
chain, therefore secondary rearrangement would not account for
triggering the loss of original chain production. Conversely, this
result indicates that loss of the original light chain could play a
role in providing the induction signal for new rearrangement. HTD8
cells continually express both RAG proteins without losing the
expression of sIg. Moreover, expression of the new light chain is
seen only in HTD8 cells, but not in its parental HB4C5 cells, although
both cell lines express RAG proteins at the same level. This pattern is
consistent with the result that loss of the production of the original
light chain is restricted to HTD8 cells, but not in the parental
HB4C5 cells. These results suggest that the loss of the original light
chain is an intermediate step providing a stimulus for new
rearrangement. We speculate that the expression of a new light
chain may represent a mechanism somewhat analogous to the receptor
editing model that has been described for autoantibody regulation.15,16 This receptor editing model proposes that B cells expressing an autoantibody undergoes a change through a
secondary rearrangement of its light chain thus becoming a
nonautoreactive cell. Compared with our HTD8 cells, significant
differences are present. During the editing process, the previously
active VJ coding joint is rendered inactive by a nested rearrangement.
This new rearrangement results in the deletion of the original coding joint. Thus, in the receptor editing model, loss of the original coding
joint is a passive process. In contrast, HTD8 subclones, which do not
produce the original light chain, nonetheless retain the originally
formed VJ coding joint for the original chain. This confirms that a
new rearrangement occurred on a previously excluded allele. We show
that the original chain mRNA level is significantly lower in cells
producing the new light chain compared with cells producing only the
original chain. This shows that the loss of production of the original
light chain is a result of the low expression level for the
original transcript, not the elimination of the VJ coding joint
formation on the chromosome. In contrast to the low amount of original
chain produced, the production level of the new chain was high.
If the significantly reduced level of the original chain transcript
was caused by a defect in the transcriptional regulatory machinery,
production of the new chain should be also low. Analysis of the
differences between the HTD8 cells and the parental HB4C5 cells will be
useful in obtaining insights into the mechanism responsible for the
highly frequent loss of rearranged light chain production in plasma cells.
We found that V genes used in secondary rearrangements in the HTD8
cells were biased towards the V4, V6, and
V10 families. It is intriguing that these V genes
belong to the least frequently expressed families.33
Recently, it has been shown that differences in the promoter of the V
gene segment is strongly associated with germline transcriptional
activity and the frequency of rearrangement, thus consequently, it may
affect the V gene usage in the expressed repertoire.44 This
finding suggests that the frequency of V gene usage is fixed by the
promoter sequence and is therefore unable to change. Our results
suggest that not only structural differences in the promoter region of
V gene segments, but also the presence of other regulatory factors, can
modulate usage of the V gene, although the mechanism of this bias
usage remains unclear. HTD8 cells will be useful in investigating the regulatory factors concerning V gene usage.
Based on these results, we have formulated the following hypothesis for
the light chain shifting in our HTD8 cells. First, the expression level
of the original chain transcript is significantly reduced. A low
level of the original light chain transcript and/or loss of
the original light chain protein caused by some impairment may
trigger the activation of VJ recombination in another locus. In the
other systems, extinction of surface IgM expression is thought to
induce secondary rearrangement.43 Thus in HTD8 cells, the
loss of original light chain protein, which fails to make a functional
Ig receptor, might be responsible for directly signaling a
locus-specific recombinase activation event. When recombinase-accessible V genes, but not recombinase-accessible heavy-chain V genes, are induced by this signal together with the
continuously expressed RAG genes, a new rearrangement occurs in the locus. If the secondary rearrangement is successful, a new chain
protein is produced replacing the original light chain. Still
maintaining allelic exclusion, this process confirms production of a
single light chain and maintains single antibody specificity,
although the original specificity might be altered. Furthermore,
although expression of the RAG genes is not terminated by production of
a new light chain and sIg expression, further rearrangements leading to
the production of another new chain have not been found. If the
expression of a new light chain signals a modulation of recombinase
activity, this finding may shed light on the recombination event, even
though the RAG genes are continuously expressed in the cells.
| |
ACKNOWLEDGMENT |
The authors thank Perry Seto for proofreading the manuscript and Kyoko
Ueda and Kousuke Morizumi for their excellent technical help.
| |
FOOTNOTES |
Submitted June 9, 1998;
accepted August 23, 1998.
Supported in part by grants from the Program for Promotion of Basic
Research Activities for Innovative Biosciences (to H. T.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hirofumi Tachibana, PhD, Department of Food
Science and Technology, Faculty of Agriculture, Kyushu University,
6-10-1, Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan; e-mail:
tatibana{at}agr.kyushu-u.ac.jp.
| |
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Abstract
Introduction