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*
Division of Molecular Immunology, Department of Pathology, Weill Medical College of Cornell University, and
The Immunology Program, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, VHDJH-C
,
VHDJH-C
, VHDJH-C
,
and V
J-C transcripts before and after Ig class switching in a
stepwise fashion. In these cells, induction of somatic mutations
required cross-linking of the surface receptor for Ag and T cell
contact through CD40:CD40 ligand and CD80:CD28 coengagement. The
induced mutations showed intrinsic features of Ig V(D)J hypermutation
in that they comprised 110 base substitutions (97 in the heavy chain
and 13 in the -chain) and only 2 deletions and targeted V(D)J,
virtually sparing CH and C. These mutations were
more abundant in secondary VHDJH-C than
primary VHDJH-Cµ transcripts and in V(D)J-C
than VJ-C transcripts. These mutations were also associated
with coding DNA strand polarity and showed an overall rate of 2.42
x 10-4 base changes/cell division in
VHDJH-CH transcripts. Transitions
were favored over transversions, and G nucleotides were preferentially
targeted, mainly in the context of AG dinucleotides. Thus, in CL-01
cells, Ig somatic hypermutation is readily inducible by stimuli
different from those required for class switching and displays discrete
base substitution modalities. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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locus 10, 11 ; and, finally, 5) a preference for
secondary Ig isotypes 12 . However, the lack of a well-defined in
vitro model of GC differentiation has limited our understanding of the
requirements for the induction, the modalities, and the mechanisms of
hypermutation. CD40:CD40 ligand (CD40L) engagement in association or not with BCR cross-linking in the presence of various cytokines has led to the induction of proliferation and isotype switching, but not somatic hypermutation 13, 14, 15, 16 . Consistent with the primary role of T cells in GC formation in vivo 1, 17, 18 , Ig somatic mutations have been induced in vitro in mouse and human B cells in the presence of T cell help and upon BCR engagement 19, 20, 21 . This, together with the finding that certain monoclonal B cell lines, such as the murine 18.81 cells 22 and a human follicular lymphoma line 23 , mutate spontaneously in vitro in the absence of specific triggers, provided impetus for the identification of the Burkitt’s lymphoma cell line BL2, which was found to accumulate somatic mutations in the expressed IgM upon BCR cross-linking and coculture with activated T cells 24 . However, these cells appear to be frozen at the surface (s)IgM+ sIgD- phenotype and are incapable of switching to downstream Ig isotypes and undergoing concomitant phenotypic differentiation. A cell line that enables analysis of the requirements and the modalities of somatic hypermutation as it relates to Ig class switching and other GC differentiative processes would constitute a more appropriate and useful model of physiological Ig hypermutation.
We have analyzed the Ig somatic hypermutation potential of our recently
identified monoclonal model of GC B lymphocyte differentiation, human
CL-01 cells. These B cells express a founder centroblast-like
phenotype, including sIgM, sIgD, CD38, and CD77 15, 16, 25, 26 .
Following engagement of CD40 by CD40L and exposure to the appropriate
cytokines, they undergo a coordinated maturation program that includes
Ig class switching to all seven downstream isotypes, progression
through phenotypic GC stages, and differentiation to memory-like B
cells and plasma cells. We show here that CL-01 cells can be induced to
not only switch to IgG, IgA, and IgE, but also to effectively mutate
the VHDJH and VJ
gene segments, while sparing the CH and C regions. CD40
and CD80 coengagement by T cell CD40L and CD28 is necessary, in
addition to BCR engagement, for the induction of Ig hypermutation.
Mutations accumulated in a stepwise fashion before and after class
switching and were distributed throughout the entire V(D)J gene
segment, indicating a lack of selection by Ag. These mutations
showed preference for transitions over transversions, biased targeting
of G within the AG dinucleotide, and evidence of strand polarity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human B cell line CL-01 has been described 15 . CL-01 cells
are sIgM+ sIgD+ and monoclonal, as shown by
blotting and Southern hybridization with labeled JH probes
and by the expression of unique VHDJH-Cµ and
VHDJH-C transcripts 27 . These cells
display on both chromosomes the switch (S)µ, 
, and S3,
S1, S1, S2, S4, S, and S2 regions in germline
configuration 15 . These cells also express the phenotype of GC
founder centroblasts, including CD38 and CD77. Upon engagement of CD40
by CD40L and exposure to appropriate cytokines, CL-01 cells undergo a
coordinated program of GC differentiation involving characteristic
phenotypic changes and switching to all downstream isotypes, i.e.,
IgG3, IgG1, IgA1, IgG2, IgG4, IgA2, and IgE, eventually giving rise to
plasmacytoid elements and memory-like B cells 15, 16, 25 .
sIgM+ sIgD+ CL-01 cells were cultured in RPMI
1640 medium (Life Technologies, Gaithersburg, MD) supplemented with
10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (FCS-RPMI) at
<105/ml.
T cells
CD4+ T cells were positively selected from normal PBMCs, prepared by fractionation through Histopaque 1077 (Sigma, St. Louis, MO) using anti-CD4 mAb-conjugated magnetic beads (Miltenyi Biotech, Auburn, CA). They were cultured in FCS-RPMI and expanded by weekly stimulation with a feeder cell mixture containing irradiated (1200 rad) PBMCs, 100 µg/ml of PHA (Life Technologies), and 100 U/ml of human rIL-2 (Genzyme, Cambridge, MA). For T:B cell coculture experiments, CD4+ T cells were used at least 2 wk after their last activation with feeder cells and were incubated for 6 h with 20 ng/ml of PMA (Sigma) and 500 ng/ml of ionomycin (Calbiochem-Novabiochem, San Diego, CA) before culture with B cells.
B:T cell cultures
CL-01 cells were cultured at 0.5 x 106 cells/well in the presence or absence of 2.5 x 106 irradiated (4000 rad) CD4+ T cells or 1 x 106 irradiated (4000 rad) human CD40L-transfected 293 cells (CD40L-293 cells) in a flat-bottom 6-well plate (5.0 ml FCS-RPMI volume). For B:T cell cocultures, plates were previously coated with 1:800 OKT3 mAb (Ortho Diagnostics Systems, Raritan, NJ) for 2 h at room temperature. To cross-link the BCR, CL-01 cells were reacted for 2 h at 4°C with Sepharose-conjugated rabbit Ab to human Ig light chain (2 µg/ml; Irvine Scientific, Santa Ana, CA) and then washed with cold PBS. After 7 days of culture, CL-01 cells were collected, freed of dead cells and debris by fractionation through Histopaque 1077 (Sigma), reacted again with anti-BCR Ab, washed, and reseeded over a new layer of irradiated T cells or CD40L-293 cells (in plates coated or not coated with anti-OKT3 mAb) in the presence or absence of cytokines. At day 14 of culture, CL-01 cells were harvested for total RNA extraction. In selected B:T cell cocultures, T cell conditioned medium (TCM) obtained from the culture fluids of T cells activated for 1 wk with 1:800 OKT3 mAb and IL-2 (100 U/ml) was used at a concentration of 1:5; IL-4 (Genzyme) and IL-10 (Schering-Plough, Kenilworth, NJ) were used at 100 U/ml and 100 ng/ml, respectively. The CD40:CD40L, CD80:CD80L, and CD30:CD30L interactions were blocked by preincubating T or B cells with saturating amounts (30 µg/ml) of mouse 24–31 mAb to human CD40L (Ancell, Bayport, MN), mouse CD28.2 mAb to human CD28 (PharMingen, San Diego, CA), mouse Ber-H2 mAb to human CD30 (Dako, Carpinteria, CA), or mouse BB1/B7-1 mAb to human CD80 (PharMingen).
PCR amplification of V(D)J transcripts
RNA was extracted from 2 x 106 cells using the
RNeasy total RNA kit (Qiagen, Cathsworth, CA). mRNA was reverse
transcribed using the SuperScript preamplification system for first
strand cDNA synthesis (Life Technologies).
VH3DJH-CH transcript cDNAs were
amplified with a VH3 leader-specific sense primer
LVH3(I) (5'-ATGGAG(CT)TTGGGCTGA(CG)CTGG(CG)TTT(CT)T-3')
27 and with the antisense-specific primers CH1-µ
(5'-GTTGCCGTTGGGGTGCTGGAC-3') (spanning Cµ nucleotides 268–288),
C (5'-TCCAGCAGTGGCGCCAAGGCGAG-3') (C, 220–242), universal primer
CH1- (5'-CAAGCTGCTGGAGGGCACGGT-3') (C, 206–226),
CH1- (5'-CTAGGCACTGTGTGCCGGCAGGGT-3') (C,
209–232), or CH1- (5'-CGAGACGGTCAGCAAGCTGATGG-3')
(C, 201–223), using Cloned Pfu DNA polymerase
(Stratagene, La Jolla, CA) and the reaction buffer provided by the
manufacturers with 30 cycles, each consisting of a 1-min denaturation
at 94°C, a 1-min annealing at 58°C, and a 1-min extension at
72°C. The amplification was completed by an additional 10-min
extension at 72°C. To amplify V1-J-C transcripts, the V1
leader-specific sense primer LV1(I)
(5'-ATG(GA)CC(TG)GCT(CT)CCCTCTCCTCCT-3') (-chain leader, 1–23) and
the C-specific antisense primer C(II)
(5'-CGTCAGGCTCAGATAGCTGCTG-3') (C, 202–223) were used with the
PCR conditions described above. The PCR cDNA products were purified
with the PCR purification kit (Qiagen) and ligated into pCR-Script SK
vector (pCR-Script Cloning Kit, Stratagene, La Jolla, CA), which was
used to transfect in XL1-Blue MRF supercompetent cells
(Stratagene). The (positive) white bacterial colonies were screened by
PCR 28 using the VH3 leader sense primer LVH3
(II) (5'-GTTGCTATTTTAAAAGGTGTCCAGTGT-3') (CL-01 heavy chain leader,
21–57) and the consensus JH antisense primer
(5'-CGGTCACCGTCTCCTCA-3') for
VHDJH-CH clones and the internal
V1 leader sense primer LV1 (II)
(5'-ACCCCTCCTCACTCACTGTGCAG-3') (CL-01 -chain leader, 23–45)
together with the internal C antisense primer C (I)
(5'-TTGGCTTGAAGCTCCTCAGAGGA-3') (C, 42–64) for VJ-C
clones. The individual colonies that had been directly used as PCR
templates were seeded onto fresh Luria-Bertani medium plates and
expanded overnight. The clones containing the
VHDJH-CH or VJ-C
transcripts were selected for single-stranded conformational
polymorphism (SSCP) analysis.
Detection of mutated VHDJH-CH
and VJ-C transcripts by SSCP
Mutated VHDJH-CH and
VJ-C transcripts were identified by SSCP analysis 29 . cDNAs
for SSCP analysis were amplified by PCR (30 cycles of a 1-min
denaturation at 94°C, a 1-min annealing at 58°C, and a 1-min
extension at 72°C) using the cloned cDNA inserted into pCR-Script SK
vector as template in a 10-µl reaction volume with Taq DNA
polymerase (Life Technologies) in the presence of 1 µCi
[-32P]dCTP (NEN Life Sciences, Boston, MA) (3000
Ci/mmol). The internal VH3 leader sense primer
LVH3(II) and CH1 antisense primer Cµ
(5'-AGACGAGGGGGAAAAGGGTT-3') (Cµ, 18–37), C
(5'-TGGGGAACACATCCGGAGCCTTG-3') (C, 8–30), universal C
(5'-GAAGACCGATGGGCCCTTGGTGGA-3') (C, 4–27), C
(5'-GACCTTGGGGCTGGTCGGGGAT3') (C, 3–24), or C
(5'-CGGAGGTGGCATTGGAGG-3') (C, 54–71) were separately used for
VHDJH-Cµ, VHDJH-C,
VHDJH-C, VHDJH-C,
or VHDJH-C analysis. To analyze the
-chain, the V1 leader sense primer LV1(II) and the C
antisense primer C (I) were used. Optimal sensitivity is achieved
when SSCP is performed using 100- to 350-bp DNA fragments. Therefore,
our amplified >400-bp fragments were digested with KpnI and
then diluted 1:15 in 10 mM EDTA, 0.1% SDS. The labeled, cleaved, and
diluted DNAs were mixed with an equal volume of sequencing stop
solution containing 95% formamide, 20 mM NaOH, 20 mM EDTA, 0.05%
bromphenol blue, and 0.05% Xylene cyanol. Samples were denatured for
10 min at 98°C, chilled on ice, and immediately loaded in 3-µl
aliquots onto a 6% acrylamide gel (20:1 acrylamide:bis) with 1x
Tris/boric acid/EDTA buffer (100 mM Tris, 90 mM boric acid, and
1 mM EDTA) containing 10% glycerol. Electrophoresis was at room
temperature for 18 h at 6 W. Gels were autoradiographed on Kodak
X-Omat AR film (Eastman Kodak, Rochester, NY).
Sequencing Ig V(D)J-C transcripts
The clones displaying an altered electrophoretic mobility in
SSCP gel were analyzed by sequenced to confirm and characterize the
nature of the mutations. Plasmids were extracted using QIAprep Spin
Miniprep kit (Qiagen) and sequenced on both strands using
Taq dideoxy terminator cycle sequencing kit and a 373
automatic sequencer (Applied Biosystems, Foster City, CA). Sequences
were compared with the unmutated CL-01
VHDJH-CH1 and VJ-C
sequence from CL-01 cells cultured in medium only using the MacVector
v.5.0 software (International Biotechnologies, New Haven, CT).
Sequencing of a total of 20 heavy chain and 10 -chain transcripts
defined as negative by SSCP analysis revealed a lack of somatic
mutations in all of them (not shown). Sequencing of a total of 15 heavy
chain and 15 -chain transcripts defined as positive by SSCP analysis
yielded 100% concordance with the SSCP analysis results and
demonstrated that a single nucleotide change in the 375 bp of the
VHDJH or the 336 bp of the VJ cDNA
sequence was sufficient to alter DNA mobility in the SSCP gel (not
shown).
Mutational analysis
The census of the somatic point-mutations was performed by
counting identical mutations in more than one transcript (of any
isotype) only once. It was assumed that identical base changes in
different transcripts were shared mutations, although some of these
mutations might have arisen as hotspots and therefore could actually be
independent mutations. The obtained values were used for the
compilation of Tables I (last column),
II, III,
IV, and V.
For the compilation of the fourth column of Table I
, all
point-mutations found in all sequenced transcripts were counted. For
the fifth column of Table I, the somatic point-mutations found in
different transcripts of the same isotype were counted only once.
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J sequences, calculated by the Inh. Sus. Calc. program version
1.0 for the Macintosh 30 and a binomial distribution model, as
reported by Chang and Casali 30 . The comparisons of the observed with
the expected number of mutations for each individual nucleotide residue
to each of the three other nucleotides were performed using a
contingency table (
2 test). The expected frequency of
mutations was calculated by taking into account the base composition of
the unmutated CL-01 V(D)J sequence; that is, it was corrected by the
frequency of occurrence of the individual nucleotides, or di-, tri-, or
tetranucleotides considered within the CL-01 V(D)J sequence assuming
randomness. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human CL-01 cells are monoclonal and express sIgM and sIgD with a
-chain. The VHDJH segment consists of a
VH26–3.7-like gene (90% identity) 31 , rearranged to DN4
and D5 genes, and an unmutated JH6c gene. The VJ
segment consists of a 1c.10.2/DPL2 V1-like gene (94% identity)
32 , rearranged to a J2 gene with a single change, probably a
point-mutation. Multiple sequences at different times of
VHDJH-Cµ, VHDJH-C,
and VJ-C cDNAs from cultured CL-01 cells revealed no somatic
mutations (not shown). To determine whether CL-01 cells could be
induced to hypermutate the expressed Ig V(D)J genes, these cells were
reacted with an anti-light chain Ab to cross-link the BCR and then
cultured with activated human T cells. After 14 days, mRNA was
extracted from the cultured cells and reverse transcribed. Ig V(D)J-C
cDNAs were amplified using the CL-01 VH or V gene leader
sense primer in combination with an antisense Cµ, C, C, C,
C, or C primer and were cloned into appropriate vectors for
further "nested" PCR amplification (in the presence of
[32P]dCTP) of V(D)J-C cDNAs. Amplified
[32P]-labeled V(D)J-C cDNAs were digested with
KpnI for SSCP analysis. Those V(D)J-C cDNAs
defined as mutated by SSCP were then sequenced (Fig. 1A).
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,
VHDJH-C-C, and
VHDJH-C-C cDNAs were readily amplified,
in addition to VHDJH-Cµ and
VHDJH-C cDNAs, from CL-01 cultured with T
cells after BCR engagement. By SSCP analysis, 16 of 112 (14.3%)
VHDJH-Cµ transcripts were mutated, i.e., they
displayed a gel mobility different from that of corresponding
transcripts from CL-01 cultured in medium alone, based on the analysis
of 50 identical VHDJH-Cµ and 50 identical
VJ-C transcripts (Table I
and Fig. 1B). Also
mutated were 20 of 110 (18.2%) VHDJH-C, 17
of 120 (14.2%) VHDJH-C, 9 of 86 (10.5%)
VHDJH-C, 3 of 24 (12.5%)
VHDJH-C transcripts, and 18 of 179 (10.1%)
VJ-C transcripts (Table I
and Fig. 1B). In contrast, no mutated transcripts were found
in CL-01 cells cultured with anti-BCR Ab in the absence of
activated T cells (50 VHDJH-Cµ, 50
VHDJH-C, and 50
VHDJH-C transcripts analyzed) or
cultured with activated T cells without BCR engagement (50
VHDJH-Cµ, 50
VHDJH-C, and 50
VHDJH-C transcripts analyzed). Thus, after
BCR engagement and exposure to activated T cells, CL-01 cells
effectively Ig class switch and mutate both primary
(VHDJH-Cµ and
VHDJH-C) and secondary
(VHDJH-C,
VHDJH-C, and
VHDJH-C) heavy chain transcripts, as well as
VJ-C transcripts. CD40:CD40L and CD80:CD28 coengagement is required for the induction of Ig somatic hypermutation
As T cell-derived soluble factors have been implicated in
sustaining Ig V gene somatic hypermutation 21 , we assessed the
ability of these factors to substitute T cells in the induction of Ig
mutation in CL-01 cells. No mutated primary or secondary Ig transcripts
were detected in CL-01 cells cultured after BCR engagement with TCM
further enriched with IL-4 and IL-10 (not shown), even upon CD40
engagement by CD40L-293 cells (50 VHDJH-Cµ,
50 VHDJH-C, and 50
VHDJH-C transcripts analyzed) (Fig. 1B). To further explore the role of CD40 in the induction of
hypermutation, CD40L on the activated T cells was blocked using a mouse
mAb to human CD40L. The failure of the CL-01 cells cultured with this
anti-CD40L mAb, but not the putatively irrelevant anti-CD30
mAb, to hypermutate the expressed Ig even upon BCR engagement (60
VHDJH-Cµ and 40 VJ-C transcripts
analyzed from anti-CD40L Ab cultures and 50
VHDJH-C and 52 VJ-C transcripts
from anti-CD30 Ab cultures) indicates that CD40 engagement by
CD40L, not mere T:B cell contact, is necessary to induce the mutational
machinery in CL-01 cells (Fig. 1B).
Because the CD28 activation pathway has been reported to be essential
for the Ab response to T cell-dependent Ags in the mouse 33 , we
addressed the role of this costimulatory molecule in the induction of
somatic hypermutation. Blocking of CD28 on the surface of activated T
cells using an anti-CD28 Ab made these T cells ineffective inducers
of hypermutation (50 VHDJH-Cµ, 50
VHDJH-C, and 50
VHDJH-C transcripts analyzed) when added to
CL-01 cells after BCR engagement (Fig. 1B). That CD28 was
required to engage CD80 (the CD28 complement on the B cell surface) to
induce Ig hypermutation was further indicated by the lack of mutations
(50 VHDJH-Cµ, 50
VHDJH-C, and 50
VHDJH-C transcripts analyzed) in CL-01 cells
cultured with activated T cells in the presence of a blocking mouse
anti-CD80 mAb (Fig. 1B). Thus, in addition to BCR
engagement, engagement of the CD40:CD40L and CD80:CD28 costimulatory
pairs is necessary for the induction of somatic Ig hypermutation in
human B cells.
The mutations induced in CL-01 cells show intrinsic features of Ig V(D)J somatic hypermutation, but not evidence of Ag-driven selection
The 38 heavy chain and 9 -chain transcripts that appeared
mutated by SSCP analysis were sequenced, and all contained somatic
point-mutations. These VHDJH-Cµ, -C,
-C, -C, -C, and VJ-C sequences were compared with
those of the VHDJH-CH1 and
VJ-C transcripts expressed by the CL-01 cultured alone
(unmutated templates) (Fig. 2). A total
of 123 mutations were found in the 38
VHDJH-Cµ, VHDJH-C,
VHDJH-C, VHDJH-C,
and VHDJH-C transcripts (575 bp each). A
total of 121 were point-mutations confined to the
VHDJH sequence (375 bp), one was a deletion of
three nucleotides in the framework region (FR) 3 of a
VHDJH-C transcript (013), and one was a
C
G S mutation at residue 87 of the CH1 sequence of a
VHDJH-Cµ1 transcript (µ015) (Fig. 2A). The 121 VHDJH point-mutations
corresponded to an overall frequency of 1.22 x 10-3
changes/base (Table I), >30-fold the PCR amplification error rate with
high-fidelity Pfu DNA polymerase (
10-6
bases/cycle, i.e., 4.0 x 10-5 changes/base in 30
cycles) (p < 0.001). The single nucleotide
change in the CH1 region of
VHDJH-Cµ1 transcript µ015 represented a
frequency of 1.35 x 10-4 changes/base, which was
threefold the PCR error rate. The 121 VHDJH
nucleotide changes comprised 96 independent mutations, 17 of which were
observed in multiple transcripts. The 96 independent point-mutations
consisted of 68 R, 27 S, and one stop codon mutations and yielded an
overall mutation rate of 2.42 x 10-4 base
changes/cell division.
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J-C transcripts
sequenced (539 bp each). Sixteen were point-mutations confined to the
VJ sequence (336 bp), and one was a 3-nucleotide deletion in the
FR2 of transcript 013. No mutations were found in the C1 sequence
(203 bp). The 16 VJ point-mutations corresponded to an overall
frequency of 5.4 x 10-4 changes/base (Table I), a
rate >10-fold the PCR error rate for 30 cycles
(p < 0.001). These mutations comprised 13
independent mutations, 3 of which were observed in multiple
transcripts. The 13 independent mutations consisted of 7 R and 6 S
mutations and yielded an overall mutation rate of 1.08 x
10-4 base changes/cell division, a value lower than that
in VHDJH (p < 0.05).
The highest load of mutations was found in the single
VHDJH-C transcript sequenced (Table I).
Overall, the VHDJH-C transcripts bore a
mutational load two- to threefold greater than that of the
VHDJH-Cµ, -C, -C, or VJ-C
transcripts (Table I). In the
VHDJH-CH transcripts, the 96
independent point-mutations targeted 83 nucleotide residues scattered
throughout the VHDJH sequence, with no
preferential segregation to complementarity-determining regions (CDRs)
or FRs (Fig. 2 and Table II). Likewise, in the VJ-C
transcripts, the 13 independent mutations targeted 13 nucleotide
residues scattered throughout the VJ sequence, with no
preferential segregation to CDRs or FRs (Fig. 2). In both the
VHDJH and VJ segments, the number of R
mutations in CDRs was lower than that theoretically expected by chance
alone, and, therefore, inconsistent with a positive selection of R
mutations by Ag (Fig. 2 and Table II).
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Randomly occurring point-mutations are expected to be one-third
transitions and two-thirds transversions, but the 96 unique
VHDJH point-mutations were equally divided
between transitions and transversions (p <
0.01) (Table III), and the 14 unique VJ point-mutations consisted
of almost twice as many transitions (n = 9) as
transversions (n = 5) (p <
0.01). In the VHDJH transcripts, G nucleotides
were mutated at a frequency (46.9% of total mutations) about 50%
higher than that expected by chance alone (33.3%) after correcting for
base composition, i.e., normalizing for the relative occurrence of G in
the unmutated VHDJH sequence template
(p < 0.05), with G A transitions
accounting for 42.2% of the total G mutations and 43.2% of the total
transitions (Table III). Preferential mutation of G was associated with
scarcity of mutations in A (p < 0.001) and T
(p < 0.001) and stochastic frequency of
mutations in C (Table III). A preference for transitions over
transversions and G nucleotide targeting could be discerned in the
VJ transcripts, but the small sample size did not allow a
meaningful statistical conclusion.
|
J sequence
was not considered because of the small number of point-mutations)
(Table IV). All 16 dinucleotides were targeted by mutations except for
TT. The AG dinucleotide and its inverse repeat CT were found to be
mutated at a frequency significantly higher than that expected by
chance alone (p < 0.001) (Table IV). This was
not due to a mere G and C preference (Table IV), as G and C were
mutated at a significantly lower frequency when occurring outside than
within AG and CT (38 and 45% vs 62 and 55% of total G and C
mutations, respectively). In addition, GA and TC, which contain the
same nucleotides in inverse order, contained 2.3- and 3.4-fold fewer G
mutations than AG and CT (Table IV).
|
We examined all occurrences of AGN, NAG, CTN, and NCT
trinucleotides to verify whether the observed concentration of somatic
point-mutations in the AG and the CT dinucleotides could in fact
reflect a preference for the trinucleotides that harbor these
dinucleotides (Table V). We also examined AGC and TAC and their inverse
repeats GCT and GTA, as these trinucleotides have been suggested to be
preferential targets of hypermutation 36 . Finally, we considered all
other trinucleotides that had been targeted by 10 or more
point-mutations in the CL-01 VHDJH sequence.
The trinucleotides most frequently targeted by mutations were AGA, GAG,
AGG, GGC, GAC, ACC, GCT, and GAC (p < 0.01).
AGC and TAC, and the inverse repeat of the latter, GTA, contained a
number of mutations not higher than that expected by chance, and so did
CTC, GGG, CTG, CCT, and CAG.
|
). In the unmutated
VHDJH sequence, G occurred 69 times outside AG,
AGA, GAG, or AGG and 36 times as part of these di- or trinucleotides,
but was the target of mutation 16 and 29 times, respectively, in
different transcripts (p < 0.001). AG was
targeted by mutations at the expected frequency whether occurring in or
outside AGA, GAG, and AGG trinucleotides. Thus, the high frequency of
mutations in AGA, GAG, and AGG trinucleotides reflected the
preferential targeting of G within the AG dinucleotide. The RGYW tetranucleotide motif is virtually spared by somatic point-mutations
The consensus RGYW motif, where R is a purine (A or G), Y is a pyrimidine (C or T), and W is A or T, has been identified as a mutational hotspot 35 . Census of the CL-01 Ig VHDJH sequence for the presence of the RGYW tetranucleotide revealed that this motif occurred 11 times in 6 different iterations (AGCA twice, AGCT once, AGTA twice, AGTT none, GGCA once, GGCT three times, GGTA twice, and GGTT none) and was mutated more frequently than expected only in the GGCT iteration (p < 0.01), in which 6 of 9 mutations targeted the terminal CT dinucleotide. The lower frequency of mutation of the AGCT iteration, also containing a CT dinucleotide, suggested that the high mutability of GGCT might be attributed to a target bias of its two intrinsic and partially overlapping GGC and GCT trinucleotides.
Somatic point-mutations accumulate stepwise concomitant with Ig class switching
The ability of sIgM+ sIgD+ CL-01 cells to
switch to IgG, IgA, and IgE allowed us to address the relationship
between somatic hypermutation and Ig class switching. A genealogical
tree was constructed using the VHDJH sequences
derived from CL-01 cells cocultured with T cells in a single 14-day
culture after BCR engagement. Identical mutations in different
transcripts were considered to be shared mutations, although some of
these might have arisen as hotspots and therefore could actually be
independent mutations. There are three branches to the genealogical
tree we generated, spanning four, three, and two generations (Fig. 3). The first branch stemmed from a
first-generation putative intermediate bearing a 56 G A transition,
which could be tracked down to two fourth-generation IgG-switched
elements bearing 5 and 10 point-mutations (013 and 002), through
a putative second-generation intermediate bearing the 56 G A and a
267 G A transition and a third-generation putative intermediate
bearing four shared mutations, flanked by an IgD intermediate bearing
the two second-generation shared mutations plus a third mutation (336 G
A). The second branch stemmed form a first-generation putative
intermediate bearing a 20 C T transition. This gave rise to two
third-generation elements, one still expressing IgD (075), the other
switched to IgE (001), both with seven point-mutations, of which two
shared the 115 C A transversion and the original 20 C T
transition. An unswitched IgD element bearing five point-mutations and
a putative IgD/IgM second-generation element with two mutations that
were conserved in the third-generation elements were also part of this
branch. Finally, the third branch consisted of a first-generation
element characterized by a 355 C A transversion, which gave rise to
IgM (µ015) and IgA (006) second-generation elements bearing three
and five point-mutations, of which only the original 355 C A
transversion was shared. Thus, the mutational machinery was active
throughout class switching in CL-01 cells, and point-mutations
accumulated in a stepwise fashion in IgG, IgA, and IgE.
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J-C
transcripts, when the appropriate stimuli are applied, i.e., BCR
engagement and T cell contact, allowing for engagement of at least the
two costimulatory molecule pairs CD40:CD40L and CD80:CD28. The ability of CL-01 cells to undergo Ig somatic hypermutation and class switching as part of an integrated differentiation program has allowed us to determine that the requirements for the induction of these two central GC maturational processes are different. As we have shown 15, 16, 25 , CD40 engagement by soluble trimeric CD40L or CD40L-expressing 293 cells in the absence of T cells effectively induces CL-01 cells, as well as freshly isolated normal human IgM+ IgD+ B cells, to switch to IgG, and IgA, and, in the presence of IL-4, to IgE. As we show here, CD40:CD40L engagement is necessary but not sufficient for the induction of Ig somatic hypermutation. CD40L-expressing 293 cells cannot substitute for T cells in inducing somatic hypermutation, even in the presence of TCM, further enriched with IL-4 and IL-10, and after BCR engagement. The failure of these switched B cells to accumulate mutations further points to somatic hypermutation and class switching as two independent and discrete processes and indicates that engagement of additional B:T cell costimulatory molecule pair(s) is indispensable to trigger the mutational machinery.
Our present findings strengthen those showing that both BCR engagement and T cell help are required to trigger Ig hypermutation 21, 24 and extend them by providing evidence that B:T cell contact allowing for CD40:CD40L and CD80:CD28 coengagement is necessary, in addition to BCR engagement, to induce this process. Furthermore, by showing that anti-CD30 mAb fails to interfere with the induction of the mutational machinery, they provide an explanation for the putatively normal somatic hypermutation process observed in CD30-deficient mouse mutants 37 and indicate that the anti-CD40L, anti-CD80, and anti-CD28 mAbs ablated Ig hypermutation not by merely reducing or abrogating B:T cell contact, but rather by specifically interfering with CD40:CD40L or CD80:CD28 coengagement and related signaling. Whether, after BCR engagement, CD40:CD40L and CD80:CD28 coengagement is sufficient to induce Ig hypermutation or the coengagement of these costimulatory pairs mediates the induction of crucial T cell surface molecules and/or factors remains to be determined.
Ig somatic hypermutation and class switching are intimately linked in the GC, where point-mutations account for a vast majority of the changes, which include only scarce insertions and deletions 38 . Ig hypermutation is thought to begin in centroblasts before class switching 15, 39 . It is not known whether high-affinity GC centrocytes that have undergone isotype switching undergo further hypermutation, although it has been suggested that isotype switching does not terminate hypermutation 40 . Our present findings at the clonal level suggest that the onset of somatic hypermutation is not related to Ig class switching to IgG, IgA, or IgE and further emphasize that these are two independent processes. They also show that the modalities of in vivo GC hypermutation are reflected in CL-01 cells, in which, vis-à-vis of 137 somatic point-mutations, only two codon deletions were found, both involving triplets and both leaving the transcripts in frame. The conservation of shared mutations among primary and secondary isotypes and the observation of nonmutated Ig secondary isotype transcripts suggest that B cells accumulate somatic mutations along cell divisions before and after isotype switching occurs.
In induced CL-01 cells, VHDJH-C and -C
transcripts bore a load of somatic mutations approximately twofold
greater than that of VHDJH-C transcripts or
their primary VHDJH-Cµ and
VHDJH-C counterparts (Table I). The higher
frequency of mutations in CL-01 cell VHDJH-C
transcripts in vitro extends findings in vivo showing, in centroblasts,
a heavier load of mutations in VHDJH-C than
in VHDJH-Cµ transcripts 3, 41 and indicates
that this differential mutational load may reflect an inherent feature
of the integrated hypermutation and class-switching processes. This
contention would be further supported by the 1000-fold higher mutation
rate in a VHDJH-C construct than in its
VHDJH-Cµ counterpart after stable integration
into an established B cell line 42, 43 . In CL-01 cells, the frequency
of mutations in VHDJH-C,
VHDJH-Cµ, and
VHDJH-C, but not
VHDJH-C, transcripts was about two- to
fourfold higher than in VJ-C transcripts. A similar higher
load of mutations in the heavy chain than the light chain of
Ag-selected Abs has been interpreted to reflect the dominant role of
the VHDJH over the VLJL
segment in providing the structural correlate for Ag binding 44, 45 .
Our in vitro findings in a system putatively devoid of nominal
antigenic pressure suggest that more efficient targeting of mutations
to the heavy chain is an intrinsic property of the hypermutation
machinery.
In vivo studies have shown that Ig V(D)J somatic point-mutations are
not generated randomly in terms of base substitution, preference, and
distribution (hotspots) 6, 36, 46 . In induced CL-01 cells, G bases
were preferentially targeted with G A transitions, which are
especially copious in all transcripts of different heavy chain
isotypes. The preferential G targeting in CL-01 cells differs from the
lack of such a bias in productive and nonproductive human
VHDJH rearrangements ex vivo 27, 47, 48, 49, 50, 51, 52, 53 and
contrasts with the preferential targeting of A in the mouse 8, 36 . A
preferential targeting of G nucleotides has been observed in vitro in
human BL2 cells 24 , Chinese hamster cells 54 , and mouse B cells
55 and in vivo in exotherms that do not have GCs 56 , suggesting the
mechanism(s) driving such a mutational preference is phylogenetically
conserved and is operational in the absence of the GC microenvironment.
A preferential G targeting has also been found in MSH2 mismatch repair
protein-deficient mice 57, 58 and may be related to the activity of
this protein, which corrects mismatches mainly at G and C 58 .
The identification of sequence-specific preferences by the somatic hypermutation machinery is important as it may provide clues in determining the molecular mechanism of this process. In addition to base targeting bias, mutational hotspots have been identified throughout Ig V(D)J gene sequences 6, 36, 46 . While some of these hotspots may reflect the application of selection force(s) on the gene product, others may be inherent to the nucleotide sequence targeted by mutations, as suggested by the analysis of "passenger" or nonselectable genes 59, 60 . The putative lack of the application of a nominal positive or negative selective pressure to CL-01 cells under the culture conditions used here should allow for an insight into the base preference(s) and hotspot(s) that are inherent to the mutational machinery. In our experiments, the striking bias for G mutations (more than half of all VHDJH point-mutations) would reflect a marked mutational preference for the AG dinucleotide, whether isolated or in the context of AGA, GAG, and AGG trinucleotides.
For convention, all the present mutations in CL-01 cells were recorded
from the coding strand, although it is not known on which strand
mutations occur. The high G:C mutation ratio (1.6) in CL-01 cells
resembles the marked bias toward mutation of purines in the coding
strand, especially G A transitions as in sheep V regions 61 .
As emphasized by Storb, one cannot detect a strand bias if there is not
also a G/C or A/T bias 8 . In CL-01 cells, the higher than expected
frequency of G mutations recorded from the coding strand is consistent
with either preferential G targeting by the mutational machinery in the
nontranscribed ("top") strand or preferential C targeting by the
mutational machinery in the transcribed ("bottom") strand. In CL-01
cells, this strand polarity is further supported by the finding that T
accumulated almost twice as many mutations as A and is in agreement
with the proposed strand polarity of somatic mutations in the
experimental mouse 2, 5, 8 , although, in the mouse, strand polarity
has been associated with an A > T bias.
The Ser encoded by AGC or AGT, particularly at position 31 of Ig V(D)J sequences, has been reported to constitute a hotspot both in vivo 2, 36 and in vitro 24 . AGC and AGT codons occur five times in the CL-01 Ig VHDJH sequence, and only two of them were mutated, neither at codon 31 (AGC). A comparable sparing of AGC and AGT codons by mutations has been found in several human Ig V(D)J sequences in vivo 38 and in vitro 20 . In general, many AGC/T sequences are not mutational hotspots, and the local targeting of mutations is not simply dependent on the two or three bases surrounding the hotspot 2 . Other structural features, such as palindromes, may well be important 62 . Likewise, a scarcity of mutations was found in the different iterations of the RGYW motif in the of CL-01 cell VHDJH gene sequence, despite the originally proposed hotspot nature of this sequence 35 . This suggests that the hypermutation process has been subjected to evolutionary pressure to yield substitutions over the whole V(D)J region and that aspects of local secondary structure are also likely to contribute to the formation of mutational hotspots. These and other mechanistic issues, including those related to the activation of the mutational machinery, can be optimally addressed by further use of CL-01 cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Paolo Casali, Division of Molecular Immunology, Department of Pathology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: GC, germinal center; BCR, B cell surface receptor for Ag; CD40L, CD40 ligand; CD40L-293 cells, CD40L-transfected human embryonic kidney 293 cells; CDR, complementarity determining region; FR, framework region; R, replacement (mutation); S, silent (mutation); SSCP, single-strand conformational polymorphism; TCM, T cell conditioned medium; s, surface.
Received for publication September 4, 1998. Accepted for publication November 13, 1998.
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)
depict deletions, and the cross (x) depicts a stop codon.
B, Sequences containing the 121 point-mutations
identified in the 38 VHDJH-CH and
the 16 point-mutations in the 9 V