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1
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Department of Immunology, The Scripps Research Institute, La Jolla, CA, 92037;
Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in somatic hypermutation,
we generated transgenic mice that express antisense RNA to a portion of
mouse REV3, the gene encoding this polymerase. These
mice express high levels of antisense RNA, significantly reducing the
levels of endogenous mouse REV3 transcript.
Following immunization to a hapten-protein complex, transgenic mice
mounted vigorous Ab responses, accomplished the switch to IgG, and
formed numerous germinal centers. However, in most transgenic animals,
the generation of high affinity Abs was delayed. In addition,
accumulation of somatic mutations in the VH genes of memory
B cells from transgenic mice was decreased, particularly among those
that generate amino acid replacements that enhance affinity of the B
cell receptor to the hapten. These data implicate DNA polymerase , a
nonreplicative polymerase, in the process of affinity maturation,
possibly through a role in somatic hypermutation, clonal selection, or
both. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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intronic enhancers (11, 12, 13), and for the
introduction of nicks or double-strand breaks (DSBs)
(14, 15, 16). Recently, a novel molecule with homology to RNA
deaminases (activation-induced cytidine deaminase, AID) was
implicated in somatic hypermutation, although its role may lie in the
modification of transcripts encoding molecules important to this
mechanism (17). These data suggest a multilayered
mechanism that includes at least 1) targeting of a lesion to the V
region by transcription or transcription-related factors, and 2)
introduction of nicks or DSBs to Ig V genes followed by error-prone
synthesis.
Studies of the pattern of V region mutations in nonselected sequences
such as in passenger transgenes, synonymous substitutions, and
nonproductively rearranged alleles reveal a bias to generate
transitions and base substitutions, the hotspot sequence RGWY, and some
degree of targeting to one of the DNA strands (18, 19, 20, 21, 22, 23).
Furthermore, sequences predicted to generate secondary structure are
frequently associated with base substitutions and the few insertions
and deletions detected in hypermutated sequences (19, 24, 25, 26, 27). In sharks, in which the pattern of mutation of new Ag
receptor genes (28) is otherwise similar to that of mice
and human Ig, doublets are frequently found even in the framework
regions (18). The mutation frequency, the enhancement of
mutability by secondary structure, and the transition bias implicate a
role for error-prone polymerase activity in somatic hypermutation, as
originally proposed by Brenner and Milstein (29). The high
frequency of doublets in the shark new Ag receptor mutants implies
extension from a mismatched base pair, and such data, together with the
predominance of base substitutions, suggest that the error-prone
activity may be dependent on DNA polymerase (Pol ) (18, 30, 31). Pol catalyzes the bypass of DNA lesions that
normally stall replication forks (32). Its ability
to recognize (or to at least "ignore") unconventional bases, such
as those chemically modified by mutagens, makes it moderately prone to
insert incorrect bases, but its most prominent characteristic lies in
extension beyond a mismatched base pair (33, 34, 35). Indeed,
it was recently demonstrated that Pol extends synthesis beyond
mismatched base pairs created by other distributive error-prone
polymerases, such as Pol 
(34) and 
(36). Furthermore, there is evidence that this polymerase
plays a role in the mutagenic repair of DSBs. In yeast, the great
majority of base substitutions introduced near a DSB are the result of
Pol activity (37), suggesting that it may also play a
role in DSB repair. Therefore, recent evidence suggesting that DNA DSBs
are intermediate products of somatic hypermutation also implicates this
polymerase in Ig hypermutation.
Knockout mice lacking Pol are early embryonic lethals
(38, 39, 40). Reasoning that a small amount of Pol may
overcome the lethality of the knockout, we have generated transgenic
(Tg) mice that express antisense transcripts to the RNA encoding the
catalytic subunit of this polymerase (mouse REV3,
mREV3). These mice appear healthy, express high levels of
antisense RNA, and display very low levels of mREV3 transcript. Here,
we examine somatic hypermutation and affinity maturation in these mice
following immunization with a hapten-protein complex. Our findings
indicate that, in these mice, the overall mutation frequency is
decreased and affinity maturation is delayed, but GC formation and
other aspects of the immune response appear unaffected.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate mice that express antisense transcripts to the mREV3
gene, 870 bp of a portion of the REV3 gene was cloned into
the XhoI site of the pCXN2 expression
vector (41) downstream of the CMV enhancer and the
chicken 
-actin promoter (Fig. 1
A). Because the
transcriptional regulation of endogeneous REV3 is unknown,
we selected a promoter-enhancer combination with constitutive
expression and a highly stable 3' untranslated region (rabbit
-globin). Furthermore, we reasoned that using a powerful expression
vector that generates highly stable transcripts would maximize the
possibility of duplex formation and degradation of the REV3
target transcript. The insert was generated by amplifying a portion of
the mouse REV3 gene with oligonucleotides Zetf
(5'-GAGATTCAGATGCACTCCTGG-3') and Zetr
(5'-GTCCTGCTTGTAAGACTTCAT3-'), digestion of the PCR product with
XhoI, and then ligation to the vector (1:5 vector:insert
ratio) with T4 DNA ligase. Competent cells (INV-
F';
Invitrogen, Carlsbad, CA) were transformed with ligation product and
grown in Luria-Bertani agar plates according to the protocol for
the original TA cloning kit (Invitrogen). To release the unit core of
the expression vector (3.3 kb), a plasmid with an insert in the
antisense orientation was digested with HindIII and
SalI, and the 3.3-kb piece containing the insert was gel
purified with the gene-cleaning kit (Bio 101, Vista, CA). The contructs
were microinjected into fertilized eggs of C57BL/6 background at The
Scripps Research Institute Transgenic Facility (La Jolla, CA) by
standard procedures. Mice were screened for the presence of the
transgene by PCR of genomic DNA and RT-PCR of tail RNA with
oligonucleotides 2eTRN (5'-TCTTGGTAGAACACCCTTTCG-3'), which complements
a portion of the mouse REV3 insert, and RBGR
(5'-TGATAGGCAGCCTGCACCTGA-3'), complementing the rabbit 3'
untranslated region in the vector.
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To assess the number of integration sites, genomic DNA from the founder was isolated from the tail by phenol-chloroform extraction and digested separately with BamHI and HindIII. Southern hybridization procedures were performed under both high and intermediate stringency conditions, as described previously (42), using the above-described PCR product for the transgene as 32P-labeled probe.
Mice and immunization
Limulus polyphemus hemocyanin (Hy) (Sigma, St Loius, MO) and BSA (Biosearch Technologies, Somerville, NJ) were conjugated to DNP and (4-hydroxy-3 nitrophenyl)acetyl (NP) haptens as previously described (3, 43). Control C57BL/6 mice, obtained from the National Institutes of Health, and Tg mice that were 8–12 wk old were immunized either i.p. with 50 µg NP10-Hy suspended in alum or i.p. and in multiple s.c. sites with 100 µg NP10-Hy suspended in CFA (Sigma).
Flow cytometry of bone marrow, spleen, and thymus
Bone marrow cells were obtained from the femur by rinsing the
cells out of the bone marrow with a syringe containing HBSS. Spleen
cells were dispersed in a culture dish with HBSS by grinding the tissue
with the back of a 3-cc syringe. Dispersed spleen or bone marrow cells
in HBSS were filtered through a Nytex (Tetko, Lancaster, NY)
membrane into a culture tube. Following one round of
centrifugation at 1500 rpm for 7 min, RBCs were lysed with 4 ml of
0.83% ammonium chloride at room temperature for 4 min. HBSS was added
to bring the volume to 15 ml, and the cells were centrifuged at 1500
rpm for 7 min. Cells were resuspended in HBSS with 0.1% BSA and 0.02%
azide to a concentration of 106 ml. Spleen cells
from naive mice were stained with anti-heat-stable Ag-FITC
(M1/69; BD PharMingen, San Diego, CA) and anti-B220-CyChrome
(RA3-6B2; BD PharMingen) at 0.5–1.0 µg/106
cells. For mutation analysis, cells from approximately half a spleen
from mice immunized with 50 µg NP-Hy suspended in alum were prepared
15 days after immunization as described above, but stained with
anti-
-FITC (R26-46; BD PharMingen) and anti-B220-PE
(RA3-6B2; BD PharMingen) Abs (0.5–1.0 µg of Ab per
106 cells). Bone marrow cells from same immunized
mice were stained with anti-CD43-PE (S7; BD PharMingen) and
anti-B220-CyChrome (RA3-6B2; BD PharMingen) at the same
concentrations used for spleen cells.
T cells were obtained from thymus by grinding as described above for spleen cells, centrifuged at 1500 rpm for 7 min at 4°C, and resuspended in degassed HBSS with 0.1% BSA and 0.02% azide. Cells were stained with anti-CD4-FITC (L3T4) and anti-CD8-CyChrome Abs (53-6.7) (BD PharMingen) at 0.5–1.0 µg of Ab per 106 cells for FACS analysis.
Quantitation of Ab concentration and relative affinities
Serum Abs were quantified using a fluorescence ELISA previously
described (43). In brief, 25 µl of a 1/50–1/8000
dilution of serum was added to microtiter plates coated with NP-BSA or
DNP-BSA and incubated overnight. The plates were washed, and bound Abs
were quantified by the addition of alkaline phosphatase-labeled goat
anti-, -, -
, or -µ, and the fluorescent substrate
4-methylumbelliferylphosphate (Boehringer Mannheim, Indianapolis, IN).
Standard curves were constructed using 1–80 µg of the purified
anti-NP hybridoma protein B-1-8 (for ) (44) and the
anti-DNP hybridoma 109.3 on DNP plates for and
(45).
To determine relative affinities, hapten inhibition assays were done as described previously (43, 44). Briefly, 25 µl of a 1/200–1/2000 dilution of serum obtained 15 days after immunization from mice immunized with NP-Hy in alum were mixed with NP cappoate (Biosearch Technologies) at concentrations ranging from 6 x 10-5 to 6 x 10-8 M and added to individual wells of NP4-BSA-coated microtiter plates. Bound anti-NP IgG Abs were quantified by fluorescence ELISA using alkaline phosphatase-labeled goat anti-mouse IgG Abs (Caltag, Burlingame, CA). The inhibition of Ab binding was calculated as described previously (43), and the concentration of free hapten yielding IC50 of Ab binding was estimated from semilog plots.
In vitro spleen fragment culture
To quantify and analyze responsiveness of memory B cells at the
clonal level, cells from immunized mice were transferred to irradiated
carrier-primed recipients for establishment of fragment cultures, as
previously described (46). Briefly, two million total
spleen lymphocytes isolated from mice immunized 30 days earlier with
100 µg NP10-Hy in CFA were injected into
lethally irradiated carrier-primed BALB/b recipients. Sixteen hours
later, recipient spleens were removed, and 1
mm3-fragments were prepared and placed in
microtiter wells with 10-6 M
NP10-Hy for 2 days at 37°C. At days 6, 7, and
12, culture fluids were analyzed by ELISA (see above) for the presence
of µ, , , and/or NP-specific Abs. Each fragment that was
positive for and Abs was dispersed, and recovered cells were
aliquoted into two samples for RT-PCR analysis of
VH186.2 (see RNA isolation, reverse
transcription, and PCR amplification).
GC staining
Spleens from immunized mice were cut in half, and one half was covered with OCT medium in a cryomold and placed on dry ice. Then 6- to 8-µm sections were cut with a Cryostat (CM1800; Leica, Deerfield, IL). The sections were fixed with cold acetone for 30 s. Each slide was incubated in a MeOH-hydrogen peroxide solution (4:1) for 20 min and then washed for 10 min in PBS. Slides were incubated in 1/500 biotinylated peanut lectin agglutinin (PNA; 5 mg/ml) (Biomedia, Foster City, CA) in PBS for 1 h at room temperature, rinsed with PBS, and incubated for 10 min with streptavidin-HRP in PBS (1/500) (Jackson ImmunoResearch, West Grove, PA). After a rinse with PBS for 10 min, chromogen (0.01% hydrogen peroxide, 1 mg diaminobenzidine, and 0.05 M Tris (pH 7.2)) was added, and slides incubated for 2–20 min.
RNA isolation, reverse transcription, and PCR amplification
To isolate RNA from
+B220+ total spleen
cells or cells isolated from fragment cultures, cells were pelleted,
resuspended in 200 µl TRIzol (Life Technologies, Rockville, MD) and
lysed by repeated pipeting. To isolate RNA from spleen tissues, small
spleen pieces were placed in 100 µl TRIzol, glass beads were added,
and the tissue was homogenized with a Mini-BeadBeater (Biospec
Products, Bartlesville, OK). RNA was isolated from all samples
according to the TRIzol maufacturer specifications. cDNA was made with
the Superscript II kit (Life Technologies) according to the protocol of
the manufacturer. For mutation analysis of
VH186.2, cDNA was amplified first for 30 cycles
(1 min at 94°C, 45 s at 58°C, and 45 s at 72°C) with
Taq polymerase (Life Technologies), primers complementing
the leader region of V genes of the J558 family (leader (forward),
5'-CATGGAATTCTTGGCAGCAACAGCTACAGG-3') and a portion of the
C1 domain (onk335,
5'-TCCCTGAAGCTTATTTTCTTGTCCACCTTG-3'). A second 22-cycle round of
PCR was done (1 min at 94°C, 45 s at 60°C, and 45 s at
72°C) with 1 µl PCR product from the first reaction using the
forward leader and an internal oligonucleotide complementing a region
proximal to the J region in the C1 domain
(onk14, 5'-TCCAAAGCTTGGGGCCAGTGGATAGAC-3').
To estimate the Taq polymerase error rate, a plasmid
containing an insert with the germline VH186.2
sequence was amplified as described above, and our estimated
Taq polymerase error rate was 1/10,000 bases.
For expression analysis, primers complementing the transgene (endogenous REV3) that do not overlap with the REV3 region in the transgene were used and are described as follows: (Zetf, described above; PolZr, 5'-GGCATTGAGCATCCGTGACAG-3'); and the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) (HPRTMf, 5'-CCTCATGGACTGATTATGGAC-3'; HPRTMr, 5'-GTCAAGGGCATATCCAACAAC-3'). These primers were used in 24-cycle reactions (1 min at 94°C, 45 s at 60°C, and 45 s at 72°C). For cloning purposes, the 5' end of the forward leader, onk335, and onk14 primers were slightly modified to acomodate restriction sites for either HindIII or EcoRI.
Cloning and sequencing of samples
PCR product was purified with the PCR purification kit (Qiagen, Chatsworth, CA) and digested with HindIII and EcoRI. Ligation and transformation of digested product into Top10F'-competent cells was done according to the manufacturer specifications for the vector (pZero cloning vector; Invitrogen). To isolate plasmids, cells from colonies were treated with lysozyme and boiled for 40 s, followed by centrifugation and precipitation of plasmid DNA with isopropanol. The insert was sequenced with 35S, a primer complementing the SP6 site in the vector, and T4 sequenase (US Bioscience, West Conshohocken, PA), and the sequenced product was run in a 6% polyacrylamide gel. Sequences were analyzed with the DNasis software (Hitachi Software Engineering, Tokyo, Japan). The entire length of the V region including the complementarity-determining region (CDR)3 (310–330 bps) was sequenced for the vast majority of the clones.
Statistical tests used to analyze mutation frequencies are described by Sokal and Rohlf (47).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of 10 antisense REV3-Tg founder mice, only 1 expressed
the transgene at high levels. Southern hybridization of genomic DNA
from this mouse revealed two integration sites, each with, at most, two
copies of the transgene (Fig. 2A). The fact that only one
line expressed the transgene and that so few Tg animals were recovered
may reflect strong selection during embryonic development for Tg
animals that did not completely lose expression of the REV3 gene (it is
possible that animals with large copy numbers of the transgene, as is
often seen during transgene integration, were embryonic lethals). A
line of mice expressing significant levels of the antisense product was
then generated through a series of backcrosses (Fig. 1
B).
All animals used in this study had at least two copies of the
transgene, either as heterozygotes at both integration sites or as
homozygotes at one of the integration sites (Fig. 2A). To
test for transgene expression in B cells, RT-PCR was done on bone
marrow and splenic B cells. A high level of expression of the transgene
in B cells was detected in all Tg animals used in this study (Fig. 2B).
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C),
whereas the levels of the housekeeping genes HPRT, cyclophilin, and
G6PD remained similar to controls. This reduction was seen in all Tg
animals with more than one copy of the transgene regardless of the
tissue analyzed (memory B cells, total spleen, Peyer’s patches, and
tail RNA; data not shown).
Mice with low levels of Pol appear healthy and of normal body
weight and do not seem to have decreased lifespans (the oldest founder
is now 23 mo), suggesting that low levels of this polymerase suffice to
overcome the embryonic defect seen in the knockout. Given the strong
defect early in hematopoeisis in the knockout (38, 39, 40), we
analyzed B cell and T cell development in Tg animals. The overall
numbers of B cells in the bone marrow were reduced (ranging from 30 to
60%), reflecting a defect that can be seen as early as in the pro-B
cell comparment but permeates all later stages of B cell development in
the bone marrow as well (Fig. 3).
Additionally, the number of B cells in the spleen were reduced in young
adult mice (20–50% reduction; Fig. 3). The nature of the defect in
the bone marrow is currently under investigation. The number of T cells
in the thymus was nearly normal at all stages of development, with a
slight decrease in the percentage (<15% decrease) of CD4
single-positive cells (Fig. 3). Histological analysis of the spleen
revealed normal morphology in all areas, including the lymphoid
compartment.
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To examine humoral immune responses in Tg mice, four Tg and four
control C57BL/6 mice were immunized i.p. with 0.05 mg
NP10-Hy in alum. The immune response of Ighb mice
to the NP hapten has been well characterized by a number of
laboratories (9, 44, 51, 52). Typically, the mice produce
high levels of -bearing Abs that predominantly use the heavy chain V
gene segment VH186.2, primarily joined to the D
segment DH16.1 and the J (JH2) segment. Affinity
of the receptor among -bearing Abs is most enhanced by a tryptophan
(W) to leucine (L) change in VH186.2 amino acid
residue 33 in first CDR (52). Late in the response, a
different set of highly mutated clones with high affinity arises that
is characterized by a glycine (G) in CDR3 residue 99, which is often
associated with a lysine (K) to arginine (R) replacement in CDR2
residue 58 (53).
The mice were bled 15 days after immunization and sacrificed for
analysis of somatic mutation, GC formation, serum Ab titers, and
relative affinities. Both groups of mice mounted a vigorous serum
anti-NP Ab response. ELISA analysis indicated that the serum of
control and Tg mice contained similar amounts of anti-NP Abs.
In addition, Tg mice produced comparable quantities of IgG anti NP-Abs
(0.55 ± 0.34 mg/ml) with control mice (0.4 ± 0.34 mg/ml),
suggesting that neither the levels of specific Ab nor isotype switching
was impaired by decreased Pol transcripts in Tg mice. The fact that
we used a promoter-enhancer combination that induces constitutive
expression of the transgene raised the possibility that the reduction
of Pol occurred in many cell types and, thus, could have exerted an
indirect effect on immune responses, particularly if this reduction
impacted the "quality" of T cell help. However, T cell development
appears relatively normal in Tg mice, and the similar levels of IgG in
Tg animals suggest that isotype switch, a mechanism that requires
effective T cell help and Ag presentation, was normal.
To examine whether decreased levels of Pol had a qualitative impact
on the Ab response, the relative affinities of serum Abs from all mice
were evaluated by hapten inhibition assays. Although Tg mice had
similar amounts of IgG serum Abs, the affinity of NP-specific IgG
attained by day 15 after immunization was considerably lower than
control mice for three of the four Tg mice (Fig. 4). Because of the close correlation of
selected mutations and affinity maturation of anti-NP Abs, this
finding can be explained either by a defect in somatic hypermutation,
in the selection for high affinity variants in the GC reaction, or in
the generation of memory B cells.
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). GCs were also abundant 30 days after
immunization in Tg mice but not in control mice. Large and persistant
GCs in antisense REV3 mice is reminiscent of mice deficient
in the recently discovered activation-induced cytidine deaminase
molecule, which appears to play a role in somatic hypermutation
(16).
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Somatic hypermutation in memory B cells of Tg mice expressing
antisense RNA to the gene encoding Pol
To determine whether the observed decrease in affinity of serum
Abs was the result of a defect in somatic hypermutation, we sequenced
the VH186.2-encoded H chain V genes from
+B220+ B cells obtained
from three Tg and three control mice that were immunized with NP-Hy in
alum and sacrificed 15 days after immunization. Almost all the clones
had at least one mutation, and to ensure that only memory B cells were
analyzed, nonmutated sequences were excluded from the analysis. A lower
frequency of somatic mutations was detected in the sequences derived
from memory B cells of Tg animals when compared with control animals
(Fig. 6). The lower frequency of mutation
appeared not to be due to an inability to generate highly mutated
clones, but rather the existence of a population of poorly mutated
cells in each of the Tg mice, which was rarely seen in the control
animals (62% of Tg clones and 21% of control clones had three
mutations or less; Fig. 6; Kolmogorov-Smirnov two-sample test,
p < 0.001). Importantly, the lower frequency of
mutation was accompanied by a highly significant decrease in the
proportion of cells that had acquired mutations associated with high
affinity to the NP hapten (17% of Tg clones and 77% control clones
had selected changes; Fig. 6; G test for goodness of fi,
p < 0.001).
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Consistent with this increase in selected mutations, the binding to NP of the serum IgG Abs at day 30 was 50% inhibited at a mean concentration of ± 0.3 x 10-5, which is comparable to that seen for day-15 Abs from control animals.
Previous studies have shown the presence of a highly mutated memory B
cell population with negligible levels of B220 on their surface
(55). Thus, to ensure inclusion of this population in our
analysis, H chains from total spleen cells were analyzed from the
fourth immunized Tg mouse (number 557) and the fourth control mouse
sacrificed on day 15. Again, only clones with at least one mutation
were considered (Tg, 14 of 16 clones were mutated; control, 17 of 17
clones were mutated). The control animal had a lower frequency of
mutation and of selected changes than the sorted
+B220+ B cells from
day-15 controls, which probably reflects the inclusion of a subset of B
cells that exit the GC reaction early and participate in the primary
response (56). Nevertheless, the Tg animal had a 43%
lower mutation frequency than the similarly treated control (Tg, 2.4
mutations per mutated clone with 0% of clones with selected changes;
control, 4.2 mutations per mutated clone with 29% of the clones
containing the selected changes).
Although the mutation frequency is lower, the pattern of mutations
generated by memory B cells of low Pol animals is nearly identical
with the pattern seen in the controls (Table I). Similarly, the majority of the
mutations was concentrated to the CDRs in both groups (although more
pronounced in the control CDR1 due to the W to L replacements). Close
to half of the mutations in both Tg and controls occurred at the
consensus hotspot motif RGYW. Furthermore, Tg mice had
replacement:synonymous (r:s) ratios that were entirely consistent with
both negative and positive selection (6). The r:s ratio in
reading frame (RF)1 was 1.4, in RF2 was 2.7, and in RF3 was 2.3,
whereas the ratio in CDR2 was 4.2, and there were 38 replacements but
no synonymous changes in CDR1. Another important indication for
selection in the NP response is the amino acid composition of CDR3
(53). Among clones in which the diversity element could be
identified, 76% of Tg origin and 78% of control origin used the
NP-response characteristic DFL16.1, mostly in RF1 and predominantly
with the JH2 segment (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in somatic hypermutation,
we generated Tg mice that express antisense RNA to a portion of the
catalytic subunit of the mouse REV3 gene. Although there is
a significant reduction in the number of B cells generated in the bone
marrow, these animals mounted vigorous humoral immune responses
following immunization, formed large GCs, and generated comparable
frequencies of memory B cells to control mice. However, by day 15, most
failed to generate high affinity Abs. The overall frequency of somatic
mutations and of selected changes in the VH186.2
gene was reduced in the six Tg animals when compared with similarly
treated controls, which was reflective of a large population of poorly
mutated clones (less than three mutations). Homozygosity of the Tg
integration site is unlikely to explain the data presented here because
animals heterozygous at both integration sites or homozygous at
different integration sites converged on the same phenotype. Evidence
for a lower frequency of overall mutation and especially of selected
changes was observed by three different experimental approaches:
sorting for +B220+
cells, analysis of total spleen cells, and in vitro boosting of memory
B cells in spleen fragment cultures.
One possible interpretation of this data is that we have interfered
with the process of selection rather than mutation by introducing a
defect in apoptosis. A reduced ability to undergo apoptosis may prevent
the elimination of cells with low affinity receptors. Indeed, the
magnitude of the decrease in selected changes and in somatic mutations
in antisense Pol mice is similar to that previously seen in mice
that overexpress BCL-2 (57). However, in contrast to Pol
-reduced mice, BCL-2-Tg mice have a significant increase in the
frequency of memory B cells (57). Additionally, although
the FR r:s ratios in both antisense REV3 mice and BCL-2-Tg
mice were consistent with negative selection against deleterious
mutations (2.0 and 1.8, respectively), the CDR r:s ratios in antisense
REV3 mice were very different from BCL-2-Tg mice (7.5 vs
2.9), suggesting ongoing positive selection in the REV3
antisense-Tg mice. Furthermore, although there is speculation that Pol
plays a role in apoptosis (35), there is no evidence
that this polymerase plays a direct role in this process and, indeed,
it would be unprecedented for a translesion synthesis polymerase to
perform such a function.
Impaired proliferation due to a defect in replication could also
account for the lower frequency of somatic hypermutation in these mice.
The early lethality of the Pol knockout may in fact reflect a
defect in the replication of rapidly dividing cells carrying a large
load of damaged DNA (39). However, yeast cells deficient
in REV3 proceed with normal kinetics through S phase after
exposure to the DNA-damaging agent cisplatin but arrest in
G2 where DSB repair via homologous recombination
can occur (58). In a separate study, REV3
mutant yeast cells were much more sensitive to damage in
stationary-phase cells than in cells in exponential phase when exposed
to both cisplatin and mechlorethamine, which generate interstrand
cross-links (59).The evidence that Pol introduces base
substitutions near DSBs in yeast (37) suggests that this
polymerase plays a role in DSB repair, and this function may be the
cause of the early lethality of the knockout. Indeed, mice exhibiting
inactivation of the DSB repair gene RAD50 also
display early lethality (60). Finally, a defect in
replication would be difficult to reconcile with the abundant and large
GCs that antisense REV3 mice formed. There is a precedent in
the literature for the impact that impaired proliferation has on
GC structure: mice deficient in mismatch repair genes generate small
and few GCs due to microsatellite instability and reduced proliferative
potential (61).
The third interpretation of these data is that Pol plays a direct
role in the process of somatic hypermutation. The fact that we did not
completely abolish Pol in these animals may have resulted in a
sluggish Ig hypermutation machinery, whereby hypermutation-introduced
DSBs may have been repaired much more inefficiently, both via
error-free synthesis or with the residual Pol molecules the B cells
may have contained, thereby reducing the rate of somatic hypermutation
and delaying affinity maturation. In support of this notion, serum Abs
from Tg mice analyzed 30 days after immunization had affinities
comparable to that of controls. It has been proposed that the
hypermutation rate needs to be sufficiently high to allow for the
acquisition of specific affinity-enhancing mutations but limited by the
probability for introduction of mutations deleterious to the structure
of the receptor or affinity (62, 63). This model predicts
that, with a decreased rate of hypermutation, the probability of
obtaining an affinity-enhancing mutation is low and, because fewer
cells would have negatively impacted receptors from mutation, GCs would
be large and persistent. This model would particularly apply to the
response to the NP hapten, wherein a single mutation (W to L) in CDR1
has such a profound impact on affinity. Thus, the lower frequency of
clones with selected changes and the larger GCs in the Tg mice may
reflect a lower rate of hypermutation. Our data may in fact represent
the first evidence that the rate of hypermutation is critical to the
kinetics of affinity maturation, at least in the case of the NP
response, and these data may be unobtainable in a case in which somatic
hypermutation is completely disrupted.
We did not observe an alteration in the pattern of mutations in
conjunction with the decreased mutation frequency, and this result may
reflect a role of Pol in extending synthesis from a mismatched
terminus, as recent studies have demonstrated (34, 36). In
fact, it is likely that what makes Pol an integral component of
error-prone synthesis is its remarkable ability to extend from a
mismatched terminus created by other more error-prone polymerases (such
as and ; 34, 36). Thus, reducing the levels of the
"mismatch extender" should impact all mutations similarly,
resulting in a reduction in the frequency of mutation without an
altering of the pattern of mutations.
Here we have tested a putative role for Pol in somatic
hypermutation of Ig V genes by generating mice with low levels of this
polymerase. However, the intimate relationship between somatic
hypermutation and affinity maturation makes it difficult to separate a
defect in somatic hypermutation from a defect in clonal selection.
Therefore, we cannot formally rule out a subtle defect in clonal
selection. However, because both negative selection and positive
selection appear intact as revealed by the CDR and RF r:s ratios and
the usage of the anti-NP prototypical CDR3, the findings presented
here are most consistent with a direct role of Pol in the mechanism
of somatic hypermutation.
|
Acknowledgments |
|---|
, and to David
Schatz for discussions on Ig hypermutation double-strand breaks. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marilyn Diaz, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: mdiaz{at}scripps.edu
3 Abbreviations used in this paper: GC, germinal center; DSB, double-strand break; Pol , DNA polymerase ; Tg, transgenic; Hy, Limulus polyphemus hemocyanin; NP, (4-hydroxy-3 nitrophenyl)acetyl; HPRT, hypoxanthine phosphoribosyltransferase; PNA, peanut lectin agglutinin; CDR, complementarity-determining region; r:s, replacement:synonymous; RF, reading frame; mREV3, mouse REV3.
Received for publication January 23, 2001. Accepted for publication April 25, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
G- and A-antibodies. J. Immunol. 96:587. light chain sequences of mouse antibody. Nature 228:1045.[Medline]
gene: critical role for the intron enhancer/matrix attachment region. Cell 77:239.[Medline]
may depend on sequences 3' of C and occurs in passenger transgenes. EMBO J. 10:2139.[Medline]
chains: a segment of the major intron hypermutates as much as the complementarity-determining regions. Proc. Natl. Acad. Sci. USA 91:12614.. Science 272:1646.[Abstract]
. Proc. Natl. Acad. Sci. USA 95:6876. and act sequentially to bypass DNA lesions. Nature 406:1015.[Medline]
and Rev1 protein in
eukaryotic mutagenesis and translesion replication. Cold Spring
Harbor Symp. Quant. Biol. In press.
in mice results in early embryonic lethality. Curr. Biol. 10:1221.[Medline]
(Rev3) leads to embryonic lethality and impairs blastocyst development in vitro. Curr. Biol. 10:1213.[Medline]
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3.3 kb and lacks internal sites for
either of the restriction enzymes used. Therefore, the two integration
sites are likely to contain a single copy of the transgene.
B, Transgene expression by RT-PCR in Tg animals used in
this study. The source of cDNA for lanes 1, 2, 4, and
5 was sorted B220+
, Clones with a K to R change at amino acid
58, which may possibly nullify affinity enhancement by the W to L
change and which, when combined with glycine at position 99, represent
a separate lineage of high affinity variants in the NP response (see
Humoral immune responsiveness of Tg mice expressing antisense RNA
to Pol