By
From the Department of Microbiology and Immunology and The Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, results obtained from mice with targeted inactivations of postreplication DNA mismatch repair (MMR) genes have been interpreted to demonstrate a direct role for MMR in antibody variable (V) gene hypermutation. Here we show that mice that do not express the MMR factor Msh2 have wide-ranging defects in antigen-driven B cell responses. These include lack of progression of the germinal center (GC) reaction associated with increased intra-GC apoptosis, severely diminished antigen-specific immunoglobulin G responses, and near absence of anamnestic responses. Mice heterozygous for the Msh2 deficiency display an "intermediate" phenotype in these regards, suggesting that normal levels of Msh2 expression are critical for the B cell response. Interpretation of the impact of an MMR deficiency on the mechanism of V gene somatic hypermutation could be easily confounded by these perturbations.
Key words: mismatch repair; germinal center; B cell response; somatic hypermutation ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Postreplication mismatch repair (MMR)1 functions to identify and correct nucleotide misincorporations in the nascent DNA strand during chromosomal replication in prokaryotes and eukaryotes (1). The genes encoding the components of this system were initially identified in Escherichia coli as mutations that resulted in increased genome-wide mutation rates, and were designated Mut genes. A central component of the postreplication MMR system in E. coli is MutS, the protein involved in the recognition of mispaired nucleotides (1, 4). Homologues of MutS have been shown to function in a similar manner in Saccharomyces cerevisiae and humans (4). Both humans and mice have at least five homologues of MutS, termed Msh2-6 (4, 5). Recent interest has focused on the human homologues of bacterial MMR genes, since mutations in these genes have been associated with a large fraction of hereditary nonpolyposis colorectal cancer kindreds (2, 3). Strains of mice deficient in several of the MSH proteins have now been generated via gene targeting technology, and most display expected defects in MMR and a propensity to develop certain types of cancer. For example, mice deficient in Msh2 are predisposed to the development of a form of pre-T cell leukemia with onset at 2-3 mo of age, with older mice developing intestinal and skin neoplasias (6). Nevertheless, Msh2-deficient mice develop normally and are fertile (6, 7, 9).
Recently, several groups have presented evidence obtained from the analysis of antibody V gene somatic hypermutation in mice deficient in MMR proteins. Three of these groups have concluded that their data support the idea that MMR proteins are involved in the incorporation or fixation of mutations in V genes during this process. Cascalho et al. have proposed that the mammalian MutL homologue Pms2 is directly involved in the introduction of mutations into V genes (10), whereas Gearhart and colleagues have argued that both Msh2 (11) and Pms2 (12) alter the spectrum of mutations resulting from the action of the V gene "mutator," by preferentially repairing certain types of lesions. Rada et al. have observed that the V genes in antigen-activated B cells of Msh2-deficient mice have a reduced V gene hypermutation frequency, but an increased frequency of mutations at sites previously identified as "hot spots" in normal mice. Therefore, they conclude that hypermutation takes place in sequential Msh2-independent and -dependent phases during immune responses in normal mice (13).
It is well documented that the somatic hypermutation of
V genes is intimately associated with, and may be a prerequisite for, memory B cell genesis in mice (14). In most
of the studies on a possible role of MMR in V gene somatic
hypermutation, the potential for an MMR deficiency resulting in pleiotropic defects in the memory B cell response
(18, 19) was acknowledged but not directly or extensively
examined. However, preliminary investigation of this issue
has provided support for this idea. Rada et al. observed a
decreased IgG1 response in Msh2 /
compared with +/
mice (13). Moreover, Frey et al. observed that B cells in
the Peyer's patches of Msh2-deficient mice that are subjected to chronic antigenic stimulation and proliferation
displayed high levels of length variation at the D6Mit59
microsatellite locus (20). Instability at several such microsatellite loci is a signature of elevated chromosome-wide
mutation rate (21).
In this report, we present data demonstrating that an
Msh2 deficiency in mice indeed results in wide-ranging defects in the B cell immune response. Among these are an
attenuated progression of the germinal center (GC) reaction, dramatically reduced levels of antigen-induced IgG
isotypes, and a greatly reduced anamnestic response. Significantly, Msh2 +/ mice display an "intermediate" phenotype in these regards, suggesting that normal levels of expression of Msh2 are crucial to antigen-driven B cell
proliferation and development. Despite the previously recognized propensity of Msh2-deficient mice to develop T
cell leukemia (6, 8), T cell numbers and proliferative function appear essentially normal in young Msh2-deficient mice. In addition, stimulation of B cells from Msh2-deficient mice in vitro revealed only subtle differences in proliferation, apoptosis, or isotype class switching compared
with wild-type B cells. We discuss how an Msh2 deficiency
might result in these phenotypic outcomes, and how such
pleiotropic effects on the B cell response in vivo could confound evaluation of a potential role of the MMR system in
V gene somatic hypermutation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Msh2-deficient Mice.
The line of Msh2-deficient mice used in this study was created by targeted inactivation of exon 11 of the Msh2 gene, and has been described previously (8). Mice used in our studies were maintained by brother-sister mating and were of a mixed C57BL/6 × 129/o/a background. Homozygous knockout, heterozygous knockout, and Msh2 wild-type offspring were identified using DNA derived from ear-clip tissue, and a previously described PCR assay (9). Age-matched mice of 7-12 wk of age were used in all experiments, and littermates were used in individual experiments when possible.Immunizations and Serology.
Preparation of and immunization with (4-hydroxy-3-nitrophenyl)acetyl chicken gamma globulin (NP-CGG) were performed as described previously (22). Mice received 100 µg i.p. of antigen in alum for primary immunization, and the same amount of antigen in PBS i.p. for secondary immunization. TNP-Ficoll was injected in PBS at a dose of 50 µg i.p. per mouse. Mice were bled via the retroorbital sinus at various times after immunization, and the levels of anti-NP, anti-CGG, or anti-TNP antibodies of various isotypes were assayed in sera obtained from these samples by previously described ELISA assays (23, 25). These same assays were also used to evaluate antibody levels in supernatants obtained from in vitro stimulation of B cells, with the exception that anti-Ig reagents were used to capture secreted antibody.Immunohistochemistry and Flow Cytometry Analysis.
Spleen isolation, flash freezing, sectioning, and immunohistochemistry were all conducted essentially as described previously (23, 24). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed on spleen sections to evaluate numbers of apoptotic nuclei using the ApopTagTM kit obtained from Oncor, according to the manufacturer's instructions. Numbers of apoptotic nuclei in GCs were counted at 400× using a compound microscope. GC sizes were determined by counting the number of PNA+ cell diameters at 100× magnification in the largest GC dimension. Size categories were as follows: small, 10- 29 diameters; medium, 30-39 diameters; and large, 40 or more diameters. GC microdissection, VIsolation and In Vitro Stimulation and Analysis of T and B Cells.
In some experiments (see legends for details), total lymph node cells were used as an enriched source of T cells for in vitro analysis. In others, splenic T cells were enriched by treating total spleen cells with anti-class II (MKD6) and anti-HSA (J11d) mAbs and guinea pig complement (Rockland), followed by Percoll gradient purification of small dense lymphocytes. The enriched T cells were incubated with various concentrations of mitogenic reagents, antigen, or with 4 × 105 allogeneic, irradiated (2,000 rads) spleen cells, at 2 × 105 cells per well in 100 µl RPMI plus 10% FCS. For plate bound anti-CD3 stimulation, 96-well tissue culture plates were coated with 25 µg/ml 2C11 mAb, followed by extensive washing with sterile PBS and blocking with media containing 10% FCS. Proliferation was evaluated 45-50 h after T cell plating on anti-CD3-coated wells via a pulse of [3H]thymidine for 6-18 h, followed by cell harvesting on glass fiber filters and scintillation counting. Small dense splenic B cells were isolated from spleens via T cell depletion using a cocktail of anti-CD4 (172), anti-CD8 (31M), and anti-Thy1 (polyclonal rabbit anti-mouse; Sigma) antibodies and guinea pig complement, followed by purification of high-density cells on Percoll gradients. The resulting cells were incubated in vitro in RPMI plus 10% FCS including various concentrations of LPS (Difco), goat anti-mouse IgM F(ab')2s (Pierce) or an anti-murine CD40 mAb (FGK45; reference 29). In some experiments, recombinant murine IL-4 (PeproTech) was included in the cultures at 50 ng/ml. To evaluate proliferation, such cultures were pulsed after 45-50 h with [3H]thymidine, cells were harvested onto glass fiber filters, and 3H incorporated into DNA was evaluated by scintillation counting. To evaluate cell cycle progression and apoptosis, cells were harvested at various times after initiation of culture, fixed and permeabilized in 70% EtOH, treated with RNase (Fisher Scientific), and stained with propidium iodide (PI; Sigma) followed by flow cytometric analysis. Numbers of G1, S, G2, and apoptotic cells were evaluated using a Coulter Epics Profile II and the Elite software. Supernatants from such cultures were also harvested at various times and evaluated for levels of antibodies of various isotypes, as described above.Analysis of Microsatellite Instability.
DNA was isolated as described (30) from in vitro cultures of B cells, and instability at the D6Mit59 locus was evaluated using a PCR-based assay essentially as described previously (9). One of the primers used for this analysis was end-labeled with 32P using T4 polynucleotide kinase; the PCR reaction products were then separated on acrylamide/urea DNA sequencing gels, and the gels were exposed to x-ray film. Microsatellite primer sequences were obtained from the Whitehead Institute for Genome Research (http://www.genome.wi.mit.edu). ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our studies were initiated, as were those of others (10,
20), to test the idea that a deficiency in MMR might directly alter the V gene hypermutation process. Since hypermutation takes place predominantly in GCs in normal
mice, we used an immunohistochemical GC microdissection PCR approach to evaluate levels of V gene mutation during the immune response to NP-CGG (16, 31). We
observed a relatively normal frequency and chemical spectrum of V somatic mutation among PCR DNA clones
isolated from
+ splenic GCs at days 12 and 15 of the primary NP-CGG response in a pilot study (data not shown).
However, during the course of this study, we found that
the GC reaction in Msh2
/
mice was abnormal. The
results of an extensive analysis of this phenomenon are
summarized in Table I. In Msh2
/
mice immunized
with 100 µg of NP-CGG in alum, antigen-specific splenic
GCs were present at day 8 after immunization at a normal
frequency compared with wild-type mice, but their average size appeared somewhat smaller, and large GCs (
40
cell diameters) were absent. At 12 d after immunization of
Msh2
/
mice, the average size of antigen-specific GCs
had not increased, resulting in continued absence of the large GCs that make up a sizable percentage of all GCs observed
in +/+ mice at this time. In addition, at this later time point
the average number of antigen-specific GCs per unit area of
spleen in Msh2
/
mice had decreased twofold compared
with day 8, whereas Msh2 +/+ mice showed no significant
difference in this frequency at the two time points. Analysis
of spleens from NP-CGG-immunized Msh2 +/
mice
revealed an intermediate phenotype with respect to these
alterations, and the intermediate level of total GCs at day
12 in such mice was statistically significant compared with
the values obtained from either +/+ or
/
mice.
|
Evaluation of levels of apoptosis in the GCs of Msh2 +/+,
+/, and
/
mice 8 d after immunization via the
TUNEL assay showed significantly higher levels of apoptotic nuclei in the small and medium size antigen-specific
GCs of Msh2
/
mice compared with +/+ mice, with
+/
mice displaying intermediate levels of such nuclei
(Fig. 1). Interestingly, the variation in number of apoptotic nuclei per GC was much larger in Msh2
/
compared
with +/
and +/+ mice. The reduced number of antigen-specific GCs at day 12 in Msh2
/
mice precluded
obtaining a statistically significant comparison of levels of
apoptotic nuclei at this time point.
|
Staining of spleen sections from mice immunized 8 and
12 d earlier with NP-CGG with an IgG-specific reagent
showed a reduction in the number of IgG+ B cells in
the GCs of Msh2 /
mice relative to +/+ mice (data not shown). Moreover, the periarteriolar lymphoid sheath
(PALS)-associated antibody-forming cell (AFC) focus reaction, a predominant component of the early primary anti-
NP-CGG response (22, 32), was also substantially reduced
in Msh2
/
spleens (Fig. 2). This was observed using
both antigen- and IgG-specific staining. Again, Msh2 +/
mice displayed an intermediate phenotype with respect to
these alterations (Fig. 2). Despite these differences, no obvious abnormalities in splenic architecture, the size of other
splenic B and T cell microenvironments, size and location
of follicular dendritic cell networks, or locales of antigen-specific B cell proliferation and differentiation were observed among Msh2 +/+, +/
, and
/
mice during
these studies.
|
These observations led us to conduct detailed
analysis of the anti-NP-CGG serum antibody responses of
Msh2-deficient mice. As shown in Fig. 3 A, at early stages of
this response, antigen-specific IgM levels were only slightly
lower in Msh2 +/ and
/
mice compared with +/+
mice. However, dramatic differences in the levels of NP-specific
,
, and IgG isotypes were apparent between Msh2 +/+ and
/
mice at all times in the primary response,
with Msh2 +/
mice displaying an intermediate phenotype
in most cases (Fig. 3, A and B). Msh2 +/
mice also
showed a delayed serum IgG1 response (peaking at day 21 instead of 14), and expressed increased levels of IgG2a,
although this isotype was a minor component of the total response in all mice. Also readily apparent was the severely
diminished or reduced secondary serum antibody response in
Msh2
/
and +/
mice, respectively. In
/
mice, this
secondary response was of a magnitude not noticeably different from peak levels in the primary response in most assays.
This result was not idiosyncratic to the anti-NP response, as
the secondary response to the carrier, CGG, was also severely
blunted in
/
mice and reduced in +/
mice (Fig. 3 C).
Analysis of the serum antibody response to TNP-Ficoll, a T
cell-independent antigen, revealed analogous IgM responses in +/+ and
/
mice, but the total (kappa) anti-TNP response decayed more rapidly in Msh2
/
mice. This more
rapid decay appeared to be accounted for by a severe deficiency in the IgG3 response, particularly at late times after
immunization (Fig. 3 D). Interestingly, total serum IgG levels were found not to differ significantly in Msh2 +/+, +/
,
and
/
mice (data not shown), suggesting that long-term
homeostatic regulation of these levels is not perturbed by an
Msh2 deficiency.
|
Alteration of the T cell-
dependent B cell response in Msh2-deficient mice could
result from preexisting abnormalities in the T cell compartment, the B cell compartment, or both. Flow cytometric analysis of splenic B lymphocytes revealed no apparent abnormalities in numbers of mature and immature
subsets in Msh2-deficient mice (Fig. 4). Similar analyses of
the T cell compartment revealed no obvious differences
between Msh2 +/+ and /
mice (data not shown).
These observations are consistent with previous publications indicating that the splenic lymphoid compartment of
Msh2-deficient mice is overtly normal (6). Analysis of
B lineage cells in the bone marrow of such mice revealed
a slight decrease in the proportion of B220+ immature
IgM+, IgD
B cells in Msh2
/
compared with +/+
mice, but a more dramatic four- to fivefold reduction in
mature "recirculating" B220+, IgM+, IgD+ B cells in
/
mice. The latter result is consistent with the previous results of Rada et al. (13).
|
To assess generic T cell activation and proliferation function, in vitro stimulations of lymph node and splenic T cells
were performed. No significant differences in anti-CD3-,
Con A-, or alloantigen-induced proliferative responses were
observed among Msh2 +/+, +/, or
/
T cells (Fig.
5). In addition, KLH-primed lymph node T cells obtained
from Msh2-deficient mice proliferated in response to restimulation with KLH in vitro to an extent that did not differ reproducibly from wild-type cells (data not shown).
|
The lack of evidence for defects in T cell
function and mature peripheral B cell numbers and maturity in Msh2 mice prompted a detailed analysis of B cell responses in vitro. Small dense B cells were purified from
Msh2 +/+, +/, and
/
mice and stimulated with
anti-IgM F(ab')2 fragments, LPS, or a mitogenic anti-CD40 mAb in vitro. The cultures were then evaluated for
cell cycle progression via [3H]thymidine incorporation.
Cell cycle progression and apoptosis were also evaluated using PI-flow cytometric DNA content analysis. Msh2
/
B
cells displayed only slightly reduced proliferation that was
not statistically significant compared with +/+ B cells, except at the highest concentrations of stimulant (Fig. 6). PI cell cycle analysis revealed only minor differences in cell
cycle progression and apoptosis induced by these same
stimuli among the various types of B cells during a 4-d culture period. Msh2
/
cultures contained only slightly
elevated levels of apoptotic cells at early time points in a
few experiments (data not shown).
|
Since our serological and immunohistochemical analysis of
the B cell response of Msh2-deficient mice revealed a severe attenuation of the IgG response, induction of isotype
class switching in vitro was examined using small dense
splenic B cells. B cells were cultured with LPS, which results in low levels of switching to IgG3 and IgG2b in normal B cells, or with LPS plus IL-4, which suppresses switching to IgG3 and drives efficient switching to IgG1 and IgE isotypes in normal B cells (33). Levels of class-switched
IgG1 and IgG3-secreted antibody were then determined by
ELISA. Msh2 +/+, +/, and
/
B cells stimulated
with LPS gave rise to levels of IgG3 that did not differ
greatly, and Msh2
/
B cells produced only slightly reduced amounts of this isotype (Fig. 7). Little or no IgG1 was
produced by any of these cell types in response to LPS. In
response to stimulation with LPS plus IL-4, Msh2 +/+, +/
, and
/
B cells gave rise to similar levels of IgG1,
and little IgG3. These data argue that the regulation and efficiency of isotype class switching in vitro are not greatly
perturbed by an Msh2 deficiency in B cells.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice lacking Msh2 display a striking defect in their ability to sustain a B cell response. Moreover, mice containing
only one copy of the functional Msh2 gene (resulting in
50% of the wild-type level of Msh2 protein expression [7])
display an "intermediate" phenotype in this regard, indicating
that even a slight reduction in Msh2 activity can perturb
this response. It should be noted that the intermediate B
cell response defect displayed by Msh2 +/ mice makes
them inappropriate controls for studies on the effects of an
Msh2 deficiency in vivo. Msh2 deficiency results in an inhibition of the progression of the GC reaction, dramatically reduced levels of IgG-producing AFCs and antigen-
induced IgG serum antibody, and a blunted anamnestic
response. We have also observed that Msh2 expression is
elevated in GCs (Vora, K.A., R. Fishel, and T. Manser,
unpublished observations), consistent with the idea that this
protein plays a critical role in B cell development in this microenvironment.
Nonetheless, why an Msh2 deficiency leads to such a severe attenuation of mature B cell responses in vivo remains
a subject of speculation. Although we found that T cell
numbers and proliferative function in vitro were normal in
Msh2 /
mice, a more detailed analysis of Th function
might have exposed differences between antigen-primed
wild-type and Msh2-deficient T cells. A complicating factor in this regard is that in an environment where B cell
function is deficient, defects in levels of T cell activation or
differentiation may arise secondarily (34). Since Msh2
/
mice are predisposed to the development of pre-T cell leukemia, it is tempting to speculate that general T cell physiology may be perturbed in these mice. Our experiments
were performed in young mice with no evidence of T cell
abnormalities. In addition, Msh2 +/
mice, which do not
frequently develop T cell leukemia (8; Cranston, A., and
R. Fishel, unpublished observations), displayed obviously
altered B cell responses, and the immune response to the T
cell-independent antigen TNP-Ficoll was reduced in Msh2
/
mice. Finally, initiation of the GC reaction, a T cell-
dependent process, appeared normal in Msh2-deficient
mice. Nevertheless, given the previous conclusions of others that the GC reaction appears to require lower levels of
T cell help than does the AFC response (35, 36), and that class switching to many IgG isotypes is driven by Th cells
(33), a primary or secondary defect in the development of
an efficient Th response in Msh2-deficient mice must still
be considered. Resolution of this issue will require the
construction of mice with selective Msh2 deficiencies in
the B and T cell compartments.
In principle, an intrinsic B cell defect due to Msh2 deficiency might result from perturbations in any of the somatic DNA alteration pathways essential to normal B cell
development. Taken together, however, our data argue
against this possibility. Analysis of B lineage cells in the
bone marrow of Msh2 /
mice revealed a major deficiency in the size of the mature, IgM+, IgD+ "recirculating"
subpopulation, but immature IgM+, IgD
B cell numbers
were only slightly reduced and peripheral B cell numbers
and maturity levels appeared normal, consistent with the idea that primary B cell development is not greatly altered
due to an Msh2 deficiency. As suggested previously (18),
an increased rate of V gene hypermutation due to the absence of a "counteracting" MMR system might dramatically increase the frequency of generation of nonfunctional
antigen receptors on GC B cells, resulting in substantial increases in GC cell death rate. However, this would not easily explain the severely attenuated PALS AFC focus and T
cell-independent B cell responses we observed, since V
gene mutation does not take place at a high rate during
these responses (26, 37). Finally, the possibility that Msh2
might be involved in class switch recombination, while intriguing due to its documented role in suppression of homeologous recombination (38), is not supported by our
finding that Msh2-deficient B cells can efficiently and accurately class switch in vitro.
On the other hand, our data strongly suggest that the effects of an Msh2 deficiency on B cell function do not become manifest until the stages of antigen-driven responses
characterized by high rates of proliferation. The early stages
of the primary T cell-dependent antigen-driven response,
including IgM production and initiation of the GC reaction, were not greatly affected by an Msh2 deficiency. We
also observed a normal early IgM response to TNP-Ficoll.
In contrast, GC expansion in Msh2 /
mice appears blocked, and this alteration is associated with a substantially higher frequency of apoptotic cells in GCs. The observation that the PALS AFC focus response is severely attenuated in Msh2-deficient mice could also be explained by the
rapid clonal expansion necessary for this response (22, 32),
or by the derivation of precursors of this response from the
GC reaction (39). The reason for the deficiency in the mature IgM+, IgD+ recirculating bone marrow B cell population in Msh2
/
mice is more difficult to explain, but
might indicate a reduced peripheral B cell life span, or derivation of a subset of this population from the memory B
cell pathway.
Interestingly, we observed more variability in the number of apoptotic nuclei per GC in Msh2 /
mice compared with controls (Fig. 1). In normal mice, intra-GC B
cell apoptosis is thought to result from negative selection of
autoreactive B cells and a lack of positive selection of B
cells with low affinities for antigen (40). The increased
and greater variability in numbers of GC apoptotic nuclei
characteristic of Msh2-deficient mice is consistent with the
superimposition of another, highly stochastic process leading to intra-GC apoptosis. A generic defect in high rate B
cell proliferation due to increased genome-wide mutation rate (the "mutator" phenotype) is a reasonable candidate for
this process. The perturbations we observed in IgG expression in Msh2-deficient B cell responses could well result
secondarily from such a proliferative defect, as class switching requires cell division and usually takes place after a period of clonal expansion (22, 33).
These considerations raise the question of why we observed only subtle changes in Msh2 /
B cell proliferation, apoptosis, and class switching during our in vitro
studies. The answer may simply relate to the fact that rates
and extents of B cell proliferation approaching those characteristic of certain stages of antigen-driven B cell development are not attained by most B cells subjected to in vitro
stimulation (45). Moreover, B cells undergoing the initial
stages of apoptotic death are probably rapidly engulfed by
phagocytic cells in vivo, but may remain intact for extended periods in vitro. Under conditions of low to moderate rates of clonal expansion of Msh2-deficient B cells,
DNA lesions normally recognized by Msh2-containing
complexes may be recognized and repaired, albeit less efficiently, by other factions of the DNA repair system. However, as cell cycle time decreases, a less efficient MMR system might well become limiting for clonal expansion.
Accumulation of point mutations in genes pivotal in regulating cell viability, or a sudden block to cell cycle progression due to more severe lesions whose repair, suppression,
or detection is normally in part mediated by Msh2 (such as
double strand breaks [46] or homeologous recombination
products [38]) would likely culminate in induction of apoptotic pathways and cell death. Indeed, analysis of MSH2-deficient Peyer's patch GC B cells, a population undergoing
chronic, high rate proliferation, revealed high levels of instability at the D6Mit59 microsatellite locus (20). In contrast,
when we performed a similar analysis of this locus in Msh2
+/+, +/
, and
/
B cells stimulated for 5 d in vitro with
anti-IgM, LPS, or anti-CD40, no instability at this locus
could be detected (data not shown). Clearly, these observations warrant a more detailed analysis of Msh2-deficient B
cell cycle progression, life span, and genome stability in vivo.
Previous conclusions regarding the effect of MMR deficiencies on V gene hypermutation have been garnered largely from the analysis of the V gene products of antigen-driven B cell responses taking place in vivo (11, 20). As our data show that the T cell-dependent B cell response, during which the hypermutation process takes place, is grossly perturbed by such a deficiency, these data mandate a reevaluation of these conclusions. Memory B cell development takes place simultaneously in a single animal in many distinct lymphoid microenvironments, the most important of which are probably the GCs (40, 47, 48). Because the B cell clonal composition of a given GC is limited (31, 49), the nature of antigen selection forces and clonal proliferation will vary in different GCs. Such differences are amplified by the rather random nature of hypermutation, resulting in very different V region mutant repertoires in different GCs. In addition, levels of V gene hypermutation have been observed to vary in GCs at the same time during an immune response (50). Thus, since GCs are "independent islands of antibody V region somatic evolution" (22, 51), the impact of an MMR deficiency on the progression of the GC reaction may vary dramatically from GC to GC, depending on the variables discussed above.
These observations make it clear that sampling biases are
likely to be problematic in the analysis of the effects of
MMR deficiencies on V gene somatic hypermutation.
Such sampling biases could result from substantial GC to
GC, and even mouse to mouse, variation in the effects of
an MMR deficiency on memory B cell genesis. For example, in our analysis of V mutation in Msh2
/
mice, we
chose to microdissect only the GCs that stained strongly
with NP for PCR recovery of V
genes. This limited analysis revealed approximately normal frequencies of GC V
gene mutation, supporting the idea that Msh2 is not involved in the hypermutation process (20, 27). However,
such NP++ GCs become progressively rare in Msh2
/
compared with +/+ mice, and those we picked might
only have been representative of a small clonal subpopulation that had not succumbed to the stochastic effects of absence of Msh2, perhaps due to a lower rate of proliferation.
Indeed, if high proliferation rate predisposes a B cell clone to death in an Msh2-deficient situation, the outcome of antigen-driven clonal selection during B cell responses may often be reversed in Msh2-deficient mice compared with normal mice, to favor those antigen-stimulated clones that exit the cell cycle earliest during the response. Somewhat analogous arguments have been presented by Frey et al. (20). Such biases might be further exacerbated when the memory B cell compartment is sampled, as even in normal mice this population is derived from a minor fraction of all clones that participate in the primary GC reaction (15, 31, 52, 53). Overall, an Msh2 deficiency may result in majority representation of responding B cells that have failed to complete or activate normal steps in memory B cell genesis, including steps in clonal expansion, selection, and V gene hypermutation. For example, Kuo and Sklar (54) have observed that the mammalian homologue of the MutM gene (8-oxyguanine DNA glycosylase) is highly induced in the rapidly proliferating B cells of GCs. This enzyme is involved in the pathway leading to repair of oxidatively damaged G nucleotides in DNA (55). Since an Msh2 deficiency attenuates progression of the GC reaction, perhaps via promoting the death of cells undergoing rapid proliferation, such a deficiency could result, secondarily, in overrepresentation of GC B cells that have not induced MutM. This could lead to a preferential sampling of mutations at germline G residues, if oxidative damage to these residues was elevated during the GC reaction. Such a scenario could explain the observations of Jacobs et al. (27), Phung et al. (11), and Rada et al. (13) that V gene mutations sampled on one strand in Msh2-deficient mice are observed predominantly at positions in which a germline G or C nucleotide was present.
Following from these arguments, the differences in hypermutation frequency and pattern observed in previous studies of MMR-deficient mice (10, 20) could well have resulted from biases in sampling of the V gene products of memory B cell responses (18, 19). Thus, while these differences are intriguing, whether MMR proteins play any direct role in the generation, fixation, or repair of DNA lesions during the hypermutation process remains an unresolved question. Clearly, if such a role is to be accurately evaluated in the future, experiments will need to be conducted under conditions where the effects of MMR deficiencies or defects on other aspects of B cell differentiation are minimized.
![]() |
Footnotes |
---|
Address correspondence to Tim Manser, Department of Microbiology and Immunology, The Kimmel Cancer Institute, Jefferson Medical College, BLSB 708, 233 South 10th St., Philadelphia, PA 19107. Phone: 215-503-4543; Fax: 215-923-4153; E-mail: manser{at}lac.jci.tju.edu
Received for publication 8 October 1998.
We would like to thank Anton Rolink (Basel Institute for Immunology, Basel, Switzerland) for the anti-CD4, anti-CD8, and anti-CD40 mAbs, Clifford Snapper (Uniformed Services University of the Health Sciences, Bethesda, MD) for TNP-Ficoll, and all members of the Manser laboratory for their indirect contributions to this work. We also thank the Department of Laboratory Animal Services for care of mice, and the members of our DNA repair group for helpful discussions.
This work was supported by National Institutes of Health grants AI23739 to T. Manser, and CA56542 and CA67007 to R. Fishel. V.M. Lentz and K.A. Vora were supported by National Institutes of Health training grants AI07492 and CA09678, respectively.
Abbreviations used in this paper AFC, antibody-forming cell; GC, germinal center; HSA, heat stable antigen; MMR, mismatch repair; NP-CGG, (4-hydroxy-3-nitrophenyl)acetyl chicken gamma globulin; PALS, periarteriolar lymphoid sheath; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Modrich, P.. 1991. Mechanisms and biological effects of mismatch repair. Annu. Rev. Genet. 25: 229-253 [Medline]. |
2. | Fishel, R., and R.D. Kolodner. 1995. Identification of mismatch repair genes and their role in the development of cancer. Curr. Opin. Genet. Dev. 5: 382-395 [Medline]. |
3. | Kolodner, R.. 1996. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev. 10: 1433-1442 [Medline]. |
4. | Fishel, R., and T. Wilson. 1997. MutS homologues in mammalian cells. Curr. Opin. Genet. Dev. 7: 105-113 [Medline]. |
5. |
Fishel, R..
1998.
Mismatch repair, molecular switches and signal transduction.
Genes Dev.
12:
2096-2101
|
6. | de Wind, N., M. Dekker, A. Berns, M. Radman, and J. te Riele. 1995. Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell. 82: 321-330 [Medline]. |
7. | Reitmair, A.H., R. Schmits, A. Ewel, B. Bapat, M. Redston, A. Mitri, P. Waterhouse, H.-W. Mittrucker, A. Wakeham, B. Liu, et al . 1995. MSH2 deficient mice are viable and susceptible to lymphoid tumors. Nat. Genet. 11: 64-70 [Medline]. |
8. | Reitmair, A.H., M. Redston, J.C. Cai, T.C.Y. Chuang, M. Bjerknes, H. Cheng, K. Hay, S. Gallinger, B. Bapat, and T.W. Mak. 1996. Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice. Cancer Res. 56: 3842-3849 [Abstract]. |
9. | Cranston, A., T. Bocker, A. Reitmair, J. Palazzo, T. Wilson, T. Mak, and R. Fishel. 1997. Female embryonic lethality in mice nullizygous for both Msh2 and p53. Nat. Genet. 17: 114-118 [Medline]. |
10. |
Cascalho, M.,
J. Wong,
C. Steinberg, and
M. Wabl.
1998.
Mismatch repair co-opted by hypermutation.
Science.
279:
1207-1210
|
11. |
Phung, Q.H.,
D.B. Winter,
A. Cranston,
R.E. Tarone,
V.A. Bohr,
R. Fishel, and
P.J. Gearhart.
1998.
Increased hypermutation at G and C nucleotides in immunoglobulin genes
from mice deficient in the MSH2 mismatch repair protein.
J.
Exp. Med.
187:
1-7
|
12. |
Winter, D.B.,
Q.H. Phung,
A. Umar,
S.M. Baker,
R.E. Tarone,
K. Tanaka,
R.M. Liskay,
T.A. Kunkel,
V.A. Bohr, and
P.J. Gearhart.
1998.
Altered spectra of hypermutation in
antibodies from mice deficient for the DNA mismatch repair
protein PMS2.
Proc. Natl. Acad. Sci. USA.
95:
6953-6958
|
13. | Rada, C., M.R. Ehrenstein, M.S. Neuberger, and C. Milstein. 1998. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity. 9: 135-141 [Medline]. |
14. | Berek, C., and C. Milstein. 1987. Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96: 23-41 [Medline]. |
15. | Manser, T., L.J. Wysocki, M.N. Margolies, and M.L. Gefter. 1987. Evolution of antibody variable region structure during the immune response. Immunol. Rev. 96: 141-155 [Medline]. |
16. | Rajewsky, K., I. Forster, and A. Cumano. 1987. Evolutionary and somatic selection of the antibody repertoire in the mouse. Science. 238: 1088-1094 [Medline]. |
17. | Stenzel-Poore, M.P., U. Bruderer, and M.B. Rittenberg. 1988. The adaptive potential of the memory response: clonal recruitment and epitope recognition. Immunol. Rev. 105: 113-133 [Medline]. |
18. |
Kim, N., and
U. Storb.
1998.
The role of DNA repair in somatic hypermutation of immunoglobulin genes.
J. Exp. Med.
187:
1729-1733
|
19. |
Kelsoe, G..
1998.
V(D)J hypermutation and DNA mismatch
repair: vexed by fixation.
Proc. Natl. Acad. Sci. USA
95:
6576-6577
|
20. | Frey, S., B. Bertocci, F. Delbos, L. Quint, J.-C. Weill, and C.-A. Reynaud. 1998. Mismatch repair deficiency interferes with the accumulation of mutations in chronically stimulated B cells and not with the hypermutation process. Immunity. 9: 127-134 [Medline]. |
21. | Bhattacharyya, N.P., A. Skandalis, A. Ganesh, J. Groden, and M. Meuth. 1994. Mutator phenotypes in human colorectal carcinoma cell lines. Proc. Natl. Acad. Sci. USA. 91: 6319-6323 [Abstract]. |
22. | Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1185 [Abstract]. |
23. |
Vora, K.A.,
J.V. Ravetch, and
T. Manser.
1997.
Amplified
follicular immune complex deposition in mice lacking the Fc
receptor ![]() |
24. |
Vora, K.A.,
K. Tumas-Brundage, and
T. Manser.
1998.
A
periarteriolar lymphoid sheath-associated B cell focus response is not observed during the development of the anti-arsonate germinal center reaction.
J. Immunol.
160:
728-733
|
25. | Fish, S., E. Zenowich, M. Fleming, and T. Manser. 1989. Molecular analysis of original antigenic sin. I. Somatic mutation, clonal selection, and isotype switching during a memory B cell response. J. Exp. Med. 170: 1191-1205 [Abstract]. |
26. | Jacob, J., and G. Kelsoe. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath- associated foci and germinal centers. J. Exp. Med. 176: 679-687 [Abstract]. |
27. |
Jacobs, H.,
Y. Fukita,
G.T.L. van der Horst,
J. de Boer,
G. Weeda,
J. Esser,
N. de Wind,
B.P. Engelward,
L. Samson,
S. Verbeek, et al
.
1998.
Hypermutation of immunoglobulin
genes in memory B cells of DNA repair-deficient mice.
J.
Exp. Med.
187:
1735-1743
|
28. | Lentz, V.M., M.P. Cancro, F.E. Nashold, and C.E. Hayes. 1996. Bcmd governs recruitment of new B cells into the stable peripheral B cell pool in the A/WySnJ mouse. J. Immunol. 157: 598-606 [Abstract]. |
29. | Rolink, A., F. Melchers, and J. Andersson. 1996. The SCID but not the RAG-2 gene product is required for the S mu-S epsilon heavy chain class switch. Immunity. 5: 319-330 [Medline]. |
30. | Alt, F.W., and D. Baltimore. 1982. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence for three D-JH fusions. Proc. Natl. Acad. Sci. USA. 79: 4118-4120 [Abstract]. |
31. | Jacob, J., J. Przylepa, C. Miller, and G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V-region mutation and selection in germinal center B-cells. J. Exp. Med. 178: 1293-1307 [Abstract]. |
32. | Smith, K.G., T.D. Hewiston, G.J.V. Nossal, and D.M. Tarlington. 1996. The phenotype and fate of the antibody forming cells of the splenic foci. Eur. J. Immunol. 26: 444-453 [Medline]. |
33. | Snapper, C.M., K.B. Marcu, and P. Zelazowski. 1997. The immunglobulin class switch: beyond "accessibility." Immunity. 6: 217-223 [Medline]. |
34. |
Macaulay, A.E.,
R.H. DeKruyff, and
D.T. Umetsu.
1998.
Antigen primed T cells from B cell-deficient JHD mice fail
to provide B cell help.
J. Immunol.
160:
1694-1700
|
35. |
Stedra, J., and
J. Cerny.
1994.
Distinct pathways of B cell differentiation. I. Residual T cells in athymic mice support the
development of splenic germinal centers and B cell memory
without an induction of antibody.
J. Immunol.
152:
1718-1725
|
36. |
Wen, L.,
W. Pao,
F.S. Wong,
Q. Peng,
J. Craft,
B. Zheng,
G. Kelsoe,
L. Dianda,
M.J. Owen, and
A.C. Hayday.
1996.
Germinal center formation, immunoglobulin class switching,
and autoantibody production driven by "non ![]() ![]() |
37. | Maizels, N., and A. Bothwell. 1985. The T-independent immune response to the hapten NP uses a large repertoire of heavy chain genes. Cell. 43: 715-720 [Medline]. |
38. | Rayssiguier, C., D.S. Thaler, and M. Radman. 1989. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 342: 397-401 . |
39. | Klinman, N.R.. 1997. The cellular origins of memory B cells. Semin. Immunol. 9: 241-247 [Medline]. |
40. | MacLennan, I.C.M.. 1994. Germinal centers. Annu. Rev. Immunol. 12: 117-137 [Medline]. |
41. | Pulendran, B., G. Kannourakis, S. Nouri, K.G.C. Smith, and G.J.V. Nossal. 1995. Soluble antigen can cause enhanced apoptosis of germinal-center B cells. Nature. 375: 331-334 [Medline]. |
42. | Shokat, K., and C.C. Goodnow. 1995. Antigen-induced B-cell death and elimination during germinal-centre immune responses. Nature. 375: 334-338 [Medline]. |
43. | Han, S., B. Zheng, J. Dal, Porto, and G. Kelsoe. 1995. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. IV. Affinity-dependent, antigen-driven B cell apoptosis in germinal centers as a mechanism for maintaining self-tolerance. J. Exp. Med. 182: 1635-1644 [Abstract]. |
44. | Hande, S., E. Notidis, and T. Manser. 1998. Bcl-2 obstructs negative selection of autoreactive, hypermutated antibody V regions during memory B cell development. Immunity. 8: 189-198 [Medline]. |
45. | Melchers, F.. 1997. Lymphoid development. Res. Immunol. 148: 426-428 [Medline]. |
46. |
Alani, E.,
R.A.G. Reenan, and
R. Kolodner.
1994.
Interaction between mismatch repair and genetic recombination in
Saccharomyces cerevisiae.
Genetics.
137:
19-39
|
47. | Nossal, G.J.V.. 1994. Differentiation of the secondary B-lymphocyte repertoire. The germinal center reaction. Immunol. Rev. 137: 172-185 . |
48. |
Thorbecke, G.J.,
A.R. Amin, and
V.K. Tsiagbe.
1994.
Biology
of germinal centers in lymphoid tissue.
FASEB J.
8:
832-840
|
49. | Kroese, F.G.M., A.S. WuBenna, H.G. Seijen, and P. Nieuwenhuis. 1987. Germinal centers develop oligoclonally. Eur. J. Immunol. 17: 1069-1072 [Medline]. |
50. | Ziegner, M., G. Steinhauser, and C. Berek. 1994. Development of antibody diversity in single germinal centers: selective expansion of high-affinity variants. Eur. J. Immunol. 24: 2393-2400 [Medline]. |
51. | Vora, K.A., and T. Manser. 1995. Altering the antibody repertoire via transgene homologous recombination: evidence for global and clone autonomous regulation of antigen-driven B cell differentiation. J. Exp. Med. 181: 271-281 [Abstract]. |
52. | McKean, D., K. Huppi, M. Bell, L. Staudt, W. Gerhard, and M. Weigert. 1984. Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. Proc. Natl. Acad. Sci. USA. 81: 3180-3185 [Abstract]. |
53. |
Blier, P.R., and
A. Bothwell.
1987.
A limited number of B
cell lineages generates the heterogeneity of a secondary immune response.
J. Immunol.
139:
3996-4005
|
54. |
Kuo, F.C., and
J. Sklar.
1997.
Augmented expression of a
human gene for 8-oxyguanine DNA glycosylase (MutM) in
B lymphocytes of the dark zone in lymph node germinal
centers.
J. Exp. Med.
186:
1547-1556
|
55. | Michaels, M.L., and J.H. Miller. 1992. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J. Bacteriol. 174: 6321-6325 [Medline]. |