By
From the Institute for Genetics, University of Cologne, 50931 Cologne, Germany
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Abstract |
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Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report
here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells.
IgM+IgD+CD27+ B cells resemble class-switched and IgM-only memory cells in terms of cell
phenotype, and comprise ~15% of PB B lymphocytes in healthy adults. Moreover, a very
small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells.
Overall, the B cell pool in the PB of adults consists of ~40% mutated memory B cells and 60%
unmutated, naive IgD+CD27 B cells (including CD5+ B cells). In the somatically mutated B
cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged V
genes. This might be due to an intrinsically lower mutation rate in
light chain
genes compared with heavy chain genes and/or result from
light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of
the CD27 cell surface antigen which therefore may represent a general marker for memory B
cells in humans.
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Introduction |
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In T cell-dependent immune responses, naive B cells are
recruited into the germinal centers (GC)1 of peripheral
lymphoid organs after antigen activation. Within these
structures, antibody mutants are generated through the
process of somatic hypermutation. Eventually, high affinity
B cells are selected either into the plasma cell or the memory B cell pool (1). In the mouse, memory B cells have
mostly switched from the initial expression of IgM to that
of other Ig classes. Therefore, it came as a surprise that in
humans, substantial numbers of IgM-expressing memory B
cells seem to occur along with the "classical" class-switched memory B cells (2, 3). We identified a population of somatically mutated IgM-bearing B cells in the peripheral
blood (PB), namely IgM+IgD (IgM-only) cells (4), that
phenotypically, and presumably also functionally (5), resemble class-switched cells. In the PB, IgM-only and class-switched cells each comprise 10-15% of all B lymphocytes.
IgM-only cells also occur at high numbers in the various
lymphoid organs (see reference 4), notably in the splenic
marginal zone (6, 7).
Despite the extensive characterization of human B cell subsets at the level of rearranged V genes in recent years, no concordant picture arose as to whether somatically mutated B cells also accumulate in the IgD-expressing compartment. Several studies on tonsillar IgM+IgD+ B cells (8) as well as IgD-expressing PB B cells (11, 12) have indicated that somatically mutated IgD memory B cells occur, if at all, at a low frequency. This implies that in humans, as in the mouse, IgD represents a marker for naive B cells. However, the recent work of Paramithiotis and Cooper (13) challenges this view: based on the findings of (a) mutated µ-transcripts in cDNA libraries generated from mature bone marrow B cells and (b) a high number of mature IgM+IgD+ cells in the marrow, they concluded that IgD-bearing memory B cells home to this primary lymphoid organ. In the study of Paramithiotis and Cooper, IgD-expressing B cells were not selectively analyzed, and because somatically mutated B cells express higher levels of Ig mRNA than naive B cells (4), it remains uncertain which fraction of the mutated µ-transcripts was indeed derived from the presumed IgM+IgD+ memory B cells in the bone marrow, and not from IgM-only memory cells or (contaminating) IgM plasma cells.
A special case represents tonsillar IgD+IgM GC B cells
that have deleted the cµ gene and harbor an exceptionally
high load of somatic mutations (12). These cells probably
differentiate into somatically mutated IgD-only plasma cells
homing to the tonsillar subepithelium (14), but descendents
of those cells have not been observed in the PB (12).
Maurer et al. (15) and Agematsu et al. (16) recently described two subsets of IgD+ B cells in human tonsils and
PB, respectively, that can be distinguished by the expression of the CD27 cell surface antigen, a member of the
TNF receptor family. This antigen is expressed on essentially all IgD, i.e., class-switched and IgM-only, PB B
cells. In vitro, IgD+CD27+ but not IgD+CD27
cells respond to activation stimuli in the same way as IgD
CD27+
B cells. Here we characterize this newly described IgD+
CD27+ B cell subset with respect to both the level of V
gene mutation and cell phenotype, and compare those cells
with class-switched and IgM-only memory B cells as well
as with IgD+CD27
cells.
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Materials and Methods |
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Cell Separation and Flow Cytometry.
Buffy coats of healthy adult donors were obtained from the blood bank of the Institut für Transfusionsmedizin of the Cologne University Hospital. PBMC were isolated by Ficoll-Isopaque density centrifugation, and CD19+ B cells were enriched to >98% by magnetic cell separation using the MiniMACS® system (Miltenyi Biotec, Bergisch Gladbach, Germany) as described (11). For the analysis of cell surface antigens, the B cell-enriched cell suspension was incubated with biotinylated goat anti-human IgD (GaH-IgD; Southern Biotechnology Associates, Inc., Birmingham, AL), with FITC- or PE-conjugated anti-CD27 (PharMingen, San Diego, CA) and with either anti-CD23-FITC, anti-CD5-FITC (both from Becton Dickinson, Mountain View, CA), or GaH-IgM-PE (Sigma, München, Germany) for 10 min on ice. After washing with PBS/ 0.5% BSA, GaH-IgD was developed with Streptavidin-CyChrome (PharMingen). The cell suspensions were analyzed on a FACScan® (Becton Dickinson). For the isolation of single IgD+CD27+ and IgD+CD27Single-cell PCR.
Single cells in PCR buffer (see above) were incubated with 0.5 mg/ml proteinase K for 1 h at 50°C. The enzyme was inactivated by denaturation at 95°C (10 min). For the first round of amplification, a primer mix consisting of six VH and four V ![]() |
Results |
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Functional studies performed on isolated IgDCD27+, IgD+CD27+, and
IgD+CD27
PB B cells revealed that IgD+CD27+ B cells
respond in a similar way to activating stimuli as IgD
B
cells (16). To examine whether IgD+CD27+ cells share
phenotypic similarities with "classical" class-switched and
IgM-only memory cells, we stained this subset for cell surface markers which are differentially expressed on memory
B cells on the one hand and naive B cells on the other,
namely CD23 and CD5 (see reference 4). B cell-enriched
fractions from several donors were stained for IgD, CD27,
and the respective antibodies against CD23 and CD5 and
analyzed on a FACScan® (Fig. 1). Like class-switched and
IgM-only cells, IgD+CD27+ B cells were predominantly
CD23
and CD5
. The IgD+CD27
fraction, on the
other hand, contained both CD23+ and CD5+ cells (Fig.
1). Staining for IgM expression revealed that the majority
of IgD+CD27+ cells
in contrast to IgD+CD27
cells
express high levels of surface IgM (Fig. 1). In this respect, IgD+CD27+ cells resemble IgM-only B cells (4). To confirm the observation of Agematsu et al. that IgD
B lymphocytes express CD27 (16), IgG+, IgA+, and IgM-only B
cells were selectively analyzed for CD27 expression. As expected, cells of those subsets were CD27+ (data not
shown).
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To determine the percentages of the IgDCD27+,
IgD+CD27+, and IgD+CD27
subsets among PB B lymphocytes, B cell-enriched fractions from the PB of eight
healthy adults were stained for IgD and CD27 and analyzed
flow-cytometrically (not shown). The PB of the donors showed considerable variation in the frequencies of the
PB B cell subsets, with 29-65% IgD+CD27
, 6.5-22%
IgD+CD27+, and 13-43% IgD
CD27+ B cells. In accord
with previous results (16), the B cell pool in the PB comprises on average 60% IgD+CD27
, 15% IgD+CD27+, and
25% IgD
CD27+ B cells. The latter population can be further subdivided in on average 15% IgG+&IgA+ and 10%
IgM-only cells.
IgD+CD27+ and IgD+CD27
PB B cells were analyzed for the level of somatic mutation
in their rearranged VH genes. After enrichment of CD19+
B cells derived from the PB of three healthy adults by magnetic cell separation, the corresponding cell suspensions
were stained for IgD and CD27 (using FITC-conjugated
liposomes; see Materials and Methods), and single
IgD+CD27+ and IgD+CD27
cells were isolated flow-cytometrically. The use of FITC-conjugated liposomes,
which stain 100-1,000 times brighter than FITC or PE, was necessary to achieve a good separation of the CD27+
and CD27
populations on the FACS® 440. Sorting gates
set for the isolation of single cells of the respective subsets
are indicated in Fig. 2. While IgD+CD27+ cells were isolated from all three donors, IgD+CD27
cells were sorted
only from donors 1 and 2. Since the vast majority of both
IgD+CD27+ and IgD+CD27
B cells coexpress IgM (see
Fig. 1), these populations are designated below as
IgM+IgD+CD27+ and IgM+IgD+CD27
B cells, respectively.
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Rearranged VH genes were amplified by PCR from the genomic DNA of the cells using a seminested approach. Negative controls consisted of reaction mixtures without cells and were always negative. From the 3 donors, a total of 156 cells were analyzed. From 69 cells a potentially functional rearrangement, and from 6 cells only a nonfunctional rearrangement, were obtained. 12 cells gave rise to 2 V gene rearrangements; in 9 cases a nonfunctional VHDHJH joint was amplified in addition to a potentially functional rearrangement; and in the remaining cases, 2 potentially functional joints were obtained. The amplification of two potentially functional rearrangements from one sample could either be due to sorting of two cells into one reaction tube or could indeed reflect the presence of two functional VH gene rearrangements in a B cell, as has been described for cases of B cell chronic lymphocytic leukemia (22). However, it is also possible that in those cells one of the potentially functional rearrangements was in reality not functional. All sequences represented unique VHDHJH joints (not shown). The results of the sequence analysis are listed in Table 1.
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Whereas all but 1 of the 32 rearranged VH genes amplified from the IgM+IgD+CD27 cells were unmutated, 63 of 67 VH genes analyzed from the IgM+IgD+CD27+ population showed somatic mutations (1-30-bp differences
compared with the most homologous germ-line genes;
Table 1). In 11 of the rearrangements of the IgM+
IgD+CD27+ fraction, deletions/insertions of variable sizes
were identified in addition to point mutations (Table 1).
This is in accord with recent findings of a considerable frequency of deletions and/or insertions in B cells undergoing
somatic mutation (20, 23). The average somatic mutation
frequencies of the IgM+IgD+CD27+ B cells (considering
only nucleotide exchange mutations) were determined to
be 3.7% for donor 1, 5.0% for donor 2, and 5.9% for donor
3 (see Table 3). These results demonstrate that in addition
to IgM-only B cells, IgM+IgD+CD27+ cells represent a
further population of IgM-bearing B cells in the PB that
express somatically mutated V genes. Furthermore, this
analysis shows that IgM+IgD+ B cells which carry unmutated V genes are CD27
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Since replacement mutations are usually counterselected within the FRs of antibody V region genes to preserve the structure of the V domain, antigen-selected B cells show on average a replacement/silent (R/S) mutation ratio between 1.0 and 1.5, i.e., considerably smaller than the value expected assuming random mutagenesis (~3.0; reference 24). For the IgM+IgD+CD27+ B cells, an R/S value of the mutations within the FRs of 1.5 was determined (not shown), which is in the same range as that typical for class-switched and IgM-only memory B cells (24).
A Minute Fraction of PB B Cells Consists of IgMIn most samples of B cell-enriched cell suspensions stained for IgM and IgD, an IgD+IgM population
could be recognized that comprised usually <1% of PB B
lymphocytes. These cells were found to be CD27+ (Fig. 3).
To determine whether such cells harbor somatically mutated V genes, single IgM
IgD+ cells were flow-cytometrically isolated from the B cell-enriched cell suspension of
donor 3 stained against IgD and IgM (not shown). IgD-only cells comprised <0.5% of PB B lymphocytes in this
case. Rearranged V genes were amplified from the genomic DNA using VH leader primers in a seminested PCR
strategy as described above. 34 cells were analyzed. From
13 cells, 1 VH gene rearrangement each was amplified. The
sequences of all amplificates were unique (not shown).
Four rearrangements were unmutated. The remaining nine
VHDHJH joints carried a high load of somatic mutations
ranging from 15 to 59 bp differences to their respective VH
germline genes (Table 2). The high load of somatic mutation within the rearranged V region genes of IgD-only B
cells might also explain why the PCR efficiency for these
cells was relatively low (13 of 34 cells positive): mutations
at the primer binding sites may have often resulted in failure of successful amplification. Six of these sequences were
potentially functional, one represented an out-of-frame rearrangement, and in two instances the functionality of the
original rearrangement before the accumulation of mutations was uncertain (Table 2). Three of the mutated rearrangements showed deletions and/or insertions.
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The unmutated sequences likely stemmed from contaminating IgM+IgD+CD27 B cells, which represent a much
larger cellular compartment than IgD-only cells. The mutated sequences show an exceptionally high mutational load
(average 15.2%) which has previously been described only
for V regions expressed by IgD-only GC B cells (~12%
mutation; reference 12) and IgD-only tonsillar plasma cells
(14). Therefore, it appears that the IgM
IgD+ B cells analyzed here represent the PB descendents of IgD-only GC B
cells. Since IgM
IgD+CD27+ cells were included in the
sorter gate set for the isolation of IgD+CD27+ cells, it is
possible that some of the (highly mutated) sequences in the
analysis of those cells (Table 1) were indeed derived from
IgD-only B cells. The low average R/S value for mutations within the FRs of the six potentially functional VH region
genes of the IgD-only cells (1.4) indicates selection of these
cells for antigen receptor expression.
The average VH gene mutation frequency of IgM+IgD+CD27+ B cells was determined to be
~5%. In our previous work, we focused on rearranged V
genes to determine mutation frequencies of PB B cell subsets (4, 11, 18). To reliably compare the average V gene
mutation frequency of the IgM+IgD+CD27+ fraction with
that of the other somatically mutated, IgM-expressing subset (IgM-only cells), we isolated single IgM-only B cells
from donor 3 and determined the level of somatic mutation
in VH regions. 32 IgM-only cells were analyzed. From 16 cells, 1 potentially functional rearrangement per cell was
amplified, and from 1 cell, both a nonproductive and a
productive VHDHJH joint (not shown). One potentially
functional VH gene rearrangement showed a 3-bp deletion
in CDRII. All sequences showed unique VHDHJH joints
(not shown). All but one of the rearrangements were somatically mutated (5-21-nucleotide differences), yielding
an average mutation frequency of 5.8% (Table 3). Thus,
the VH gene mutation frequency determined for IgM-only
cells is in the same range as that of the IgM+IgD+CD27+
cells analyzed from the same donor (5.9%; Table 3).
To determine whether the V genes of
IgM+IgD+CD27+ and IgM-only B cells also harbor a similar load of somatic mutations, rearranged V
genes were
amplified from the genomic DNA of IgD+CD27+ B cells
in which mutated VH regions had already been identified. From the 3 donors, a total of 44 IgD+CD27+ B cells, from
which VH gene rearrangements had been obtained, were
analyzed for V
rearrangements. From 20 cells, 1 rearrangement was obtained per cell; 3 cells gave rise to 2, 1 cell to 3 V
J
joints (Table 4). From 12 cells only a potentially functional, from 9 cells 1 or 2 nonfunctional, and from 3 cells
both a productive and a nonproductive rearrangement
were amplified (Table 4).
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All nonproductive V rearrangements were found to be
unmutated. This is most likely due to the fact that in
-
(and some
-) expressing B cells,
loci harboring nonproductive V
rearrangements are usually inactivated by a deletion of both the C
gene and the
-enhancers (25). V
J
joints are often retained on the chromosome, and such V
J
joints appear not to be subject to somatic mutation (26,
27). Therefore, it seems reasonable to consider only potentially functional V
regions for the determination of the average V
gene mutation frequency in IgM+IgD+CD27+
cells.
14 of the 15 cells that carried a potentially functional V
rearrangement showed somatic mutations (1-11-bp differences; Table 4) in the respective V
J
joints. The average mutation frequency was 2.0% (Table 4). The corresponding VH gene mutation frequency of the cells in which a
mutated V
region could be identified amounts to 6.1%
(see Table 4). Thus, IgM+IgD+CD27+ cells show a level of
somatic mutation in their V
regions that is in the same
range as that seen in IgM-only cells (2%; reference 4), and
in both populations of B cells, the V
regions are considerably less mutated than the VH region genes.
To further confirm that the lower level of somatic mutations in versus heavy chain genes is a general feature of
human B cells, we also amplified VH and V
region genes
from single class-switched,
-expressing B cells of a fourth
adult donor. Indeed, the average mutation frequency of 18 VH region genes amounted to 6.1%, whereas the average
mutation frequency of 8 V
region genes was twofold
lower, namely 3.0% (not shown).
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Discussion |
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Almost all IgM+IgD+CD27+ B cells analyzed carried somatically mutated V region genes, in contrast to IgM+
IgD+CD27 B lymphocytes (Table 1). IgM+IgD+ CD27+
cells phenotypically resemble both class-switched and
IgM-only cells in that they are CD27+, CD23
, and CD5
(Fig. 1; reference 4), and generally appear to be larger
than CD27
B cells (15, 16; our unpublished observations). Unlike IgM+IgD+CD27
cells, they express high
levels of membrane IgM (Fig. 1), similar to IgM-only cells
(4). That IgM+IgD+CD27+ cells may represent memory B
cells is further supported by the following observations. (a)
Upon stimulation in in vitro assays, both IgD
CD27+ and
IgD+CD27+ cells
in contrast to IgD+CD27
cells
are
quickly activated and secrete large amounts of Ig (15, 16).
This is consistent with one of the key features of memory B cells, as reported for splenic marginal zone and tonsillar
subepithelial memory B cells (28, 29). (b) A small fraction
of tonsillar GC B cells expresses IgD, and half of those cells
carry somatically mutated V region genes (30). These GC
B cells might be the precursors of the IgM+IgD+CD27+
PB B cells. A second potential precursor population for the
IgM+IgD+CD27+ B cells is represented by a rare type of
tonsillar GC which are populated by somatically mutated
IgD+CD70+ GC B cells (31; Küppers, R., and C. van Noesel, unpublished observations). IgM+IgD+CD27+ PB B
cells are also CD70+ (32; our unpublished observations). (c)
CD27-expressing B cells are almost absent from cord blood
(16, 33), which is devoid of memory B cells. Although it
presently cannot be ruled out that IgM+IgD+CD27+ B
lymphocytes are generated in a GC-independent pathway
(34), these observations collectively suggest that these cells,
besides class-switched and IgM-only cells, represent a third
phenotypically defined memory B cell subset in human PB.
Regarding the mutated µ-transcripts identified in cDNA
libraries generated from mature bone marrow B cells (13),
it seems likely that those transcripts were derived from both
IgM-only and IgM+IgD+CD27+ memory cells homing to
or circulating through the bone marrow.
We observed a minute fraction of IgD-only B cells in the PB, usually representing <1% of PB B cells (our unpublished observations). They are CD27+ (Fig. 3) and express V region genes with an exceptionally high load of somatic mutation (Table 2), as has been described for IgD-only GC B cells (12) and tonsillar IgD-only plasma cells (14). Based on these similarities, it seems likely that the IgD-only cells in the PB represent the descendents of IgD-only GC B cells. Thus, it appears that IgD-only GC cells can differentiate not only into tonsillar plasma cells, but also into recirculating sIgD+ cells. The fact that these highly mutated cells express surface Ig and that the average R/S mutation value for the FRs of the potentially functional VH region genes amplified from IgD-only cells (1.4) is in the range typical for antigen-selected memory B cells indicates that these cells have been selected within the GC for antigen receptor expression.
In Human Memory B Cells, VH Region Genes Harbor on Average a Two- to Threefold Higher Load of Somatic Mutations than VIn our previous V gene analyses on
B cell subsets derived from healthy adults, we repeatedly
determined somatic mutation frequencies of ~2% for IgM-only and 4% for class-switched cells (4, 11, 18). Those values were derived from analyzing rearranged V genes. The
average VH gene mutation frequency of ~5% in IgD+
CD27+ B cells (Tables 1 and 3 made us wonder whether
VH regions may in general carry a higher load of somatic
mutations than V
regions. To clarify this, we sequenced
rearranged VH genes from IgM-only cells as well as V
regions from IgD+CD27+ cells. Furthermore, from one donor VH and V
region genes were amplified from single
class-switched B cells. Indeed, within a given cell population, VH regions show a two- to threefold higher average
mutation frequency than V
regions (Tables 3 and 4. The
same conclusion can be drawn from the results of both a V
gene analysis of sporadic Burkitt's lymphomas (n = 9; V
: 1.8% average mutation frequency, VH: 3.4% [35]) and a
single cell study of rearranged VH as well as V
genes expressed by IgM-bearing PB B lymphocytes (36). The
present data also demonstrate that IgD+CD27+ B cells
show a level of somatic mutation in the same range as that
of IgM-only cells (2% for V
[Table 4, and reference 4];
~5% for VH [Table 3]).
The simplest interpretation of the lower mutation frequency of V than VH genes is that the intrinsic mutation
rate is higher in the latter. However, the lower mutation
load of V
genes could also be due to novel V
gene rearrangements in GC B cells (37, 38), which would have gone
through fewer rounds of somatic mutation than the corresponding VH gene rearrangements.
The VH gene mutation values discussed above hold true
for adults. For µ- and -transcripts derived from tonsillar
and PB memory B lymphocytes of children, we and others
previously reported mutation frequencies of 2 and 4%, respectively (9, 10). What could be the explanation for the
discrepancy between the mutation frequencies of memory
cells in children and in adults? Perhaps the hypermutation mechanism is not yet fully active in GC B lymphocytes of
children. Alternatively, memory B cells may be driven repeatedly into GC reactions where they acquire additional
somatic mutations. However, the mutation load does not
appear to increase considerably in adults with advancing
age (4, 11).
On the basis of
the present data, B cells that express unmutated V genes are
IgM+IgD+CD27 and comprise ~60% of PB B cells.
Lipsky and colleagues find a similar fraction (55%) of unmutated or slightly mutated V genes amplified from individual CD19+ PB B lymphocytes (39). IgM+IgD+CD27
B cells can be further distinguished into a large population of CD5
cells and a smaller subset of CD5+ B cells (18).
The latter are thought to belong to a separate B cell lineage
(for a review, see reference 40) and presumably do not regularly participate in T cell-dependent immune responses (41, 42). They comprise 10-20% of B cells in the adult PB. CD5-negative IgM+IgD+CD27
cells are termed naive B
cells as they represent the presumed precursors of GC B
cells in T cell-dependent immune responses. These cells
make up between 40 and 50% of PB B lymphocytes (Fig. 4).
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About 40% of PB B cells represent memory B cells (Fig. 4). There is evidence that such cells occur at similar numbers also in secondary lymphoid organs (summarized in reference 4). The large fraction of memory B cells in humans contrasts with the situation in the mouse, where the frequency of memory B cells (in old, nonintentionally immunized mice) is in the range of 5% of all peripheral B lymphocytes (43). This difference might largely be explained by the longer life span of humans, leading to the accumulation of a much larger fraction of memory B cells. IgD and/ or IgM-expressing memory B cells were described in rodents and chickens already 20 years ago (24, 44). However, these cells were not phenotypically or molecularly characterized in detail, and there is no information on the size of this memory compartment in those species. In models of T cell-dependent immune responses in the mouse, memory B cells almost exclusively express isotypes other than IgM and IgD, indicating that the memory B cell pool shows a different composition in mice and in humans. In humans, IgM-bearing memory cells seem to predominate over class-switched memory cells (4; Fig. 4): human memory B cells can be distinguished into (on average) 40% class-switched, 20% IgM-only, and 40% IgM+IgD+CD27+ cells (and probably a small population of IgD-only cells). A common characteristic of these subsets is the expression of the CD27 cell surface antigen, which thus may represent a general marker for memory B cells in humans. CD27-expressing B cells can be activated through interaction with the CD27 ligand, CD70 (47), a molecule belonging to the TNF receptor family which is found on peripheral T cells, and recently it was shown that B cells stimulated via CD27- CD70 interaction acquire a plasma cell phenotype in vitro (48). This finding suggests that memory B cells, activated by antigen in the context of T-B cell interaction, may quickly, as a result of an additional CD27 stimulation, differentiate into Ig-secreting cells. Although in vitro the differentiation of memory B cells into plasma cells can be achieved without CD27 stimulation (29), it is tempting to speculate that in vivo CD27-CD70 interaction represents the key signal to bias memory B cells to the plasma cell differentiation pathway.
Kindler and Zubler note that in vitro-activated IgM-only PB B cells differentiate into plasma cells, but do not change isotype even under conditions that promote switching in a large fraction of IgM+IgD+ B cells (5). This indicates that IgM-only memory B cells are committed to secrete IgM. Such behavior of IgM-only cells may be explained by an internal rearrangement between the 5' and 3' ends of the sµ regions, resulting in deletion of this region (49, 50). Thus, further switching to downstream isotypes in IgM-only (and potentially also IgM+IgD+CD27+) B cells may be abolished.
Despite the prevalence of IgM-expressing over "classical" class-switched memory B cells, the role of IgM- expressing memory cells in T cell-dependent immune responses remains elusive. Are IgM-only and IgM+IgD+ CD27+ cells as a rule generated in the course of a T cell- dependent immune response from a fraction of GC B cells that do not undergo class switching? Or are they generated specifically against particular pathogens? Do IgM+IgD+ CD27+ memory cells upon antigen stimulation secrete IgD in addition to IgM? Pentameric IgM enables efficient cross-linking of antigen and permits a strong activation of the complement system. These features are advantageous in the defense against bacteria. In this regard, it is interesting to note that the splenic marginal zone, the entry port of blood-borne antigens, is mainly populated by IgM-only memory B cells (6, 7). Perhaps these cells have been generated to quickly respond to bacteria that invade the bloodstream.
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Footnotes |
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Address correspondence to Ralf Küppers, University of Cologne, University Hospital, LFI E4 R706, Joseph-Stelzmannstr. 9, 50931 Cologne, Germany. Phone: 49-221-478-4490; Fax: 49-221-478-6383; E-mail: rkuppers{at}mac.genetik.uni-koeln.de
Received for publication 1 July 1998 and in revised form 13 August 1998.
We are most grateful to Michaela Fahrig for excellent technical assistance and Alexander Scheffold for providing FITC-conjugated liposomes. We thank Christoph Göttlinger for help with the FACS®, Julia Jesdinsky for sequencing work, and Andreas Thiel for discussions.This work was supported by the Deutsche Forschungsgemeinschaft through SFB243 and SFB502.
Abbreviations used in this paper FR, framework region; GC, germinal center(s); PB, peripheral blood; R/S, ratio of replacement to silent mutations.
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