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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 226-234
Marginal-Zone B Cells in the Human Lymph Node and Spleen Show Somatic
Hypermutations and Display Clonal Expansion
By
Anne Tierens,
Jan Delabie,
Lieve Michiels,
Peter Vandenberghe, and
Chris De Wolf-Peeters
From the Departments of Pathology and Hematology, University
Hospitals of Leuven; and the Experimental Genetics Group, Flemish
Institute for Biotechnology, Center for Human Genetics, University of
Leuven, Leuven, Belgium.
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ABSTRACT |
Splenic marginal-zone B cells, marginal-zone B cells of Peyer's
patches in the gut, and nodal marginal-zone B cells (also identified as
monocytoid B cells) share a similar morphology and immunophenotype.
These cells likely represent a distinct subset of B cells in humans and
rodents, but their precise ontogenetic relationship as well as their
origin from B cells of the germinal center is still debated. To study
this, we performed a mutation analysis of the rearranged immunoglobulin
variable genes (VH) of microdissected single nodal and
splenic marginal-zone cells. In addition, we investigated the presence
of proliferating cells and B-cell clones in the human splenic and nodal
marginal zone as well as adjacent germinal centers. This was performed
by immunohistochemical staining for the Ki-67 antigen and denaturing
gradient gel analysis of amplified immunoglobulin heavy chain genes'
complementarity determining region 3 of microdissected cell clusters. A
variable subset of nodal and splenic marginal-zone B cells showed
somatic mutations in their rearranged VH genes, indicating
that both virgin and memory B cells are present in the nodal and
splenic marginal zone. Nodal and splenic marginal-zone B cells
preferentially rearranged VH3 family genes such as DP47,
DP49, DP54, and DP58. A preferential rearrangement of the same
VH genes has been shown by others in the peripheral
CD5 IgM+ B cells. These data
suggest that the splenic and nodal marginal-zone B cells are closely
related B-cell subsets. We also showed that marginal-zone B cells may
cycle and that clones of B cells are frequently detected in the nodal
as well as the splenic marginal zone. These clones are not related to
those present in adjacent germinal centers. These data favor the
hypothesis that clonal expansion occurs in the marginal zone. Whether
the somatic hypermutation mechanism is activated during the clonal
expansion in the marginal zone and which type of immune response
triggers the clonal expansion need to be elucidated.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MARGINAL-ZONE B cells represent a
distinct subset of B cells present in the spleen and ileal Peyer's
patches where an abundant influx of antigens occurs.1 The
cells are medium sized with irregular nuclei and abundant pale
cytoplasm, and typically express IgM and CD25 but not CD5, CD10, CD23,
and usually not IgD.1,2 B lymphocytes with a similar
morphology and phenotype are observed in the lymph node and are called
monocytoid B cells or nodal marginal-zone B cells. These
IgM+, IgD B cells are inconspicuously
intermingled with the small lymphocytes of the lymphocytic
corona.2 However, in some reactive conditions such as
toxoplasmic lymphadenitis or human immunodeficiency virus infections, a
more prominent monocytoid B-cell proliferation previously known as
"immature sinus histiocytosis" may be present.2
Studies in rodents on the migration of hapten-specific B cells provided
evidence that memory B cells, generated in the immune response both to
T-cell-dependent (TD) and T-cell-independent (TI) type 1 antigens,
colonize the marginal zone of the spleen.3-6 Another
subpopulation of marginal-zone B cells does not represent memory B
cells and is involved in the immune response to TI-2 antigens. In
addition, rodent marginal-zone B cells are noncirculating and do not
cycle, indicating that clonal expansion does not occur in the rodent
marginal zone.7,8 In humans, mutation analysis of
rearranged immunoglobulin variable (VH) genes of
microdissected marginal-zone B cells in the spleen and Peyer's patches
has shown mutated VH genes in the majority of the
cells.9,10 A large population of marginal-zone B cells
therefore likely represent memory B cells. Marginal-zone memory B cells
may be related to the IgM-expressing B cells in the
peripheral blood, in which somatic hypermutation of the rearranged Ig
genes has been documented as well.11 The presence of
somatic hypermutations, which until now have only been documented to
occur in the germinal center in the course of TD immune reactions, may
indicate that human marginal-zone memory B cells are generated in such
an immune response. Indeed, there is as yet no evidence that the
somatic hypermutation mechanism is activated in the TI immune reaction
in humans. In addition, a minor population of marginal-zone B cells
display rearranged VH genes without somatic mutations and
therefore may represent the memory cells of a TI immune response or
naive B cells.
The morphology and the immunophenotype of nodal marginal-zone B cells
suggest that these cells are similar to those in the splenic marginal
zone. To test this hypothesis, we performed a mutation analysis of
rearranged VH genes of microdissected single monocytoid B
cells. For comparison, a similar analysis was performed on splenic
marginal-zone B cells.
In the human, it is not known whether nodal marginal-zone B
cells may proliferate and clonally expand similar to the
germinal-center B cells. Therefore, we investigated the presence of
B-cell clones in the nodal marginal zone using denaturing gradient gel
analysis of amplified immunoglobulin heavy chain genes'
complementarity determining region (IgH CDR3) products of
microdissected nodal marginal-zones B-cell areas and compared these
results with those obtained from adjacent germinal centers. A similar
analysis was performed on splenic marginal-zone B cells for comparison.
Cell proliferation was analyzed by the expression of the
proliferation-associated nuclear antigen Ki67.
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MATERIALS AND METHODS |
Case selection.
Four lymph nodes with the morphology characteristic of lymphadenitis
were selected for the analysis of monocytoid B cells.12,13 Cases of reactive lymphadenitis with a prominent proliferation of
monocytoid B cells have been selected. The cause of the lymphadenitis was not identified in three of the patients, whereas one showed serological evidence of toxoplasma infection. We studied spleen tissue
obtained from two patients diagnosed with idiopathic thrombocytopenia and one patient in which the spleen was removed in the course of
surgery for severe diverticulitis. Histological examination of these
spleens showed a normal morphology including a prominent marginal zone.
The patient characteristics are summarized in Table 1.
Immunostaining of frozen sections.
Five-micrometer frozen tissue sections were stained with a mixture of
anti-IgD (Dakopatts, Glostrup, Denmark) and anti-CD23 (Dakopatts)
antibodies using the Avidin-Biotin-peroxidase complex (ABC) method as previously described.12 The slides were
counterstained with hematoxylin. Splenic and nodal marginal-zone B
cells were easily recognized in these tissue sections. Indeed, the
marginal zone in the spleen is a distinct zone surrounding the white
pulp. In the lymph nodes, a prominent marginal zone surrounded the
lymphocytic corona or mantle zone and may locate along the marginal
sinus. Immunostaining for IgD and CD23 highlighted the lymphocytic
corona and the dendritic cells of follicle centers, respectively, and therefore unequivocally allowed the identification of the surrounding marginal zones.
To highlight the proliferating cells in the lymph node and spleen,
5-µm frozen tissue sections were stained with an antibody against the
proliferation-associated nuclear antigen Ki67 (mib1; Biogenex, San
Ramon, CA) using the ABC method.
Microdissection of cells.
The immunostained sections were digested with 10 mg/mL collagenase H
(Boehringer Mannheim, Mannheim, Germany) in phosphate-buffered saline
(PBS) for 1 hour at 37°C. Subsequently, nodal and splenic marginal-zone B cells were microdissected and aspirated using a
hydraulic micromanipulator (Narishige MMW-202D, Tokyo, Japan) as
previously described.13 Either single cells or cell
clusters containing a similar number of cells (±500) were isolated.
For the single-cell analysis and for the analysis of entire nodal and
splenic marginal zones and germinal centers, cells or tissue were
dropped in 10 µL of polymerase chain reaction (PCR) buffer (50 mmol/L
KCL; 10 mmol/L Tris-HCL, pH 8.4; 0.01% gelatin) containing 200 µg/mL
of proteinase K (Qiagen, Hilden, Germany). The isolated cells were
covered with 50 µL of mineral oil, and digested at 37°C for 16 hours.
PCR amplification of the rearranged IgH genes.
The rearranged IgH genes from single cells were amplified using a
seminested PCR method as previously described.13,14 In the
first round of the PCR a mixture of six framework I (FR I) VH family-specific primers and three primers complementary
to all JH genes were used. The second round of the PCR was
performed in six separate reactions with one of the six VH
FR I primers and internal JH primers.
After digestion of the cells, 30 µL of a mastermix (200 µmol/L
dNTPs, 2.5 mmol/L MgCL2, 10 nmol/L of each primer in PCR
buffer) was added and heated at 94°C for 10 minutes. While at 94°C,
1.5 U of Taq polymerase diluted in 10 µL of dH2O was
added to the reaction mixtures. The PCR conditions of the first round
consisted of 1 cycle at 95°C for 2 minutes, 59°C for 4 minutes, and
72°C for 80 seconds followed by 34 cycles at 95°C for 90 seconds,
59°C for 30 seconds, and 72°C for 80 seconds and 1 final cycle of
72°C for 5 minutes. In the seminested PCR reaction, which was
performed in a 50-µL volume, 2 µL of the first round
product were used as the template. In the second round of amplification
the same reagent concentrations were used except for MgCL2,
which was at 1.5 mmol/L, and for the primers, which were at 100 nmol/L.
The second PCR consisted of a total of 35 cycles using annealing
temperatures of 65°C and 61°C for VH3, VH4
and VH1, VH2, VH5, VH6
primers, respectively. The denaturing and extension temperatures as
well as the cycling times were identical to those of the first round of
PCR. All PCR reactions were performed in a Perkin Elmer 480 thermocycler (Perkin Elmer, Norwalk, CT). Positive controls consisted of the Namalwa cell DNA, whereas negative controls consisted of single
T cells from CD3-stained serial sections of our cases, as well as DNA
obtained from peripheral blood cells of healthy donors. An aliquot of
10 µL of the PCR products was size fractionated through a 1.5%
agarose gel in 1 × TBE buffer.
For the analysis of dissected, entire nodal, and splenic marginal
zones, and adjacent germinal centers, a seminested PCR method was used
to amplify the IgH CDR3 of B cells, as previously
described.15,16 In the first round of the PCR consensus
primers to the 3 end of the third framework region of VH
genes and to the 3 end of the antisense JH segment were used. The
second round of PCR was performed with the same VH primer
and an internal JH primer with a 40-bp GC clamp attached to
the 5 end of the primer. After digestion of the cells, 40 µL of a
mastermix (100 µmol/L dNTPs, 0.2 µmol/L of each primer, 2 mmol/L
MgCL2, and 1 U Taq polymerase in PCR buffer) was added. The
PCR conditions consisted of denaturing for 40 seconds at 94°C,
annealing at 55°C for 40 seconds, and extension at 72°C for 40 seconds. Thirty cycles of PCR were performed. Two microliters of the
PCR product of the first round was used as the template for the second
round in a total volume of 100 µL. With the exception of the internal
JH primer, the same reagents were used for the second round
of PCR. The PCR conditions of the second round were identical to those
of the first round. Positive controls were from the B-cell
line Namalwa, whereas negative controls consisted of peripheral blood
cell DNA.
Denaturing gradient gel electrophoresis (DGGE).
To sensitively detect small clones of B cells, IgH CDR3 PCR products
were analyzed by DGGE.15
Forty microliters of the PCR GC-clamped products were concentrated by
vacuum drying (DNA concentrator, Model 100; Savant Instruments, Holbrook, NY). The dried products were resuspended in 5 µL of loading
buffer. These PCR products were then electrophoresed through a 40% to
70% denaturing gradient at 60°C and 160 V for 4.5 hours in
TAE buffer (40 mmol/L Tris; 40 mmol/L sodium acetate;
1 mmol/L EDTA, pH 7.4) using the Biorad DGGE system (Biorad,
Hercules, CA) as previously described.15 The products were
visualized with ultraviolet light after staining with ethidium bromide.
Sequencing of the PCR products.
The PCR products obtained from the single-cell analysis were gel
purified before sequencing. The PCR products were concentrated by
vacuum drying. The dried products were resuspended in 10 µL of
loading buffer and size fractionated through a 1.5% agarose gel. The
appropriate bands were excised and the PCR products were extracted from
the gel with Qiaquick spin-columns (Qiagen) following the
manufacturer's recommendations. The comigrating bands by DGGE analysis
of IgH CDR3 PCR products in case 4 (see Fig 4) were eluted from the
polyacrylamide gel and reamplified using the seminested PCR method
previously described, except for the use of a nested consensus
JH primer without GC-clamp. These PCR products were gel
purified as outlined above, before sequencing.
An aliquot of the purified DNA was directly sequenced on both strands
using Sanger's chain-termination method and fluorescent dideoxynucleotide chain terminators.17 The sequencing
primers were identical to the primers used for the second round of the seminested PCR of the rearranged VH genes. The
JH primer used for the reverse sequencing reaction was
determined based on the sequence obtained in the forward sequencing
reaction. The products of the sequencing reaction were analyzed using
the Applied Biosystems 373A sequenator (Applied Biosystems, Foster
City, CA).
Sequence analysis.
The identification of the VH, IgH diversity genes
(DH), and IgH junction genes (JH) germline
sequences was performed by sequence comparison with the June 1997 update of V Base, which is a comprehensive database of human
immunoglobulin germline gene sequences compiled from published
sequences (V BASE sequence directory, Tomlinson I.M., MRC Centre for
Protein Engineering, Cambridge, UK). Mac Vector 5.0 sequence analysis
software (Oxford Molecular Group Inc, Campbell, CA) was used for the
sequence analysis. For the germline DH gene attribution,
the longest stretches with the highest homology were retained with a
minimum of six successive matches or seven successive matches
interrupted by one mismatch.
Somatic mutation analysis.
The probability that somatic mutations in the rearranged VH genes
resulted from antigen selection was analyzed according to Chang and
Casali.18 The probability that an excess of replacement (R)
mutations in VH complementarity determining regions (CDRs) or FRs occurred by chance only was calculated using the binomial distribution model: P = (n!/(k!(n  k)!) × qk × (1 q)n k, where n is the total number of
observed mutations, k is the number of observed R mutations in the CDRs
or the FRs, and q is the probability that an R mutation will localize
to CDRs or FRs (q = CDR rel (or FR rel) × CDR Rf [or FR Rf]).
The inherent susceptibility of R mutations of the CDRs and FRs (CDR Rf
and FR Rf, respectively) has been calculated for each of the identified
germline genes and is based on the chances of the occurrence in each
codon of an amino acid replacement given any single nucleotide change
not resulting in a termination codon.
| |
RESULTS |
Sequence and mutation analysis of rearranged IgH genes of single
marginal-zone cells.
A total of 76 nodal and splenic marginal-zone B cells were analyzed by
PCR amplification, and 31 PCR products were obtained originating from
30 cells (Tables 2 and 3).
Eight of the 31 rearranged genes (26%) likely represent unexpressed
alleles because they are out of frame or contain a termination codon.
Both nodal and splenic marginal-zone B cells preferentially rearranged
genes of the VH3 family, and to a lesser extent, of the
VH4 family. A marked overrepresentation of some
VH genes such as DP 47 (5/31), DP 49 (5/31), and DP 54 (4/31) was observed. DP 58 was used in 2 cells. No preferential
rearrangement involving any particular DH gene was observed
except for the D21-9, a member of the DXP family, which was represented
in 5 of the 31 sequences. In 16% (5/31) of the rearranged genes a
recombination of 2 DH genes occurred. In addition, a marked
predominance of JH4 and JH6 gene rearrangements (27/31) was observed.
The mutation analysis data are summarized in Tables 2 and 3. The
out-of-frame rearranged genes as well as those containing stop codons
have not been analyzed for evidence of antigen selection. The deduced
amino acid sequences of the rearranged VH genes are given
in Figs 1 and 2.
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| Fig 1.
Deduced VH protein sequences of monocytoid B
cells. Lowercase letters indicate replacement mutations with regard to
the germline sequences. These sequence data are available from the
European Molecular Biology Laboratory (EMBL) under
accession numbers AJ 227721 to 227725, AJ 227727, AJ 227728, AJ 227729, AJ 227730, AJ227745, AJ 227726, AJ 227750, AJ 227738, AJ 227741, AJ
227751, AJ 227749, respectively. FR, framework; CDR, complementarity
determining region; *, stop codon.
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| Fig 2.
Deduced VH protein sequences of splenic
marginal-zone B cells. Lowercase letters indicate replacement mutations
with regard to the germline sequences. These sequence data are
available from EMBL under accession numbers AJ 227736, AJ 227742, AJ
227740, AJ 227739, AJ 227731, AJ 227746, AJ 227743, AJ 227735, AJ
227732, AJ 227733, AJ 227737, AJ 227734, AJ 227744, AJ 227747, AJ
227748, respectively. FR, framework; CDR, complementarity determining
region; *, stop codon.
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In all but two cases, the sequences of the rearranged Ig VH
genes contained point mutations with respect to the closest germline gene sequences. It cannot be excluded that some of the so-called mutations may actually represent germline polymorphisms. However, these
are likely few because most of the human VH germline genes have been identified.19 In addition, some of the so-called
mutations could be attributed to Taq polymerase-induced errors.
However, these errors are infrequent in our experience; in our lab, Taq polymerase-induced errors occur at a frequency of 0.095/1,000 bp.
Mutation analysis of the rearranged VH genes showed a
mutation pattern that differed between the splenic marginal-zone B
cells and the nodal marginal-zone B cells with regard to the mutation frequency and the pattern of somatic mutation. In nodal marginal-zone B
cells, the mutation frequency of the rearranged VH genes
ranged from 0% to 9.5% with a mean of 2.6%. One cell showed a
germline configuration of its rearranged VH gene whereas
six cells showed one or two point mutations in their VH
genes. All but one nodal marginal-zone B cell showed a random
distribution pattern of mutations in their rearranged VH
genes.
In contrast, the marginal-zone B cells of the spleen displayed a
mutation frequency of 4.2% in their rearranged VH genes, ranging from 0% to 8.2%. The rearranged Ig VH genes of
eight splenic marginal-zone B cells showed a mutation pattern
characteristic of antigen selection. Of these, seven rearranged
VH genes showed fewer R mutations in the FRs than expected
by chance only. This is suggestive of negative antigen selection. One
rearranged VH gene displayed R mutations in the CDRs
suggestive of positive antigen selective pressure, as reflected by the
obtained P value (.03). A random pattern of somatic mutations
was observed in the rearranged VH genes of four cells. Two
cells (7-VH3 and 9-VH3 of case 7) showed zero
or two point mutations in their VH genes, respectively. Codon 52b was
missing in one sequence when compared with the closest germline gene
(4-VH3 of case 4). This sequence may likely represent a
germline polymorphism with two instead of three three ATG repeats at
codons 52 to 52b with respect to the published closest germline
sequence.
Immunohistochemistry for Ki67.
As expected, germinal-center B cells showed a prominent expression of
the proliferation-associated nuclear antigen Ki67. In addition,
clusters of B cells within the marginal zone of the lymph
node also showed a distinct nuclear staining for Ki67, indicating that
a large number of these cells are cycling (Fig
3). In contrast, the splenic marginal zone
contained only a few Ki67-expressing B cells. The absence of T cells or
histiocytes as stained on serial sections showed that the
Ki67-expressing cells were B cells.
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| Fig 3.
Ki67 staining of a lymph node. (A) The germinal center of
the secondary follicle (bottom) contains numerous Ki67-expressing
cells. A smaller but impressive number of Ki67-expressing cells are
also observed in the marginal zone (top). (B) Detail of the marginal
zone.
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DGGE analysis of IgH CDR3 products from microdissected nodal and
splenic marginal-zone B-cell clusters.
We performed a DGGE analysis of the amplified IgH CDR3 products from
microdissected clusters of splenic and Ki67-positive and -negative
nodal marginal-zone B cells, and from adjacent germinal-center B cells.
DGGE analysis showed the following two patterns according to the zone
analyzed: a smear, indicating polyclonal IgH gene rearrangements, and
distinct bands, indicating IgH gene rearrangements originating from
clones of B cells in the marginal zone. The majority of the splenic and
nodal marginal zones analyzed harbored small clones of B cells, whereas
the minority of the marginal zones consisted purely of polyclonal B
cells (Figs 4 and 5).
Interestingly, when analyzing marginal zones containing numerous
proliferating B lymphocytes, our results invariably showed the presence
of clonal IgH gene rearrangements. However, clonal IgH gene
rearrangements could also be observed when analyzing marginal zones
containing only few or no cycling cells.
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| Fig 4.
DGGE analysis of IgH CDR3 PCR products from
microdissected nodal and splenic marginal zones. Lane 1, lanes 2 and 3, lanes 4 to 6, lanes 7 to 8, lanes 9 to 10, and lane 11 represent cases
5, 6, 2, 4, 7, and 1, respectively. Two patterns are observed on the
denaturing gradient gel: a smear indicating polyclonal IgH gene
rearrangements in lanes 1, 5, 6, and 9; and one or multiple bands
indicating clonal IgH gene rearrangements in lanes 2 to 4, 7, 8, 10, and 11. Lanes 7 and 8 display comigrating bands indicating the presence
of the same B-cell clone in the respective marginal zones. Sequence
analysis of these IgH CDR3 products confirmed the presence of the same
clone.
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| Fig 5.
DGGE analysis of IgH CDR3 PCR products from
microdissected marginal zones and adjacent germinal centers. Lanes 1 to
4, lanes 5 to 10, lanes 11 to 14, and lanes 15 to 18 represent cases 4, 1, 5, and 7, respectively. The even and odd numbers represent the
analysis of the marginal zones and adjacent germinal centers,
respectively. Two patterns are observed: a smear indicating polyclonal
IgH rearrangements in lanes 6, 7, 8, 10, and 12; and a pattern with one
or multiple bands indicating clonal IgH rearrangements in lanes 1 to 5, 9, 11, and 13 to 18. The analysis of the IgH CDR3 PCR products from
Ki67-expressing marginal zones are observed in lanes 2, 4, 6, and 10.
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All but one of the microdissected germinal centers harbored clonal
B-cell proliferations (Fig 5). Interestingly, DGGE analysis of the
amplified IgH CDR3 products from microdissected germinal centers and
adjacent splenic or nodal marginal zones never showed comigrating
bands. In contrast, the IgH CDR3 products of two nodal marginal zones
obtained from the same lymph node (case 4) displayed a comigrating band
(Fig 4). The identical migration pattern of these bands on DGGE
suggested the presence of the same B-cell clone in these monocytoid
B-cell areas. Sequence analysis of these IgH CDR3 products confirmed
the presence of the same clone (not shown).
| |
DISCUSSION |
Our study shows that the marginal-zone B cells of the lymph node and
the marginal-zone B cells of the spleen predominantly rearrange
VH3 family genes, and to a lesser extent, VH4
family genes. The preferential usage of VH3 family genes is
accounted for by the frequent rearrangement of some VH3
family genes such as DP47, DP49, DP54, and DP58. These genes
represented 39% (12/31) of the productively rearranged VH
genes. Similarly, there was a slight preferential rearrangement of the
D21-9 gene, and a striking preferential rearrangement of the
JH4 and JH6 genes. No difference in
VH gene usage was noted between the splenic and nodal
marginal-zone B cells. Why these particular genes are preferentially
rearranged in nodal and splenic marginal-zone B cells is not clear. The
restricted use of a small number of VH genes by the nodal
and splenic marginal-zone B cells might reflect a case bias. However,
this does not seem likely for nodal marginal-zone B cells because the
lymph nodes selected for this study were obtained from patients
presenting with different conditions. Although two of the three spleens
studied were obtained from patients with idiopathic thrombocytopenic
purpura (ITP), it is also unlikely that case bias may have accounted
for the limited VH gene repertoire of splenic marginal-zone
B cells. Indeed, in 90% of the patients diagnosed with ITP, antibodies against various platelet membrane glycoproteins have been
shown.20 Because these antibodies are almost exclusively of
the IgG isotype, the marginal-zone B cells are not likely the effector
cells in this condition accounting for the VH gene bias.
Interestingly, a preferential usage of the same VH3
germline genes as observed in the nodal and splenic marginal-zone
B cells studied here has been shown in circulating
CD5IgM+B cells.21 DP 47 is the
most frequently rearranged VH gene segment, observed in
12.1% of the CD5 IgM-expressing B cells,
whereas other genes of the VH3 family such as DP49, DP54,
and DP58 are observed in 5.8%, 5.8%, and 4.4% of the productive
rearrangements, respectively. In addition, a preferential usage of the
D21-9 gene and the JH4 and JH6 genes is also a
striking characteristic of the CD5IgM+ B
cells in peripheral blood. Taken together, these findings indicate that
the splenic and nodal marginal-zone B cells as well as the circulating
CD5IgM+ B cells likely belong to the same
subset of B cells.
The majority of the splenic and nodal marginal-zone B cells in our
study showed somatic mutations in their rearranged VH
genes. This indicates that memory B cells constitute the majority of B
cells in the splenic and nodal marginal zone. In addition, marginal zones also harbor naive B cells. These findings are in agreement with
those previously published for the human marginal-zone B cells of the
spleen and the Peyer's patch.9,10 Interestingly, the lymph
node marginal-zone B cells showed a lower mutation frequency of their
rearranged VH genes than the splenic marginal-zone B cells.
In contrast to only 2 out of 14 marginal-zone B cells in the spleen, 7 of the 16 nodal marginal-zone B cells showed a germline or
near-germline configuration of their rearranged VH genes.
In addition, all but 1 of the rearranged VH genes of the
marginal-zone B cells in the lymph node showed a random distribution of
R mutations in the CDRs and FRs. In contrast, 8 marginal-zone B cells
of the spleen showed a mutation distribution pattern in their
rearranged VH genes indicative of an antigen selective
pressure. Thus, at least in our study, the majority of the nodal
marginal-zone B cells seem to be naive or early memory B cells, whereas
the majority of splenic marginal-zone B cells represent late memory B
cells. Interestingly, the number of mutations in the rearranged
VH genes of the marginal-zone B cells of Peyer's patch is
even higher than that of the marginal-zone B cells of the
spleen.10 These differences in the number of mutations
observed in nodal marginal-zone B cells and marginal-zone B cells of
the spleen and Peyer's patch are likely caused by differences in
antigen exposure. Alternatively, these findings may indicate that the
nodal and splenic B cells located in the marginal zone represent
different subsets of B cells. However, the latter seems less likely in
view of the similarities in morphology, immunophenotype, and
VH gene usage of these B cells in the lymph node and the
spleen. Interestingly, the majority of IgM-expressing peripheral blood
B cells, representing 10% of the circulating B cells, also
carry mutated VH genes.11,22,23 They do not
express CD10, CD38, CD77, or activation markers such as interleukin-2
receptor (CD25) or transferrin receptor (CD71), and are usually
negative for CD23, which is predominantly expressed by naive B
cells.11 These data indicate that IgM+
peripheral blood B cells also represent memory B cells. In addition to
the pattern of VH gene usage, these data are also in
agreement with the hypothesis that nodal and splenic marginal-zone B
cells and circulating IgM+ B cells belong to the same
B-cell compartment.11 Whether the memory B cells of this
compartment result from a TD or TI immune reaction or both, or whether
this may vary according to anatomic location is not known. It is clear
that mutations arise in the rearranged VH genes of B cells
generated through a TD immune reaction, but as yet there is no evidence
that the hypermutation mechanism is activated in the TI immune
reaction. However, somatic mutations may occur independent of a TD
antigen stimulation in Xenopus and sheep,24,25 although in
these species somatic mutations serve to diversify the primary antibody
repertoire.
Interestingly, it is documented here for the first time that both the
nodal marginal zone as well as the splenic marginal zone may harbor B
cell clones. This is in agreement with the previous finding of the
presence of B-cell clones in the marginal zone of Peyer's
patch.10 The presence of B-cell clones in the marginal zone
may indicate either local clonal expansion or the migration of clonally
related memory B cells, possibly from nearby germinal centers. The
latter is less likely because no identical clones could be identified
in the marginal zones or adjacent germinal centers of the lymph node
and the spleen. In addition, our data show that an important fraction
of the nodal but not the splenic marginal-zone B cells are cycling.
Taken together, these findings favor the hypothesis that clonal
expansion occurs in the marginal zone and that clones may persist
thereafter. From our findings it seems likely that some of these cells
may then enter the circulation as CD5IgM+
circulating B cells. The fact that cycling marginal-zone B cells were
frequently observed in the lymph node but only rarely in the spleen may
be explained by the different phases of the immune response in which
the cells were analyzed. Indeed, marginal-zone B cells in the spleen
are resident cells, whereas those in the lymph node are only
conspicuous when the lymph node is acutely inflamed. Whether the
somatic hypermutation mechanism is activated during clonal expansion in
the marginal zone and whether clonal expansion follows triggering
through TI antigenic stimulation is speculative and needs to be further
elucidated.
In the mouse as well as in the human B-cell repertoire, three B-cell
subsets, B-1a, B-1b, and B-2, have been recognized by their surface
markers and the location, timing, and pathway of development.26-29 Conventional B-2 cells constitute the
major population of the adult B-cell repertoire whereas B-1 cells
represent 15% to 30% of the circulating, splenic, and tonsillar
B lymphocytes. B-1 cells mainly produce naturally occurring
polyreactive IgM antibodies whereas B-2 cells produce antigen-induced
monoreactive IgG. Polyreactive B cells akin to B-1 cells may localize
in the splenic marginal zone in rodents.30 In addition, B-1
cells display a phenotype similar to marginal-zone B cells as well as a
similar range of somatic mutations in their rearranged VH
genes. However, marginal-zone B cells do not express surface CD5 as
B-1a cells. Whether marginal-zone B cells belong to the B-1b
cell subset, which is CD5 but CD5 mRNA+,
seems possible but still needs to be shown by the expression of CD5
mRNA.
In conclusion, we have provided evidence in this study that
marginal-zone B cells of the lymph node comprise virgin as well as
memory B cells, similar to marginal-zone B cells of the spleen. In
addition to similar morphological and immunophenotypical
characteristics, these data indicate that nodal and splenic
marginal-zone B cells belong to the same B-cell compartment.
Our study also shows that B-cell clones are present both in the nodal
and splenic marginal zone that are unrelated to those present in
adjacent germinal centers and that B cells may cycle in the marginal
zone. These data indicate that clonal expansion takes place in the
human marginal zone. Our combined data suggest that somatic
hypermutation occurs during clonal expansion in the marginal zone.
Alternatively, the somatic hypermutations may have been acquired in
germinal centers at distant sites before migration of the cells to the
marginal zone and the occurrence of clonal expansion.
| |
ACKNOWLEDGMENT |
We thank Monique Pattou, Suzanne Taelemans, and Erna Van Dessel for
excellent technical assistance, and Michel Pipeleers for photography.
| |
FOOTNOTES |
Submitted May 18, 1998;
accepted August 31, 1998.
Supported by grant G.0311.97 of the Fonds voor Wetenschappelijk
Onderzoek, Vlaanderen. J.D. and P.V. are postdoctoral research fellows
of the Fonds voor Wetenschappelijk Onderzoek, Vlaanderen.
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 Jan Delabie, MD, PhD, Department of
Pathology, University Hospitals of Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium.
| |
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Abstract
Introduction