From Schering-Plough, Laboratory for Immunological Research, 69571 Dardilly, France
Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells
in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's
bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently
cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre-B cells, germinal center (GC) B cells, circulating memory cells, or
plasma blasts. This situation reflects what has been a general problem in cancer research for
years: how to compare a tumor with its normal counterpart. Although several studies have
demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a
biased
light chain expression and a Cµ-C
isotype switch. Using surface markers, we have
previously isolated a population of surface IgM
IgD+CD38+ GC B cells that carry the most
impressive somatic mutation in their IgV genes. Here we show that this population of GC B
cells displays the two molecular features of IgD-secreting myeloma cells: a biased
light chain
expression and a Cµ-C
isotype switch. The demonstration of these peculiar GC B cells to
differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro
suggests that IgD-secreting plasma and myeloma cells are derived from GCs.
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Introduction |
Immunoglobulin D (IgD) is the major antigen receptor
isotype coexpressed with IgM on the surface of mature
naive B cells (1). Strikingly, while membrane IgD on
human B cells is preferentially associated to
light chain (1,
10), secreted IgD from myeloma cells is preferentially associated to
light chain (11, 12). The ability of myeloma
cells to secrete IgD appears to be the result of an unusual
Cµ to C
switch mediated by DNA recombination between sequences within JH-Cµ intron and Cµ-C
intron
(13).
One question has been which B cell differentiation window corresponds to the stage where IgD myeloma cells
were originated. The answer for this will clarify the long
standing controversial issues (17, 18) of whether the myeloma precursors are hematopoietic stem cells (19), pre-B
cells (20), germinal center (GC)1 B cells (21), circulating
memory cells (22, 23), or plasma blasts (24). Although several studies have demonstrated somatically mutated Ig variable region genes in multiple myeloma including IgD myeloma (23), it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Here, we report a
population of IgM
IgD+ GC B cells that share three unique
molecular features of IgD myeloma cells: (a) most impressive somatic hypermutation in IgVH genes, (b) Cµ-C
isotype switch, and (c)
light chain expression. These GC B
cells were shown to differentiate into plasma cells but not
memory B cells, suggesting that IgD-secreting myeloma are
derived from B cells at GC stage but not at memory stage.
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Materials and Methods |
Assay for Sµ-
/
Recombination.
Genomic DNAs were extracted from 3 × 107 EBV transformed cells or 105 fresh purified
cells, according to the standard procedure. Genomic DNA was
submitted to PCR amplification using the 5' primer P3 (5'-CGGCAATGAGATGGCTTT-3') and the 3' primer P4 (5'-GGCAAACTGTCATGG GTT-3'), as shown in Fig. 1A. All
PCR reactions were performed with Taq polymerase (Perkin-Elmer Corp., Norwalk, CT) using the reaction buffer provided
by the manufacturers and a DNA thermal cycler (Perkin-Elmer
Corp.) with 35 cycles of 1 min denaturation at 94°C, 2 min
primer annealing at 60°C, and 3 min extension at 72°C. Complete extension of the products was then performed by a final 10-min incubation at 72°C. For DNA sequencing, PCR products were cloned using the TA cloning kit (Invitrogen, Carlsbad, CA). Individual bacterial colonies were randomly picked and extracted plasmids were sequenced in an automated DNA sequencer (Applied Biosystems, Foster City, CA) on both strands.

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Fig. 1.
Cµ-C switch recombinations in IgM IgD+CD38+ GC B cells. (A) Schematic representation of the Sµ- / and /µ- /µ recombinations
(14). (B) PCR amplification of Sµ- / recombination. Lane A, IgM+IgD+CD38+ GC founder cells; lane B, IgM IgD+CD38+ GC B cells; lanes C,
D, and E, EBV clones from IgM IgD+CD38+ cells; lanes F, G, and I, EBV clones from IgM+IgD+CD38+ cells. (C) DNA sequences of Sµ- / junctional regions from IgM IgD+CD38+ cells. Upper strings, germline sequences of the Sµ region; lower strings, germline sequences of the /µ- / intron.
The central string represents the sequences of cloned PCR products. Homologous nucleotides are linked by vertical bars. Arrowed figures, base numbers,
base No. 1 being the first 3' of the VDJ region. (D) Schematic representation of the break points.
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Measurements of Ig Secretion.
IgG, IgA, and IgM measurements
were performed using ELISA as previously described (34). For
IgD measurement, flat-bottomed 96-well plates were coated with
2 µg/ml of monoclonal anti-IgD antibodies (Nordic Immunological, Tiburg, The Netherlands) overnight at 4°C. After six
washes, plates were first saturated for 3 h at 37°C with RPMI
(GIBCO BRL, Gaithersburg, MD) containing 10% fetal calf serum (GIBCO BRL), and then incubated overnight at 4°C with
appropriate dilutions of the assays and the standard purified myeloma IgD (The Binding Site, Birmingham, UK). Plates were
washed six times and incubated with goat anti-IgD-biotin (Sigma
Chemical Co., St. Louis, MO) at 2 µg/ml for 2 h at room temperature. After six washes, streptavidin-alkaline phosphatase (Sigma
Chemical Co.) diluted 1/10,000 was added for 1 h at room temperature and enzymatic activity was revealed by p-nitrophenyl phosphate (Sigma Chemical Co.) and read at 490 nm on a Vmax
spectrophotometer. The absence of cross-reactivities with human
IgG, IgA, IgM, IgE, Ig
, and Ig
was checked, and values were
reported to a standard curve using a purified myeloma IgD.
Analysis of Ig Light Chain Expression.
5 × 103 B cells were
transformed by EBV during a 2-wk culture in a CD40 system
with 10 µl of EBV containing B95-8 supernatant in a round-bottomed 96-well plate. Cloning was performed by culturing 1 cell/
well. The clones derived from surface (s)IgM
IgD+CD38+ GC
B cells were selected by their sIgM
IgD+ phenotype and clones
derived from sIgM+IgD+CD38+ B cells were selected according
to their sIgM+ phenotype. VH gene expression by EBV clones
was analyzed by VH-C
PCR amplification and sequence analyses using primers specific for each VH family and C
region.
Among the 76 EBV clones, the V gene usage of 12 EBV clones
derived from sIgM
IgD+CD38+ GC B cells were determined.
One VH1, two VH5, fourVH3, and five VH4 were identified.
Furthermore, light chain expression by EBV clones was analyzed
by flow cytometry with anti-Ig
-FITC and anti-Ig
-FITC (Kallestad, Austin, TX).
Isolation of Tonsillar Plasma Cells.
In brief, tonsillar cells were
centrifuged through 1.5% BSA at 10 g for 10 min. CD20
CD38++ plasma cells were then isolated by cell sorting. To isolate
IgD+ and IgD
plasma cells, after centrifugation through 1.5%
BSA, cells were first stained with anti-CD38-PE (Becton Dickinson, Mountain View, CA). They were permeabilized by an overnight incubation with 1% paraformaldehyde at 4°C. Intracellular
IgD was stained with a mouse anti-IgD antibody-FITC (Dako
Corp., Carpinteria, CA). CD382+ plasma cells were finally separated into intracellular IgD+ or IgD
plasma cells by cell sorting.
Sequence Analysis of the VH5 Transcripts.
This was done according to our established methods (35). In brief, messenger
RNA was extracted from 2.5 × 104 plasma cells and cDNA was
obtained by reverse transcription. Full-length VH5-
transcripts
were amplified with 5'LVH5 primer (5'-CCCGAATTCATGGGGTCAACCGCCATCCT-3') with 3' primer HC
(5'-GGCGGCCGCTGGCCAGCGGAAGATCTCCTTCTT-3'), HCµ
(5'-TGGGGCGGATGCACTCCC-3'), or HC
(5'-CAGGGGAAGACCGATGG-3') with Taq polymerase (Perkin-Elmer Corp.).
PCR reaction was 35 cycles of 1 min denaturation at 94°C, 2 min
of primer annealing at 60°C, and 30 min at 72°C. The frequency
of Taq error in our lab is <2%. The PCR products were cloned,
using the TA cloning kit (Invitrogen). Plasmids extracted from
individual bacterial colonies were sequenced.
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Results |
Hypermutated sIgM
IgD+CD38+ GC B Cells Have Undergone Cµ-C
Switch by Recombination between Sµ and the
Pentamer-rich
/
Region.
We have previously identified a
population of sIgM
IgD+CD38+ GC B cells that contain
extensively mutated Ig variable region genes (36). An intriguing link between these B cells and IgD-secreting myeloma cells is the rare single surface expression of IgD isotype of Igs. Such a phenotype can only be explained by
either Cµ gene deletion as observed in IgD myeloma cells
(14, 15) and in unfractionated cells from normal tonsils (16)
of alternative splicing of µ-
messenger RNAs, as observed
in sIgM+IgD
B cells. To clarify this issue, PCR primers
were designed for probing recombination events between
the 442-bp
/µ region and the 443-bp
/µ region or between Sµ and the pentamer-rich region
/
(Fig. 1 A). In
three tonsillar samples, Sµ-
/
recombination but not
µ-
µ recombination was detected in sIgM
IgD+CD38+
GC cells and their derived EBV transformed clones, but
not in sIgM+IgD+CD38+ GC founder cells (37) and their
EBV-derived clones (Fig. 1 B presents the result from one
tonsil sample). To determine the Sµ-
/
break points, PCR-generated DNA products were cloned and sequenced. Fig.
1 C shows the sequences of four Sµ-
/
junctions obtained from freshly isolated sIgM
IgD+CD38+ GC B cells
and their EBV clones. The four break points, which are
presented in a schematic diagram in Fig. 1 D, demonstrate that the Cµ-C
switch had occurred in sIgM
IgD+CD38+
GC B cells.
Hypermutated sIgM
IgD+CD38+ GC B Cells Express
Light Chains.
Since the second feature of IgD secreting
myeloma was its preferential Ig
light chain expression (11,
12), we analyzed the light chain expression of a panel of
EBV transformed clones derived from discrete B cell subsets of two tonsil samples (Table 1). Although 39 out of 83 EBV clones from sIgM+IgD+CD38+ GC founder cells and
17 out of 53 EBV clones from sIgD
CD38+ GC B cells
express
light chains, 75 out of 76 EBV clones from sIgM
IgD+CD38+ GC cells express
light chains. VH sequence analysis showed that most clones were clonally independent (see Materials and Methods). These data demonstrate that sIgM
IgD+CD38+ GC B cells display the
second feature of IgD myeloma cells: preferential expression of
light chain.
Hypermutated sIgM
IgD+CD38+ GC B Cells Display Poor
Ability to Undergo Further Isotype Switch In Vitro.
As sIgM
IgD+CD38+ GC B cells had lost a major part of the Sµ region after Cµ-C
switch (Fig. 1 D), it was anticipated that
they would not undergo further isotype switch. Indeed,
sIgM
IgD+CD38+ GC B cells differentiated mainly into
IgD-secreting cells after 10 d of culture on CD40 transfected L cells with IL-2 and IL-10 (Fig. 2), a culture condition under which human naive B cells undergo isotype
switch to IgG and differentiate into IgG-secreting cells (38,
39). Thus, sIgM
IgD+CD38+ GC B cells display two common features with IgD secreting myeloma cells, i.e., the
Cµ-C
isotype switch and the preferential
light chain
expression, and they could differentiate into normal IgD-secreting cells in vitro.

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Fig. 2.
Differentiation of
IgM IgD+CD38+ B cells into IgD+
plasma cells in vitro. IgD, IgG, IgA,
and IgM secretion (A) and IgD immunostaining (B and C) of IgM
IgD+CD38+ B cells cultured for 2 wk in the presence of IL-10, IL-2,
and CD40 ligand transfected murine
fibroblasts. Ig contents were measured by ELISA and results are given
in µg/ml. Original magnifications of
microphotographs are 100 (B) and
1,000 (C).
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IgD-secreting Plasma Cells Represent a Major Population of
Plasma Cells in Human Tonsils.
We have previously demonstrated that hypermutated sIgM
IgD+CD38+ GC B
cells could not give rise to circulating memory cells in blood (36). However, IgD+ plasma cells were previously
identified in human tonsils by immunohistology (40, 41),
suggesting that sIgM
IgD+CD38+ GC B cells may differentiate into IgD-secreting plasma cells. An immunohistochemistry analysis performed on four randomly selected tonsillar samples with anti-IgD showed that IgD+ plasma
cells represent an average of 16% (range 6-20%) of total CD382+ plasma cells. They were found either within GCs
(Fig. 3 A) or within mucosal epithelium (Fig. 3, B and C),
as reported earlier for IgA+ plasma cells (42). To further
characterize IgD+ plasma cells, tonsillar plasma cells were
isolated by cell sorting according to their CD382+CD20
phenotype as previously described. In agreement with the
immunohistological analyses on tissue sections, plasma cells
isolated from five tonsil samples contains 17% (3-48%)
IgD+ and only 2-5% IgM+ cells (Fig. 3 D). Double anti-IgD and anti-IgM staining showed that plasma cells contain
either IgM or IgD, but never both isotypes (Fig. 3 E). Furthermore, IgG, IgA, and IgD were the major Ig isotypes secreted by these plasma cells during overnight cultures
(Table 2). The question is do IgD-secreting plasma cells in
tonsils indeed represent the progeny of IgM
IgD+ GC B
cells and the normal counterpart of IgD-secreting myeloma cells?

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Fig. 3.
Ig heavy chain expression by tonsillar plasma cells. Immunoenzymatic staining of tonsillar tissue sections (A, B, and C) or plasma cell cytospin
preparations (D and E). (A) Double red anti-IgD and blue anti-CD38 staining showing purple IgD+ plasma cell within a GC and red IgD+ follicular
mantle (FM) B cells. Original magnification: 400. (B and C) Double red anti-IgD and blue anti-Ki67 staining showing red IgD2+ plasma cells under the
tonsillar epithelium (EP). Original magnifications are 100 (B) and 400 (C). (D) Single red anti-IgD staining showing IgD+ plasma cells among purified
tonsillar plasma cells. Original magnification: 1,000. (E): Double blue anti-IgD and red anti-IgM staining showing exclusive expression by plasma cells of
each isotype. Original magnification: 1,000.
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IgD-secreting Plasma Cells Have Undergone Extensive Somatic
Mutation and Display Striking Clonal Relatedness.
The first important feature of sIgM
IgD+CD38+ GC B cells is their
extensively mutated IgV genes. Thus, VH5-
, VH5-µ, and
VH5-
transcripts were amplified from 10,000 plasma cells
of each of the three tonsil samples. The PCR products
were then sequenced. Consistent with the surface or cytoplasmic Ig expression of different cell subsets, VH5-
transcripts could only be amplified from IgD+CD38
naive B
cells and CD382+CD20
plasma cells, but not from IgD
CD38+ GC B cells and IgD
CD38
memory B cells (data
not shown). The 19 VH5-µ sequences had an average of
four mutations per sequence and four sequences displayed clonal relatedness (Table 3). The 62 VH5-
sequences had
an average of 10 mutations/sequence and three sequences
displayed clonal relatedness. These mutation frequencies
are similar to that of the VH5-µ and VH5-
transcripts of
GC B cells and memory B cells previously described (35,
43), indicating the GC origin of these plasma cells. The 52 VH5-
transcripts had accumulated an average of 21 mutations/sequence. 43 out of 52 sequences displayed clonal relatedness. (Clonal relatedness means that more than two sequences within the same tonsil sample are derived from
one cell by somatic mutation.) The VH5-
sequences of
plasma cells from one representative tonsil (Fig. 4 A) display three features previously observed in the VH5-
sequences of sIgM
IgD+CD38+ GC B cells (36): (a) their
mutation frequency being two- to threefold higher than
that of µ and
transcripts, (b) replacement mutations not
being concentrated within complementarity determining regions (CDRs), and (c) a high frequency of clonal relatedness. The genealogical trees deduced from the clonally related sequences (Fig. 4, B and C) indicate that somatic mutations have been accumulated during the extensive clonal
expansion of IgD plasma cell precursors, the sIgM
IgD+
CD38+ GC B cells. Since an average of 16% of tonsillar
plasma cells secrete IgD and only ~2-5% of human B cells
use VH5 genes, each tonsil sample may contain only 30-80
IgD-VH5-expressing plasma cells. These cells may represent the descendents of a single GC and may explain the
observed restricted V gene usage.

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Fig. 4.
Analysis of Ig heavy chain variable region genes of IgD+ plasma cells. (A) Schematic representation of VH5-C sequences from plasma cells
of one representative tonsil. Leader, CDR1, CDR2, and D/J regions are boxed. Sequence names are listed on the left (Seq.) and boxed names represent
clonally related sequences. Total mutation numbers are listed on the right. Germline sequences of D regions were not assigned. Mutations are represented
as replacement (circle with stem) and silent (stem). (B) Nucleotide sequences from the largest clone. The upper string gives the nucleotides of the VH5-1
germline sequence, the D region from the less mutated sequence (192), and the JH6 germline sequence. CDR1, CDR2, and CDR3 are boxed. Dashes,
matches to the first string. Mutated bases are indicated. (C) Genealogical tree from this clone. Each analyzed sequence is indicated by its circled name,
whereas common predicted intermediates are boxed. The number of mutations between sequences are indicated on the line joining them, and total sequence mutations are within brackets.
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IgD-secreting Plasma Cells Have Undergone Cµ-C
Switch.
To determine whether IgD-secreting plasma cells have undergone Cµ-C
switch, CD382+ total tonsillar plasma cells
were separated into intracellular IgD+ and intracellular
IgD
subsets by a two-color immunofluorescence cell sorter.
Sµ-
/
junctions were amplified from DNA of 10,000 cells
of each subset. Fig. 5 A shows that Sµ-
junction can be
amplified from IgD+ plasma cells of three tonsil samples,
but not from IgD
plasma cells. Fig. 5 B shows the sequences of three examples of Sµ-
/
junctions from IgD+
plasma cells. The corresponding break points are depicted
in Fig. 5 C.

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Fig. 5.
Cµ-C switch recombinations in IgD+ plasma cells of three
tonsil samples. (A): PCR amplification of Sµ- / recombinations of genomic DNA from IgD+ (lanes D+) and IgD (lanes D ) plasma cells. (B)
DNA sequences of Sµ- / junctional regions from IgD+ plasma cells.
Upper strings, germline sequences of the Sµ region; lower strings, germline
sequences of the /µ- / intron. The central string represents the sequences of cloned PCR products. Homologous nucleotides are linked by
vertical bars. Arrowed figures, the base numbers, base No. 1 being the first
3' of the VDJ region. (C) Schematic representation of the break points.
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IgD-secreting Plasma Cells Preferentially Express
Light
Chains.
To determine the light chain expression of normal IgD-secreting plasma cells, double staining with anti-IgD (blue) and anti-Ig
(red) as well as anti-IgD (blue) and
anti-Ig
(red) were performed on serial sections of three
tonsil samples. Although few IgD+ plasma cells expressed
Ig
light chain (most cells are single stained blue; Fig. 6, A
and B), >90% were shown to express Ig
light chain (double stained purple; Fig. 6, C and D).

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Fig. 6.
Ig light chain expression by tonsillar plasma cells. (A and B) Double blue anti-IgD and red anti-Ig staining showing many single stained blue
Ig IgD+ plasma cells and a few double stained purple Ig +IgD+ plasma cells. (C and D) Double blue anti-IgD and red anti-Ig staining on a serial section, showing many double stained Ig +IgD+ plasma cells in purple and a few Ig IgD+ plasma cells single stained in blue. Original magnifications: 100 (A and C) and 400 (B and D).
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IgD-secreting Myeloma Cells Have Undergone Somatic Mutation in Their Ig Variable Region Genes.
A previous study by
Kiyoi et al. showed that four cases of IgD-secreting myeloma contained somatically mutated IgV genes (32). We
analyzed the VH sequences of two well-characterized human IgD-secreting myeloma. VH sequence from patient 1 (15) displays 40 nucleotide differences from the three closest germline sequences (VH3-33/DP50, VH3-30.3/DP46,
and VH3-30.5/DP49; Fig. 7). VH sequence from patient 2 (14) displays 85 nucleotide differences from the closest germline sequence (VH1-69/DP10; Fig 7). Exhaustive analyses
and searches through different Ig databases from two different laboratories failed to identify other closest germline sequences. These data further suggest the GC origin of IgD-secreting myeloma cells.

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Fig. 7.
Analysis of Ig heavy chain variable region genes of IgD myeloma cells. Schematic representation of VH sequences from IgD myeloma cells of two samples. Patient 1 (15) shows 40 nucleotide substitutions, and patient 2 (14) shows 85 nucleotide differences plus one 9-bp
deletion (between brackets). Mutations are represented as replacement (circle
with stem) and silent (stem).
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Discussion |
IgD was first discovered by Rowe and Fahey as a unique
myeloma protein (33). IgD-secreting myeloma cells were
found to display two unusual features that could not fit into
the current model of antigen-driven B cell development
(44). First, while the membrane IgD on B cells shows a
predominance of the Ig
type, more than two thirds of all
known IgD myeloma proteins were shown to belong to
the lambda type (11, 12). Second, IgD-secreting myeloma
cells had undergone a unusual Cµ-C
switch. This raises the question of whether the features of IgD-secreting myeloma cells represent only a malignant event or reflect a
normal B cell maturation pathway.
Here we demonstrate that hypermutated sIgM
IgD+
CD38+ GC B cells, which represent 2-5% of normal tonsillar B cells (36), may represent the precursors of normal
and malignant IgD-secreting plasma cells. First, significant
numbers of normal IgD-secreting plasma cells were identified in human tonsils (40, 41). In particular, both sIgM
IgD+CD38+ B cells and IgD+ plasma cells could be found
within the same GCs. Second, CD40-activated sIgM
IgD+CD38+ GC B cells were shown to directly differentiate into IgD-secreting cells when cultured with IL-2 and
IL-10. Third, both sIgM
IgD+CD38+ GC B cells and
normal IgD-secreting plasma cells displayed a similar somatic
hypermutation rate. Fourth, both sIgM
IgD+ CD38+ GC
B cells and normal IgD-secreting plasma cells had been
originated from a few cells that had undergone impressive
clonal expansion and somatic mutation within GCs. Fifth,
like IgD-secreting myeloma cells, both cell types preferentially expressed the Ig
light chain and had undergone
Cµ-C
switch.
Previous studies have shown that IgVH and IgVL genes
of IgG, IgA, and IgD myeloma contain extensive somatic
mutation (23). These findings strongly suggest that
IgD-secreting myeloma cells are not derived from the transformation of stem cells (19) or pre-B cells (20), but from GC
B cells or post-GC memory B cells. Our previous study
demonstrated sIgM
IgD+CD38+ GC B cells did not mature into blood memory B cells. This, together with the
present finding that sIgM
IgD+CD38+ GC B cells differentiate into IgD-secreting plasma cells, suggests that IgD-secreting myeloma cells are derived from B cells at the GC
B cell stage, but not at the post-GC memory B stage.
The identification of sIgM
IgD+CD38+ GC B cells and
IgD+ plasma cells defines a novel GC B cell development
pathway in human, characterized by (a) a nonclassical isotype switch from Cµ to C
, (b) a light chain shift from
to
, (c) the impressive oligoclonal expansion and somatic hypermutation, and (d) generation of IgD-secreting plasma
cells. The molecular triggers and functional implications of
the Cµ-C
switch, the
-
light chain shift, and the enormous clonal expansion and somatic mutation in sIgM
IgD+
CD38+ GC B cells are currently unknown. The
-
light
chain shift may result from a secondary light chain rearrangement (receptor editing; references 45, 46) in GCs, as
recently demonstrated in mouse GC B cells (47).
The identification of a significant number of IgD+
plasma cells in human tonsils also challenges the previous
hypothesis that IgD functions simply as an antigen receptor,
but not as a secreted antibody. This, together with recent
identification of IgD+ memory B cells in human bone marrow (51) and virus-specific IgD-secreting plasma cells in
the spleen of mice (52), strongly suggests that IgD plays an
important role in certain types of humoral immune responses.
Address correspondence to Yong-Jun Liu, DNAX Research Institute, 901 California Ave., Palo Alto, CA
94304-1104. Phone: 650-852-9196; Fax: 650-496-1200; E-mail: yliu{at}dnax.org
We thank J.-L. Preud'homme and E. Levievre (CNRS 1172, Poitiers, France) for helpful standardization of
the IgD ELISA assay, M.-P. Lefrance (University Montpelier II, Montpelier, France) for help in mutation
rate analyses of myeloma cells, E. Bates (Schering-Plough, Dardilly, France) for critical reading of the manuscript, and S. Bourdarel and M. Vatan (Schering-Plough, Dardilly, France) for editorial assistance. This paper
is dedicated to Dr. J. Chiller, the late President of DNAX Research Institute, Palo Alto, CA.
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