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*
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612; and
Department of Pathology, Program in Immunology, Tufts University School of Medicine, Boston, MA 02111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3, µ
, and µ
, but not
µ1 CSR at its endogenous loci. The 1.B4.B6 cell line was used
in combination with plasmid-based isotype-specific S substrates in
transient transfection assays to test for the presence of trans-acting
switching activities. The 1.B4.B6 cell line supports µ3, but
not µ1 recombination, on S substrates. In contrast, normal
splenic B cells activated with LPS and IL-4 are capable of
plasmid-based µ1 CSR and demonstrate that this S plasmid is
active. Activation-induced deaminase (AID) was used as a marker to
identify existing B cell lines as possible candidates for supporting
CSR. The M12 and A20 cell lines were identified as AID positive and,
following activation with CD40L and other activators, were found to
differentially support µ and µ plasmid-based CSR. These
studies provide evidence for two new switching activities for
µ1 and µ CSR, which are distinct from µ3 and
µ switching activities previously described. AID is expressed
in all the B cell lines capable of CSR, but cannot account for the
isotype specificity defined by the S plasmid assay. These results are
consistent with a model in which isotype-specific switching factors are
either isotype-specific recombinases or DNA binding proteins with
sequence specificity for S DNA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, C3, C1, C2b,
C2a, C, and C, which are each
(except ) coupled with unique switch (S) regions. Isotype switching
focuses on S DNA and produces new hybrid S DNA combinations. The
composite Sµ-Sx DNA configuration is formed on the chromosome while
the intervening genomic material is looped-out and excised as a circle
(1, 2, 3, 4). The presence of double strand breaks (DSBs) in S
DNA (5) and the dependency of CSR on the DNA-dependent
protein kinase catalytic subunits (6), Ku80
(7) and Ku70 (8), components of the DNA-PK
complex involved in DSB repair, strongly suggest that CSR is resolved
through a nonhomologous DNA-end joining process (reviewed in Ref.
9). Activation-induced cytidine deaminase (AID), a putative RNA editing enzyme, has been implicated in both CSR and somatic hypermutation in mouse and human (10, 11). AID expression is restricted to activated germinal center B cells that undergo CSR and somatic hypermutation. AID protein sequence is homologous to the mammalian RNA editing deaminase, APOBEC-1 (12, 13). APOBEC-1 is the catalytic component of the complex that edits apolipoprotein-B mRNA by changing a single ribo-cytidine to uridine (14). This RNA editing event generates a stop codon and causes the production of a truncated apoB polypeptide by cells of the small intestine. AID has deaminase activity when tested for deamination of deoxycytidine (13). It remains unclear whether AID functions directly or indirectly in mediating CSR and somatic hypermutation (15).
The isotype specificity of the CSR process is achieved in part through
production of germline transcripts (gts; reviewed in Ref.
16). Accumulating evidence suggests that in addition to
gts, there are other factors that contribute to CSR. For example, B
cells lacking the transactivation domain of c-Rel are capable of
expressing both and gts, but are able to carry out only
µ and not µ CSR (17). Similarly, B cells
that are deficient in the p50 subunit of NF-
B can produce 1, ,
and 3 gts, but switch only µ1 and not µ
(18) or µ3 (19). Recent analyses that
assay for CSR on extrachromosomal S plasmids demonstrate the existence
of distinct switching activities that mediate µ3 and µ
CSR and are independent of gt expression (20). However,
questions remain regarding the number of isotype-specific S factors
that function in CSR, the nature of their recognition of S DNA, and
their relationship to AID.
In this study, we report studies using a plasmid-based transient
transfection assay for CSR to test for the presence of isotype-specific
transacting factors. We provide evidence for two new switching
activities that mediate µ1 and µ CSR. These activities
are distinct from each other and from the previously described
activities for µ3 and µ CSR and they indicate a high
degree of DNA sequence specificity in the mechanism of CSR. We found
that in switching B cell lines, endogenous CSR was strictly correlated
with the coordinate expression of the appropriate gt and
isotype-specific switching activity, indicating that the transacting S
factors are integral to the process of CSR. All the B cell lines
capable of supporting CSR also express AID. Thus, AID expression per se
does not fully explain the isotype specificity detected in the S
plasmid assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transformation of BALB/c splenocytes was conducted by
coculturing LPS-activated B cells with a fibroblast line expressing the
J2 virus as previously described (21, 22). Stable
transformants were cloned by limiting dilution and were assayed for
their phenotypes by FACS staining (data not shown). One of these clones
was designated 1.B4.B6. Culture conditions for 1.B4.B6, A20.3, M12,
BalI7 (20), I.29µ (23), CH12.LX
(24), TIB114 (25), J558, J558L, and MPC11
(26) were previously described. Splenic B cells were
prepared and activated with LPS as described previously
(20) in the presence or absence of rIL-4 (1000 U/ml, a
gift from C. Snapper). 1.B4.B6 cells were stimulated with LPS
(50 µg/ml; Sigma-Aldrich, St. Louis, MO), CD40 ligand (CD40L), in the
presence or absence of rIL-4 or human rTGF
1 (1 ng/ml; R&D Systems,
Minneapolis, MN). CD40L is the rCD40L-CD8 fusion protein secreted by
the J558L mouse myeloma cell line (27). Supernatant
containing CD40L was used at 1/2 dilution (v/v) for stimulation. CD40Lc
is the supernatant collected from untransfected J558L cells. FACS
analysis was conducted using 1.B4.B6 cells washed twice in ice-cold PBS
buffer (1x PBS with 1% FBS and 0.1% sodium azide). Cells were
resuspended in 100 µl of PBS buffer and were incubated on ice with
rat IgG2b FcR block (anti-mouse CD32/CD16; BD PharMingen, San
Diego, CA) for 5 min at a concentration of 5 µg/ml. Surface IgG3 was
detected by incubating the cells with 5 µg/ml FITC-conjugated goat
anti-mouse IgG3 Ab (Southern Biotechnology Associates, Birmingham,
AL) on ice for 30 min. Flow cytometry analysis was performed on a
FACSCaliber flow cytometer (BD Biosciences, Mountain View, CA). Viable
cells were gated by exclusion of propidium iodide and were analyzed for
surface phenotype. Transfection and genomic DNA isolation was conducted
as previously described (20).
Construction of plasmids
p218 and pG3.
IEP were described previously (20, 28) and p218 and pG3.IEP are referred to in this study
as pA.1 and pG3.1, respectively. To build additional S
substrates, a 300-bp fragment corresponding to residues 1813 to 2101 of
mouse S3 (M12182.1) was excised from pS3A (our unpublished
data), a 0.78-kb fragment corresponding to residues 3878 to 4661 of
S1 was isolated from the p1/B.V1 plasmid (M12389; Ref.
29), and an 0.8-kb fragment corresponding to residues 405
to 1170 of S (M17012.1) was derived from p 3PK (a gift from J.
Stavnezer). The S region fragments were individually cloned into
the XbaI site of Bluescript KS-,
orientation was determined, and then fragments were excised by a
BamHI-NotI digestion. The
BamHI-NotI fragments were cloned into gel
purified pA.1 from which the S fragment had been excised in a
BamHI-NotI digestion. The new S substrates
pG3.1-S, pG1.1, and pE1.1 were generated by introduction of
the cloned S3, S1, and S fragments into the prepared p218,
respectively.
RT-PCR and digestion circularization (DC)-PCR
RT-PCR for gts was conducted as described (18) with
modifications. Primers for 3, 1, , and gts and GAPDH were
previously described (18, 19, 30). RT-PCR primers for
AID were previously described (13). A PCR
protocol for AID amplification of 30 cycles consisting of 95°C for 1
min, 58°C for 1 min, and 72°C for 1 min was followed by a final
7-min elongation at 72°C. All PCR contained 2.5 U of Taq
polymerase (Boehringer Mannheim, Indianapolis, IN), 0.2 mM each dNTP,
2.0 µCi [-32P]dCTP (3000 Ci/mmol; NEN,
Boston, MA), 1x PCR buffer with Mg2+ provided by
the manufacturer, and 0.2 µM each primer. The PCR products were
purified by phenol and chloroform extraction and were separated by
electrophoresis on 7% polyacrylamide gels. The incorporation of
radioactive dCTP was quantified by phosphorimaging using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
PCR amplification and cloning of Sµ/S3 hybrid molecules
The PCR amplification and cloning of Sµ/S3 molecules was
conducted as previously reported (25) except that primer
µ-1.2 (5'-GCTGGGGTGAGCTCAGCTATGCTACGC-3'), which anneals to
positions 5307–5333 at the 5' end of the germline Sµ (MUSIGCD07),
was used. The C gene was analyzed by PCR amplification
using C-1 (5'-CATCTGGAGGTGCCTCAGTCGTGTGC-3') and C-2
(5'-ACATTCCCCAAGCCCCAGAGTTTCC-3') primers, which anneal to
positions 1030–1055 and 2398–2422 on the C gene
(MUSIGKAF3), respectively, and gave rise to a 1.4-kb product. The
C PCR began with denaturation at 95°C for 3 min,
followed by 30 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C
for 2 min, and a final incubation at 72°C for 7 min. PCR
amplification of the S1 cis flanking DNA was conducted
using primer Rg1 (5'-TTCAGCCTGGATGCCTACATTAAGAC-3') and dc-µ.1 or
dc-µ.2 primers (19) using the PCR amplification
conditions as for C amplification.
Endogenous and plasmid specific DC-PCR and the bacterial transformation assay
DC-PCR analysis for endogenous µ3 and µ1 CSR was
performed as described (19). DC-PCR analysis for
endogenous µ and µ CSR was performed as described
(18) with modifications. Two rounds of PCR using nested
primer sets were performed where the Sµ primers were dc-µ.1 and
dc-µ.2, as previously described (19). The S primers
were dc-.1 (5'-ACACCGATGCAGGATACACCCCAGACC-3') and dc-.2
(5'-CCCCAGACCTTCCAAGACTATG-3'), and the S primer was dc-.1
(5'-CCCTCTGATGCACACCCTCACAGG-3'). In the second round of PCR for
µ DC-PCR, the dc-µ.2 and dc-.1 were used. The DC-PCR
products for µ and µ CSR were 585 and 499 bp,
respectively. DC-PCR analysis for the S plasmids was performed as
described previously (20) with modifications. Transfected
genomic DNA (500 ng) was digested overnight with SacI (MBI
Fermentas, Amherst, NY) in the presence of 1 µM spermidine in a total
volume of 100 µl. Digested DNA (10 ng) was ligated overnight in the
presence of 200 ng of untransfected genomic DNA in a total volume of
100 µl using 6 U of T4 DNA ligase (MBI Fermentas), and 2 µl of the
ligated DNA was used for PCR amplification. The 510-bp fragment was
amplified with primers P1 and P4 in the presence of 3 µCi of
[-32P]dCTP for 26 cycles each consisting of
95°C for 1 min, 60°C for 40 s, and 72°C for 1 min 30 s,
followed by a final step at 72°C for 7 min. The 81-bp fragment was
amplified using plasmid-specific primers P2 and P31
(5'-CCTGTTACCCAGGTTACCTTGT-3') and the 110-bp fragment was amplified
using primers P51 (5'-CCTGTTACCCAGGTTACCTTGT-3') and P6
(5'-GACCGACGCCGTTCTGGCTCCTCATA-3'). Amplification of the 81- and 110-bp
fragments was as described above, except that PCR programs consisted of
36 and 34 cycles, respectively. The bacterial transformation assay was
conducted as previously described (20). Further
experimental details are available upon request.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a paucity of B cell lines that can be induced to undergo CSR. We have derived B cell lines by transforming mitogen-activated murine splenic B cells with the J2 retrovirus carrying v-raf-v-myc oncogenes (see Materials and Methods; Refs. 21, 22). One cell line, 1.B4.B6, was taken for further analysis and was found to express surface markers characteristic of mature, naive B cells; IgM+, IgD+, and B220+. Two rearranged JH bands were observed in each of the subclones of 1.B4.B6 by Southern analysis indicating clonality. Clonality was verified by sequencing the VDJ regions from PCR products obtained from cDNA templates generated from each of the subclones. The VH of 1.B4.B6 cells differed at only a single nucleotide residue from Q52, the D is identical with DFL16.2, and the J is identical with J3. There were several N insertions at both the VD and DJ joins (data not shown).
CSR can be stimulated in 1.B4.B6 cells
J2-transformed B cell lines were previously shown to respond to
the presence of a TH2 type T cell line and
Staphylococcal enterotoxin B by proliferation and secretion of very low
amounts of IgG1 (30–70 ng/ml) (21). We reasoned that T
cell signals such as soluble CD40L alone or in combination with other
mitogens and cytokines might stimulate robust isotype switching in
1.B4.B6. To test this hypothesis, 1.B4.B6 cells were grown in the
presence or absence of inducers and were analyzed for membrane IgG3
expression by FACS analysis. Surface IgG3 expression was essentially
undetectable in unstimulated 1.B4.B6 cells and was only marginally
detectable in cells stimulated for 4 days by CD40L or LPS (Fig. 1
A). In contrast, 15.3% of
the cells stimulated with CD40L and LPS expressed membrane IgG3,
demonstrating a synergistic induction by this combination of
stimuli.
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3, 1, , and gts (Fig. 1
B, lane 1). Production of the 3 gt was
induced following treatment with CD40L and LPS in the presence or
absence of TGF or IL-4 (Fig. 1B). Individual activators
or CD40L control (CD40Lc) supernatant (see Materials and
Methods) only slightly augmented 3 gt expression (Fig. 1B). Stimulation of 1.B4.B6 cells with CD40L and LPS in the
presence of IL-4 induced 1 and gts expression (Fig. 1B, lane 8), whereas CD40L and LPS in the
presence of TGF induced gt (Fig. 1B, lane
7). These observations demonstrate that gt production is inducible
in 1.B4.B6 cells and parallels the cytokine requirements for gt
induction found in normal splenic B cells (reviewed in Ref.
16).
CSR is a deletional process that occurs between Sµ and one of the
downstream S regions to generate a complex mixture of hybrid Sµ/Sx
molecules of various sizes. To determine whether µ3 CSR could
be induced in 1.B4.B6 cells, a PCR assay for detection of composite
Sµ/S3 molecules was used (25). Multiple Sµ/S3
hybrid molecules were detected in DNA isolated from 1.B4.B6 cells
following stimulation with LPS and CD40L, but were not found in
unstimulated cells (data not shown). To verify that the Sµ/S3
composite molecules amplified by PCR represent bona fide CSR events,
the Sµ/S3 hybrid molecules were cloned and 12 clones containing
inserts of different sizes were randomly chosen for automated DNA
sequence analysis. All 12 clones were found to contain Sµ and S3
sequences, and eight clones were confirmed to contain Sµ/S3 S
junctions (Fig. 1C). In all of these clones, the S junctions
showed the same characteristics as previously described breakpoints
found in physiological CSR events (31).
1.B4.B6 cells were analyzed for their capacity to switch to each of
four isotypes using the DC-PCR analysis (32) summarized in
Fig. 2A. The nonrearranging
acetylcholine receptor (nAChR) gene was used as a control
for digestion and ligation reactions, and all samples tested were
positive for the nAChR DC-PCR product (Fig. 2, B–D). DNA from TIB114 served as a positive
control for the Sµ/S3 DC-PCR product (Fig. 2B,
lane 7). DC-PCR product was only evident in DNA from cells
induced with LPS and CD40L (Fig. 2B). These findings
directly demonstrate the induction of µ3 CSR in 1.B4.B6 cells
and are consistent with the observation of surface IgG3 expression,
3 gt induction, and the presence of Sµ/S3 composite fragments
in LPS- and CD40L-stimulated cells.
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or CD40L
and LPS and IL-4, which induce the gt, and the 1 and gts,
respectively, and they were then analyzed by DC-PCR (Fig. 2, C and D). DNA from the IgA-producing myeloma cell
line, J558, served as a positive control for the Sµ/S DC-PCR
product. The Sµ/S DC-PCR product was detected only in DNA from
1.B4.B6 cells stimulated with CD40L and LPS and TGF, indicating the
induction of µ CSR (Fig. 2C). DNA from LPS and
IL-4-treated splenic B cells, which switch µ1 and µ,
served as a positive control for the Sµ/S1 and Sµ/S DC-PCR
products (Fig. 2D). No Sµ/S and Sµ/S1 DC-PCR
products were detected in unstimulated 1.B4.B6 cells or cells
stimulated with CD40L and LPS (Fig. 2D). 1.B4.B6 cells
stimulated with CD40L and LPS and IL-4 gave rise only to Sµ/S but
not Sµ/S1 DC-PCR products (Fig. 2D). No Sµ/S1
DC-PCR product was detected from CD40L and LPS and IL-4-stimulated
1.B4.B6 cells even after additional cycles of PCR (data not shown).
Priming sites for Sµ/S1 DC-PCR were intact in 1.B4.B6 cells
because the primers dc-1.1 or dc-1.2 in combination with a
downstream primer R1 (see Fig. 2A) amplified a PCR
product of the expected size and appropriate DNA sequence (data not
shown). The priming sites located at 5' end of Sµ are intact because
the dc.µ-1 and dc.µ-2 primers were able to participate in
amplification of Sµ/S3, Sµ/S, and Sµ/S DC-PCR products
in activated 1.B4.B6 (Fig. 2, B–D). Genomic
Southern analysis demonstrated that the overall S1 region in 1.B4.B6
cells is intact (data not shown). Taken together, these findings
indicate that 1.B4.B6 cells can be induced to switch µ3,
µ, and µ, but not µ1. However, it is possible
that other combinations of stimuli are capable of inducing µ1
switching in 1.B4.B6 cells.
Distinct factors mediate µ3 and µ1 plasmid-based
CSR
One hypothesis to explain the absence of endogenous µ1 CSR
in 1.B4.B6 cells is that these cells fail to express the transacting
µ1 switching activity. We previously demonstrated that distinct
switching activities independently mediate µ3 and µ CSR
using extrachromosomal S substrates that are capable of detecting
transacting factors (20). To test the hypothesis that a
µ1 switching factor is absent in 1.B4.B6 cells, a new S plasmid
was constructed to assay for µ1 CSR and is referred to in this
study as pG1.1 (Fig. 3A).
CSR on pG1.1 and pG3.1, which assays µ3 switching, was
compared in various cell lines by transient transfection, isolation of
DNA from nuclei, and transformation of bacteria to recover S/S
recombinant plasmids as previously described (20).
Recombination between S regions leads to deletion of TK and
loss of a unique EcoRI restriction site, and those plasmids
that are resistant to EcoRI digestion are likely to be S/S
recombinants (Fig. 3A). To control for possible S/S
recombination in bacteria, in the same experiments, pG3.1 and
pG1.1 DNA were untreated or digested with EcoRI, and the
frequency of ampicillin resistant colonies was determined. The
efficiency of transformation with intact plasmid was 5 x
107 colonies/µg. Following transformation, we
found no ampicillin-resistant colonies, indicating that
EcoRI resistance arises as a consequence of transfection
into cell lines. Table I shows the
cumulative numbers of colonies screened and S/S recombinant plasmids
recovered and verified by restriction mapping from several transfected
samples, as indicated. PG3.1 supported µ3 switching in
1.B4.B6 cells and LPS-activated splenic B cells, confirming previous
studies (Table I) (20). No S/S recombinant pG1.1 were
recovered from LPS-activated splenic B cells, unstimulated 1.B4.B6,
1.B4.B6 stimulated with the combination of CD40L and LPS and IL-4, or
unstimulated I.29µ, A20, or M12 cells (Table I). In contrast, 10 S/S
recombinants derived from pG1.1 were recovered from LPS and
IL-4-activated splenic B cells. The 
2 analysis
indicates that all the cell lines and LPS-activated B cells are
significantly different from the LPS and IL-4-activated B cells with
respect to the incidence of S/S recombinant plasmids recovered (Table I). The 10 S/S recombinant pG1.1 plasmids were taken for DNA
sequence analysis and the S junctions were demonstrated to lie within S
region DNA. The Sµ/S1 junctions were located for four plasmids
(4BG-121, 142, 157, and 190; Fig. 3B) and showed direct
joining of Sµ and S1 DNA without deletion, duplication, or
nucleotide insertion at the breakpoints. The microhomology at the
Sµ/S1 junctions was zero to four bases and is similar to that
usually observed (31). In the remaining six clones, CSR
occurred in the S regions; however, the junctions were too far from the
ends to be located by automated DNA sequencing. These findings indicate
that pG1.1 is capable of CSR and that the µ1 switching
factor is induced by IL-4 in LPS-activated B cells, whereas the
µ3 switching factor is found in B cells activated with LPS
alone. These results also demonstrate that 1.B4.B6 cells can produce
the µ3 but not the µ1 switching factor, and they
confirm our hypothesis that the µ3 and µ1 switching
factors are distinct.
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1 in pG1.1 is 0.78 kb as compared with the 2.0 kb
of S3 DNA in pG3.1. To determine whether the length of S DNA in
the S plasmid contributes to the frequency of CSR events, we
constructed pG3.1-S, which contains 300 bp of S3 DNA (Fig. 3A) and compared its switching frequency to that of pG3.1
in 1.B4.B6 cells. S/S recombinant pG3.1-S and pG3.1 were
recovered at essentially equal frequencies (Table I). This result
demonstrates that 300 bp of S3 containing only five tandem repeats
are sufficient to support plasmid-based CSR and suggests that the
length of S1 present in pG1.1 is sufficient for
plasmid-based CSR.
To further evaluate the expression profile of µ1 switching
activity, a previously described semiquantitative DC-PCR assay was
adapted to assess CSR on pG1.1 (20) and is shown in
Fig. 3C. The linear range of detection for the
vector-associated 510-bp fragment was established using 2-fold serial
dilutions of pG1.1 into 0.5 µg of genomic DNA followed by DC-PCR
analysis in the presence of radiolabled deoxynucleotides (Fig. 3D). The addition of increasing amounts of pG1.1 (0.25–2
ng/ml) resulted in a linear increase of vector-specific 510-bp product
(Fig. 3D). One standard sample (0.5 ng/ml) was taken from
this titration and was included in all subsequent studies to assure
that the amplification product derived from the plasmid backbone was
always in the linear range of detection. To confirm that the conditions
chosen for intramolecular ligation were also valid for the
Sµ/S1-associated 81-bp DC-PCR product, we tested for the presence
of this fragment using a range of concentrations of plasmid digested
with SacI (Fig. 3D). At high concentrations of
input plasmid (2 ng/ml), intermolecular ligation occurred and produced
the 81-bp product, whereas at dilute plasmid concentrations (<1
ng/ml), no 81-bp product was detected, demonstrating that fragment
ligation was intramolecular. A standard sample, 0.5 ng, was taken from
this titration and was included in all subsequent studies to assure
that the amplification product derived from the plasmid backbone was
always in the linear range of detection.
DC-PCR analysis of CSR on the pG1.1 plasmid was undertaken in
activated splenic B cells and in switching and nonswitching B cell
lines. The intact standard sample of pG1.1 plasmid (0.5 ng/ml) was
included as a control and showed no evidence of the 81- or 110-bp
DC-PCR products, but was positive for the 510-bp DC-PCR product as
expected (Fig. 3E, lane 1). A S/S recombinant
derivative of pG1.1 (0.5 ng/ml) was included as a positive control
and gave rise to the 510-bp fragment associated with the vector
backbone and the 81-bp fragment associated with S/S recombinant
molecules, but no 110-bp fragment associated with the circular excision
product because it was previously lost (Fig. 3E, lane
2). The 81- and 110-bp fragments were detected in B cells induced
with LPS and IL-4, but not with LPS alone and this detection confirms
that pG1.1 is recombinogenic in B cells that undergo physiological
switching. Recombination of pG1.1 is undetectable in all the B cell
lines tested, including 1.B4.B6. Taken together, these results
demonstrate isotype-specific CSR profiles of the pG3.1 and pG1.1
plasmids and support the conclusion that µ3 and µ1 CSR
are mediated, at least in part, by S region-specific factors.
S plasmid assays for µ and µ recombination
S and S regions share considerable homology to each other.
This raises the question whether µ and µ recombination
are mediated by distinct factors. A S plasmid, pE.1, carrying S
was constructed and is identical with pG3.1, pG1.1, and pA.1
in all respects except for the identity of the downstream S region
(Fig. 3A). The facility with which pE.1 and pA.1
support CSR in LPS-activated splenic B cells and a panel of B cell
lines was compared using the bacterial transformation
assay (Table II). Both pE.1 and
pA.1 were found to recombine at similar frequencies in LPS-activated
B cells, I.29µ, and 1.B4.B6 cells, suggesting that factors supporting
µ and µ CSR are present. In contrast, very rare
recombinant pA.1 and pE.1 were found in unstimulated A20 and M12
cells, indicating that the activities supporting plasmid-based CSR are
very poorly expressed in these cells. 2
analysis indicates that pA.1 and pE.1 S frequencies in 1.B4.B6,
I.29µ cells, and LPS-activated splenic B cells are essentially
identical and significantly different from those found in M12 and A20
cells. Furthermore, switching on the pE.1 and pA.1 plasmids was
constitutive in 1.B4.B6 and I.29µ cells, confirming previous
observations for pA.1 (20). DNA sequence analysis of S
junctions derived from the pE.1 and pA.1 plasmids and the S
junctions are shown (Fig. 4, A
and B). Six recombinant pE.1 plasmids were sequenced, and
four S junctions were found. Sixteen recombinant pA.1 plasmids were
sequenced, and six S junctions were found. All the junctions had
characteristics associated with previously defined CSR breakpoints
(31). The simultaneous presence of the µ and
µ switching activities in I.29µ and 1.B4.B6 could arise from
coexpression of two distinct activities or from a single activity with
specificity for both S and S DNA. To distinguish between these
alternatives, additional B cell lines are required that can be induced
to undergo µ or µ CSR.
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Recent studies show that the AID gene is specifically
expressed in mature normal B cells and is required for CSR in vivo
(10, 11). We reasoned that cell lines that express
AID might have the ability to undergo CSR, and we screened a
panel of B cell lines to assess AID expression by RT-PCR.
GAPDH expression was used as an internal control for cDNA
template input for all samples tested (Fig. 5, A and C). The
AID RT-PCR product was detected in LPS-activated splenic B
in the presence or absence of IL-4, as previously reported
(13), and in the mature B cell lines, 1.B4.B6, CH12.LX,
I.29µ, M12, and A20 (Fig. 5A). In contrast, 70/Z3 and
8A5.4A5.II.88, pre-B cell lines, TIB114 and B1.8-, hybridomas, and
J558, a myeloma, did not express detectable levels of AID
transcript. In the same experiment, a 2-fold serial dilution of cDNA
from LPS and IL-4 B cells and CH12.LX cells (taken from the same cDNAs
used in Fig. 5A, lanes 2 and 4) was
used to prepare standard curves (Fig. 5B). The arrows shown
in the linear regression plots indicate that the signal intensities for
the AID RT-PCR products (for the same dilution of cDNA used in Fig. 5A, lanes 2 and 4 and C,
lanes 6, 14, and 15) are within the
linear range of detection.
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C) was not correlated with the induction
of CSR (for M12 and A20 see below) because some stimulation mixtures
led to CSR and to a concomitant reduction of AID
expression. Forexample, CD40L and LPS induction of 1.B4.B6
cells leads to stimulation of µ3 CSR (Fig. 1), but to reduction
of AID expression (Fig. 5C, compare lanes 7 and
8). Similarly, in normal B cells stimulated with LPS or LPS
and IL-4, CSR was induced µ3 and µ1, respectively,
(data not shown), but the level of the AID transcript decreased in
response to LPS as compared with cells induced with LPS and IL-4 (Fig. 5C, lanes 5 and 6). Thus, the level of
AID transcript is not directly related to the facility with which CSR
occurs. In contrast, gt expression is directly related to the level of
CSR (16).
Among the AID-expressing cell lines, M12 and A20,
IgG+ B cell lymphoma cell lines have not been
reported to switch. To explore the switching potential of M12 and A20,
these cell were stimulated with various combinations of activators and
they were tested for their ability to express gts and to engage in CSR.
Unstimulated I.29µ cells, which constitutively express and
gts, were used as positive controls in the RT-PCR analysis (Fig. 6A, lane 8). In M12
cells, gts were induced by each combination of activators tested,
but were not found in unstimulated cells (Fig. 6A,
lanes 2–4). In contrast, gts were expressed
in unstimulated M12 cells (Fig. 6A, lane 1), and
treatment of the cells with any combination of inducers reduced gt
expression (Fig. 6A, lanes 2–4).
DC-PCR was used to assess CSR status, and the IgA expressing J558 and
splenic B cells induced with LPS and IL-4 were used as positive
controls for Sµ/S and Sµ/S DC-PCR products, respectively,
(Fig. 6B, lanes 8 and 9). In M12
cells, no µ switching could be detected under any conditions
tested (Fig. 6B, lanes 1–4), whereas
µ switching was induced with CD40L and IL-4 or CD40L and LPS
and IL-4, but not in unstimulated cells or cells treated with CD40L
alone (Fig. 6B, lanes 1–4). In A20
cells, the gt was not detected under any of the conditions tested,
but the gt was found in all of the conditions analyzed, albeit at
different levels (Fig. 6B, lanes
5–7). However, neither µ nor µ CSR was
evident under any circumstances (Fig. 6B, lanes
5–7).
|
and µ
recombination
To examine the profile of plasmid-based switching activities, M12
and A20 were transiently transfected with either pA.1 or pE.1 and
were analyzed using the bacterial transformation assay. In M12 cells,
the pA.1 plasmid underwent CSR following CD40L activation only
(Table II). In contrast, the pE.1 plasmid underwent CSR following
CD40L, LPS, and IL-4 stimulation, but not in unstimulated cells or
cells activated with CD40L (Table II). The p values derived
from the 2 analysis indicate that the
µ and µ switching factors are differentially inducible
in M12 cells. It is notable that in M12 cells, the same activation
conditions that gave rise to endogenous µ CSR (Fig. 6B) also supported pE.1 recombination.
In A20 cells, S/S recombinant pA.1 was detected in cells activated
with CD40L and TGF or with CD40L and IL-4, but not in unstimulated
cells or cells activated with CD40L alone (Table II). Furthermore, in
A20 cells, no significant pE.1-associated recombination events were
found in any activation condition tested. The
2 analyses confirm these conclusions, which
are summarized in Fig. 6, C and D. In A20 cells,
the absence of endogenous µ CSR following induction with CD40L
and IL-4 or CD40L and TGF is most likely due to the absence of the
gt (Fig. 6A, lanes 5–7) because
S/S recombinant pA.1 was detected under these conditions. In these
cells, the absence of endogenous µ CSR is correlated with the
absence of pE.1-associated recombination because gts are present
in these cells (Fig. 6A, lanes
5–7).
It is important to note that our S plasmids do not contain an origin of
replication (28), whereas the reporter plasmids for VDJ
recombination undergo episomal replication (33). Switching
frequencies derived from the bacterial transformation assay for the
pE.1 and pA.1 S plasmids transfected into M12 and A20 cells
ranged from 8.3 x 10-4 to 2.3 x
10-4 (Table II) and are similar to previously
reported results (20). The frequency of S/S recombinant
plasmids found in switching B cells is similar to that found for a VDJ
plasmid that carries a defective origin of replication and is higher
than the incidence VDJ recombination in the absence of an origin of
replication (33).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3
and µ CSR demonstrated that unique switching activities
mediated these CSR events. However, the tandem repeats in the S3 and
S regions are relatively unrelated and there are numerous sequence
differences and overall length differences between them. Our new
studies indicate that two distinct activities mediate µ3 and
µ1 switching events based on the differential capacity of the
µ3 and µ1 S substrates to support recombination in
1.B4.B6 cells and in normal mitogen-activated splenic B cells. These
findings are striking because there are only 12 nucleotide differences
between the 49-bp consensus S1 and S3 tandem repeats
(34). The S3 and S1 regions differ structurally
where S1 is the longest and the most complex. However, the S1 and
S3 DNA segments located in the pG3.1 and pG1.1 S substrates
are composed only of simple tandem repeats (34). Thus,
global structural differences are unlikely to contribute to the
differential recognition of S1 and S3 regions by the µ1
and µ3 switching activities found in 1.B4.B6 cells and in
mitogen-activated splenic B cells. Our studies also indicate that there
are distinct activities that mediate µ and µ S events.
This is based on the differential induction of plasmid-based µ
and µ switching in the IgG+ B cell
lymphoma cell lines, M12 and A20. The S and S regions are members
of the same subfamily of S regions and are most similar to Sµ
(34). Therefore, it is surprising that S is uniquely
recognized as compared with both Sµ and S. Our studies demonstrate
that relatively small differences in S DNA sequence can confer isotype
specificity to the switching reaction.
In B cell lines and in mitogen-activated normal B cells, the detection
of isotype-specific switching activities using plasmid-based assays was
paralleled by the ability of the endogenous loci to switch. There were
two exceptions to this rule. A20 and M12 cells could each be induced to
express plasmid-based µ switching activities, but did not
switch µ at their endogenous loci. The absence of gts in
A20 and M12 cells under conditions that provide for the expression of
µ switching activities is the simplest explanation for this
endogenous switching deficit. Our studies indicate that the µ1
switching activity is expressed only in B cells activated with LPS and
IL-4, whereas µ activity is present in LPS B cells. However, at
the endogenous locus, switching to is more sensitive to IL-4
induction than is switching to 1. The IL-4-dependent switching
phenomenon most likely arises from the differential induction of the e
and g1 gts by IL-4 (35). The absence of the µ1
switching activity in LPS B cells and its induction by IL-4 ensures
that µ1 switching occurs only when both the 1 gt and the
µ1 switching activities are coexpressed. Similarly, the absence
of the gt in LPS B cells, even in the presence of the µ
switching activity, assures the IL-4 dependence of this event.
Together, our studies indicate that CSR at endogenous loci is
strikingly correlated with the coordinated expression of both
isotype-specific gts and isotype-matched switching activities such that
endogenous CSR is absent if either element is deficient or limiting.
Furthermore, the expression of AID transcript led to the
successful identification of M12 and A20 cells as containing CSR
potential, suggesting that this marker may indicate a unique stage of B
cell differentiation.
Several groups have previously reported DNA constructs designed to
assay CSR. These S substrates and accompanying assay systems had a key
limitation in that recombination was not limited to B cells in general
or switching B cells in particular (36, 37, 38, 39, 40). Recently, a S
substrate assayed in stable transfection experiments and using a single
switching cell line, CH12, reported that CSR was not dependent on S
region identity (41). Although the CH12 cell line switches
predominantly µ, it is also capable of occasional µ3
and µ1 switching in a subclone-dependent fashion
(24), implying that several isotype-specific switching
activities can be expressed in these cells. Kinoshita et al.
(41) did not further test the specificity of their S
constructs in other switching cell lines with well-defined switching
profiles. Therefore, the ability of several isotype-specific S
substrates to undergo recombination in CH12 cells may not be
inconsistent with our results. Additional work is required to clarify
this issue.
S regions vary in length from 1 to 10 kb. Surprisingly,
comparison of S substrates with full-length S3 containing 44 tandem
repeats or a short version of S3 containing five tandem repeats
indicated no discernable differences in CSR frequency as assessed by
the bacterial transformation assay. Recently, a mouse was constructed
in which the Sµ region, composed of
(GAGCT)nGGGGT motifs, was removed by targeted
homologous recombination (42). In the
Sµ-/- mouse, 15 GAGCT motifs remained in regions
flanking the original Sµ region and were sufficient to support
endogenous CSR, albeit at a reduced frequency. These studies suggest
that the number of S region tandem repeats may influence the efficiency
of CSR, and are consistent with our findings. Together, these studies
indicate that a limited S DNA target is sufficient to support CSR and
they raise the interesting question of how many tandem repeats
constitute a minimal S region.
Our studies indicate the existence of at least four independent
isotype-specific switching activities. It is possible that switching
activities detected by the S plasmids result from the absence of a
suppressor activity rather than from a positive regulator. In this
scenario, isotype-specific suppressors are present and are selectively
turned off to give rise to isotype-specific switching. Alternatively,
the switching activities could be positive regulators of CSR that
function either at the cleavage step to generate nicks or
DSBs in S DNA or in the resolution of the recombination
intermediates. We favor a model in which the isotype-specific switching
activities are either distinct S recombinases or complexes composed of
a general recombinase and a docking protein with specificity for a
specific S region. The docking protein would function as a DNA binding
protein with specificity for a single S region and with the ability to
recruit the S recombinase to the S region. This would provide for
protection against inappropriate DNA cleavage because the endonuclease
would be unable to directly recognize DNA except in the presence of the
docking protein. We previously identified DNA binding proteins specific
for the tandem repeats of Sµ, S3, S2b, and S1 DNA (19, 43, 44, 45); however, additional investigation is required to
determine whether these proteins are directly involved in CSR and
function as the postulated docking proteins predicted in our model.
AID was originally identified in a subtractive hybridization screen for genes activated upon induction of CSR in a subclone of the CH12 B lymphoma cell line (13). In our studies, plasmid-based switching activities were documented in five mature B cell lines that express AID. Thus, AID expression in mature B cell lines may have strong predictive value with respect to a given cell lines switching potential. It is possible that AID is the endonuclease or regulates the expression of the endonuclease through its putative RNA editing activity. However, the AID-positive B cell lines differ with respect to their pattern of isotype specificity. This strongly implies the there are other factors in addition to AID that are important to CSR and that these additional activities confer isotype specificity to the CSR reaction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Amy L. Kenter, Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612-7344. E-mail address: star1{at}uic.edu
3 Abbreviations used in this paper: CSR, class switch recombination; AID, activation-induced cytidine deaminase; DSB, double strand break; gts, germline transcripts; CD40L, CD40 ligand; DC-PCR, digestion circularization-PCR; CD40Lc, CD40L control; nAChR, nonrearranging acetylcholine receptor; S, switch.
Received for publication October 25, 2001. Accepted for publication December 27, 2001.
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A. L. Kenter, R. Wuerffel, C. Dominguez, A. Shanmugam, and H. Zhang Mapping of a Functional Recombination Motif that Defines Isotype Specificity for {micro}->{gamma}3 Switch Recombination Implicates NF-{kappa}B p50 as the Isotype-specific Switching Factor J. Exp. Med., March 1, 2004; 199(5): 617 - 627. [Abstract] [Full Text] [PDF]
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