| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Medicine, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
light chain rearrangements in the BM and spleen. In an in vitro
IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence
of soluble HEL down-regulated surface IgM expression and also showed
induction of new endogenous light chain rearrangements. Using a
panel of soluble protein ligands with reduced affinity for the HEL-Ig
receptor, the editing response was shown to correlate in a
dose-dependent fashion with the strength of signaling through the B
cell receptor. The finding that the level of B cell receptor
cross-linking sufficient to induce anergy in B cells is also capable of
engaging the machinery required for receptor editing suggests an
intimate relationship between these two mechanisms in maintaining B
cell tolerance. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our laboratory recently showed that transgenic (Tg) overexpression of
the anti-apoptotic protein Bcl-xL allowed
self-reactive bone marrow (BM) B cells to escape central deletion in
mice Tg for both a BCR recognizing hen egg lysozyme (HEL-Ig) and
membrane-bound HEL (mHEL) (10). In the course of these
studies, we noted that recombination-activating gene
(Rag)-2 was strongly induced in BM B cells of
HEL-Ig/mHEL animals in the absence of new light chain
rearrangements. Since these mice showed nearly complete central
deletion of B cells, this suggested an abortive attempt at receptor
editing in response to extremely high levels of BCR cross-linking by
membrane-bound self-Ag. We also found that
bcl-xL allowed HEL-Ig B cells to escape
deletion in the mHEL environment. In the periphery, these "escaped"
B cells were profoundly anergic in both in vitro and in vivo assays,
and showed evidence of extensive receptor editing with up-regulated
Rag-1 and Rag-2 mRNA expression and new
endogenous rearrangements (10).
Based on these results, we were interested in further exploring the
relationship between anergy induction and receptor editing. In anergic
HEL-Ig/soluble HEL (sHEL) double Tg mice, the lysozyme self-Ag is
present in the fluid phase and the level of Ig receptor cross-linking
is significantly lower than in HEL-Ig/mHEL mice (6). The
data presented here indicate that anergic B cells in HEL-Ig/sHEL mice
indeed exhibit evidence of receptor editing, as determined by the
detection of endogenous V-J1 light chain rearrangements. In
addition, new endogenous V-J1 rearrangements could be induced in an
IL-7-driven in vitro BM culture system following incubation of HEL-Ig B
cell cultures with exogenous sHEL or several lysozyme variants with
lower affinity for the HEL-Ig BCR. These data have interesting
implications for our understanding of the nature of B cell tolerance
induction in the BM.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
MD4 HEL-Ig and ML5 sHEL Tg mice (3) were kindly provided by C. Goodnow (Australian National University, Canberra, Australia). Mice were genotyped by PCR of genomic DNA obtained from tail biopsy at the time of weaning. All animals were maintained in specific pathogen-free isolation at the University of Minnesota animal facility. Mice used for experiments were generally 4–8 wk of age.
PCR and RT-PCR
Genomic DNA and mRNA were isolated from fresh spleen and BM
cells or IL-7 culture-derived cells as described previously
(11). Endogenous V-J1 rearrangements were detected
using a semiquantitative PCR assay, essentially as previously described
(10, 11). Control experiments determined that the number
of cycles used for each PCR (28 for V-J1 and 23 for CD14
loading control) were within the linear range of amplification. The
V-J1 PCR was performed with an upstream V degenerate primer:
5'-GGCTGCAG(G/C)TTCAGTGGCAGTGG(A/G)TC(A/T)-3' and a primer that
annealed just downstream of J1: 5'-GCCACAGACATAGACAACGGAAGAA-3'
(12). Amplification conditions were as follows: genomic
DNA (from 4 x 104 cells) in 1x PCR buffer
(Boehringer Mannheim, Indianapolis, IN), 2.5 mM
MgCl2, 200 µM dNTPs, 200 ng V degenerate
primer, 100 ng J1 primer and 1.25 U Taq polymerase
(Boehringer Mannheim), with cycling conditions: 97°C for 45 s,
70°C for 1 min, and 72°C for 2.5 min for 5 cycles, followed by
94°C for 45 s, 70°C for 1 min, and 72°C for 2.5 min for
another 23 cycles, and final extension at 72°C for 6 min. PCR
amplification of C was as described elsewhere (13).
CD14 control PCR amplifications were performed using the
forward primer 5'-GCTCAAACTTTCAGAATCTACCGAC-3' in combination with a
reverse primer, 5'-AGTCAGTTCGTGGAGGCCGGAAATC-3' (14).
Amplification conditions were similar to that of the V-J1 PCR except
that the reactions contained 100 ng of each primer, products were
amplified with AmpliTaq Gold polymerase (Perkin-Elmer, Norwalk, CT),
the denaturing temperature was 66°C, and 23 total cycles were used.
RT-PCR was performed using oligo(dT)-primed RT of total RNA, followed
by PCR with Rag-2 and ß-actin primers
as described previously (10). PCR products were separated
on 1% agarose gels, transferred to nitrocellulose, probed with
radiolabeled internal oligonucleotides, and visualized by
autoradiography. Densitometry was performed using a Bio-Rad GS-700
Imaging Densitometer and Bio-Rad Molecular Analyst software (version
2.1; Bio-Rad, Richmond, CA). In some instances blots were also directly
analyzed using a Storm 840 PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) and ImageQuant software (version 4.1), with comparable
results.
IL-7 BM cultures
BM cells were flushed from tibiae and fibulae of HEL-Ig mice.
Cell suspensions were depleted of RBC by lysis with 0.15 M
NH4Cl, 1 mM KHCO3, and 0.1
mM Na2EDTA and cultured in complete media (45%
Enriched Eagle’s Medium (Biofluids, Rockville, MD), 45% RPM1 1640
(Mediatech, Washington, DC), 10% heat-inactivated FCS,
L-glutamine, penicillin, and streptomycin) at a
concentration of 2 x 106 cells/ml.
Cultures were performed in the presence of
recombinant mouse IL-7 (R&D Systems, Minneapolis, MN) at 16 ng/ml for 5
days at 37°C in 5% CO2 (first-stage culture).
In some experiments, freshly prepared HEL (Sigma, St. Louis, MO)
(sterile filtered in complete media) was added at a concentration of
200 ng/ml, unless otherwise noted. For second-stage cultures, cells
were harvested at day 5, washed extensively with PBS, and then
recultured in complete media at a concentration of
106 cells/ml without IL-7 and in the presence or
absence of HEL. Cells were then harvested after 2-day cultures for
further analysis. To measure proliferation, quadruplicate samples
(1 x 105 cells in 200 µl media in 96-well
flat-bottom plates) were cultured with IL-7 and HEL for 5 days. Cell
counts were performed using trypan blue exclusion at day 4, and
parallel cultures were pulsed for 16 h with 1 µCi of
[3H]thymidine before harvest and scintillation
counting. Cells from first-stage cultures were also harvested and
washed, then recultured in complete media in the absence or presence of
10 ng/ml IL-4 (R&D Systems), 200 ng/ml HEL, or 10 µg/ml
F(ab')2 goat anti-mouse IgM Abs (Jackson
ImmunoResearch, West Grove, PA). [3H]Thymidine
incorporation was measured for the last 16 h of a 2-day culture.
Duck egg lysozyme was custom produced by Rockland Immunochemical
(Gilbertsville, PA) and was >98% pure by SDS-PAGE. J558 cells
engineered to secrete site-directed HEL mutants were kindly provided by
F. Battista and M. Neuberger (London, U.K.) (15). Cells
were grown in complete RPMI 1640 media, and recombinant proteins were
purified from culture supernatants by ion exchange chromatography on
CM-52 cellulose as described elsewhere (15). Protein
purities were 
50% for
HEL
RD and 25% for
HELRDGN by SDS-PAGE.
Total protein was quantitated using the bicinchoninic acid protein
assay (Pierce, Rockford, IL).
Flow cytometry
Splenocytes and in vitro cultured cells were stained in FACS buffer (1x PBS, 2.5% FCS, 0.1% sodium azide) with FITC-, PE- or Cy-Chrome-conjugated mAbs to B220 (RA3-6B2), IgMa, IgDa, CD43, CD21, CD23, and isotype controls (all Abs from PharMingen, San Diego, CA). Three-color flow cytometry was performed as described (10) using a FACScalibur (Becton Dickinson, Mountain View, CA) and analyzed using CellQuest (Becton Dickinson) or Flowjo (Treestar, San Carlos, CA) software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether anergic B cells exhibit evidence for light
chain editing in vivo, we prepared single-cell suspensions of spleen
and BM from young (4- to 6-wk old) HEL-Ig and anergic HEL-Ig/sHEL mice,
and isolated genomic DNA and total RNA. Since the HEL-Ig Tg light chain
is the product of a V-J2 rearrangement (16), we used
degenerate V framework primers along with a primer downstream of the
J1 gene segment in PCR of genomic DNA to detect endogenous V-J1
joints (10, 12). Fig. 1
, A and B, illustrates the control experiments that
were performed to validate this semiquantitative assay.
|
C, spleen and BM cells of single Tg HEL-Ig
(Ig) mice showed almost no detectable endogenous V-J1
rearrangements, consistent with tight allelic exclusion by the Tg light
chain. In contrast, V-J1 rearrangements were easily detected in both
spleen and BM cells of anergic HEL-Ig/sHEL (Ig/sHEL) mice, with some
enrichment in the spleen. In four pairs of animals examined, there was
an overall 11-fold increase in the abundance of V-J1 rearrangements
in the spleen of HEL-Ig/sHEL anergic mice compared with HEL-Ig mice as
determined by densitometry. RT-PCR analysis demonstrated
Rag-2 mRNA expression in the BM but not the spleen of
HEL-Ig/sHEL mice (Fig. 2), suggesting
that the endogenous rearrangements occurred centrally at the time
when IgM receptors were first expressed on immature B cells.
Rag-2 mRNA levels in the BM of HEL-Ig/sHEL mice were
2-fold higher than in HEL-Ig BM (data not shown).
|
1
rearrangements and Rag-2 expression observed in HEL-Ig/sHEL
anergic mice were not due to the presence of an expanded population of
endogenous nontransgenic B cells. In vitro HEL-Ig BM B cell cultures
We next turned to an IL-7-driven in vitro BM culture system
(17, 18, 19, 20) to examine the response of immature HEL-Ig B
cells to incubation with soluble self-Ag in vitro. Unfractionated BM
cells from HEL-Ig mice were cultured for 5 days with recombinant IL-7
to expand the population of precursor B cells. At the end of this
first-stage culture period, >80% of lymphoid-gated cells in the
culture were B220+, indicating a selective
outgrowth of B cells. Fig. 3 compares the
cell surface phenotypes of splenocytes from HEL-Ig and HEL-Ig/sHEL mice
(upper two rows) with those of HEL-Ig BM B cells generated
in 5-day IL-7 cultures in the absence or presence of 200 ng/ml soluble
HEL (lower two rows). Strikingly, HEL-Ig BM B cells
cultured with HEL showed reduced surface IgMa
expression similar to that observed on HEL-Ig/sHEL splenocytes (the
"a" allotype is specific for the Tg IgM receptor). In this system,
the levels of IgDa were significantly lower on
the cultured cells than the splenic cells of either Ig or Ig/sHEL mice,
consistent with an immature phenotype (IgM+,
IgDneg-low) of the majority of cultured B cells.
Additional evidence for an immature phenotype of the cultured cells
included the persistence of the pro-B cell marker CD43 and low surface
levels of the maturation markers CD23 and CD21 (Fig. 3). The levels of
IgMa and IgDa expression
correlated with HEL binding as determined by a flow-cytometric HEL
sandwich assay (3) (data not shown).
|
A). Significant reductions in
DNA synthesis were also observed at the end of the first-stage culture
in the presence of self-Ag (Fig. 4B). These data were
consistent with a modest Ag-mediated growth inhibition rather than
Ag-induced death, since there were no consistent differences in the
number of apoptotic B220+ cells as determined by
7-amino-actinomycin D staining, either in the presence or absence of
self-Ag (data not shown). Cells were next harvested at the end of the
first-stage culture, washed, and recultured in the presence or absence
of IL-4, soluble HEL, polyclonal anti-IgM Abs, or combinations of
these reagents for another 2 days. In these second-stage cultures,
cells incubated with HEL during the first stage showed reduced
proliferation following stimulation compared with cells not exposed to
Ag in the first-stage culture (Fig. 4C). These differences
were less striking than the defects observed in anergic HEL-Ig/sHEL
splenic B cells analyzed in parallel (Fig. 4C), likely due
to residual IL-7 cytokine effects in the BM cells. Together, these
results indicated that exposure of HEL-Ig BM cells to HEL in an
IL-7-driven BM culture system leads to lowered levels of surface IgM
associated with modestly reduced proliferative responses following
subsequent BCR cross-linking.
|
We next tested whether HEL-Ig B cells in the BM culture
system underwent endogenous light chain rearrangements in response to
BCR ligation by the soluble self-Ag HEL. In first-stage IL-7 cultures,
HEL-Ig B cells showed low background levels of endogenous V-J1
rearrangements in the absence of Ag (Fig. 5, lanes 4 and 6),
similar to background levels described previously in another Ig Tg
system (12). Incubation of first-stage cultures with HEL
(1 µg/ml) induced modest levels of new V-J1 rearrangements
(lanes 5 and 7). However, in second-stage
cultures, following a wash to remove IL-7 and reculturing in the
presence of HEL, there was a consistent and strong induction of
endogenous V-J1 rearrangements by HEL compared with cells cultured
in the absence of HEL (compare lanes 8 and 12
with lanes 9 and 13, respectively). Background
levels of Rag-2 mRNA were quite high in this in vitro system
(see also Ref. 21), and the strong induction of new V-J
rearrangements in second-stage cultures was associated with only about
a 2-fold induction of Rag-2 mRNA (data not shown).
|
The data shown in Figs. 1C and 5 were consistent with
the hypothesis that the level of receptor cross-linking sufficient to
induce anergy in HEL-Ig B cells was also sufficient to induce editing.
Thus, we next investigated the influence of lower affinity ligands for
the HEL receptor on editing in the BM culture system. Two of the
low-affinity ligands tested were site-directed mutants of HEL
(15). The first HEL mutant,
R21D101
(HELRD), contains two
alanine substitutions in the region of lysozyme that binds to the
HyHEL10 receptor of HEL-Ig B cells.
HELRD has an overall
12-fold reduced affinity compared with HEL, as determined by BiaCore
measurements (15). The second HEL mutant,
R21D101G102N103
(HELRDGN), has four
alanine substitutions and a 100-fold lower affinity for the
HEL-Ig receptor than HEL (15). We also tested duck egg
lysozyme (DEL) with an 3500-fold reduced affinity
(22).
We first tested the influence of the various Ags to modulate
HEL-specific surface IgMa levels in first-stage
BM cultures. As shown in Fig. 6A, the ability of each of the
ligands to down-regulate IgMa levels correlated
with their affinity for the anti-HEL receptor, with HEL showing the
strongest down-regulation of IgM levels and DEL the least. These
results were consistent with previous studies in the HEL system, which
have shown that the level of IgMa down-regulation
is proportional to the strength of BCR signaling (23).
|
RD,
HELRDGN, or DEL at 100,
500, or 2500 ng/ml for 5 days. At the end of the first-stage culture,
cells were harvested for determination of V-J1 rearrangements (Fig. 6B). Significantly, the levels of V-J1 rearrangements
observed were generally proportional to the affinity and concentration
of the various Ags tested. At the higher concentrations, the various
soluble ligands induced stronger levels of endogenous rearrangements,
and even DEL, a low-affinity ligand for the HyHEL10 receptor, could
induce weak editing.
In second-stage cultures, the induction of editing showed less of a
dose response, particularly with the higher affinity ligands HEL and
HELRD (Fig. 6C, lanes 2–7). It appeared that cells in the
second-stage cultures following withdrawal of IL-7 were somewhat
"primed" to edit, compared with first-stage cultures. This was
clearly observed following Ag challenge (e.g., compare lanes
2–4 of Fig. 6, B and C), and was also seen
in control cultures in the absence of Ag (lane 1 of
Fig. 6, B and C). A similar brisk induction of
editing after IL-7 withdrawal was also noted in in vitro 3-83 Tg B cell
cultures following BCR cross-linking (12, 20).
Finally, we analyzed cells from each of the first-stage culture
conditions shown in Fig. 6B by flow cytometry to determine
the extent of IgMa down-regulation. Plotting of
the mean fluorescence intensity (MFI) of IgMa
levels for the various first-stage cultures against the densitometric
determination of V-J1 rearrangements normalized to
CD14 showed an interesting relationship between these
two variables (Fig. 6D; each data point represents the
average of three separate experiments). Cells receiving the lowest
levels of BCR signaling (high IgMa MFI) showed
the lowest levels of editing (Fig. 6D, bottom
right portion of the graph), whereas cells receiving the strongest
levels of signaling (low IgMa MFI) demonstrated
the highest levels of editing (Fig. 6D, upper
left portion of the graph). Thus, the magnitude of the editing
response in this in vitro system was correlated with BCR signaling
strength.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 rearrangements. 2) Some of the
features typical of HEL-Ig/sHEL anergic B cells were reproduced in
immature HEL-Ig B cells incubated with soluble HEL in an IL-7-driven in
vitro BM culture system, including down-regulation of surface IgM
levels, reduced proliferation following BCR cross-linking, and
induction of endogenous light chain rearrangements. 3) Lowered affinity
ligands for the HEL receptor were also found to be capable of inducing
endogenous editing responses in vitro, with the level of editing
observed generally correlated with the strength of signaling through
the BCR.
One conclusion from these data is that the level of BCR cross-linking
sufficient to induce anergy, as classically defined in HEL-Ig/sHEL B
cells, is also sufficient to induce new endogenous light chain
rearrangements. In this system, the detection of V-J1 rearrangements
in HEL-Ig Tg B cells really serves only as a marker for the receptor
editing response. This is because in this Tg system "genuine" light
chain receptor editing, defined as editing that leads to a change in
the heavy and/or light chain composition of the BCR, does not occur.
The HEL-Ig animals used in these experiments are conventional Tg mice,
where the heavy and light chain Tg cassettes are present at multiple
copies and are cointegrated in the genome, but not at the normal
endogenous heavy and light chain loci (3). Because of
this, induction of new rearrangements at the endogenous loci in
HEL-Ig Tg B cells fails to remove or inactivate the autoreactive V-J
gene segment, like that which occurs in normal B cells through nested
V-J rearrangements (24) or RS-type recombinations
(25). Thus, receptor editing in this system can be
considered a form of "frustrated" editing. The coexistence of both
anergy and editing in HEL-Ig/sHEL B cells indicates that these cells
are attempting to edit away from "anergic" levels of
self-reactivity. Thus, anergy is one potential outcome for B cells that
fail to productively edit.
Another conclusion from this work is that the level of BCR
cross-linking sufficient to induce editing occurs over a broad range of
BCR signaling strengths. An advantage of the lysozyme Tg system is that
the level of surface IgMa expressed on HEL-Ig B
cells provides a reliable readout for the level of BCR signaling
strength experienced by the cells (23). Our data indicate
that the level of IgMa found on HEL-Ig/sHEL
anergic splenic B cells was approximately equivalent to the
IgMa levels observed on in vitro cultured BM B
cells grown in the presence of 200 ng/ml HEL (Fig. 3). We also found
that editing was induced in the in vitro system after exposure to very
low-affinity soluble ligands for the HEL receptor, most notably by DEL
with 3500-fold lower affinity than HEL. Editing in response to
low-affinity ligands was previously reported in the 3-83 anti-class
I MHC Tg system (26); however, because the class I
molecules with low affinity for the 3-83 BCR were membrane bound and
present at very high concentrations, it was difficult to estimate the
true signaling strength experienced by the B cells. Taken together,
these studies suggest that the BCR signaling threshold for B cell
editing in immature cells may indeed be quite low. Furthermore, these
findings suggest that developing B cells are likely to attempt to edit
away from levels of self-reactivity that are far below the upper
threshold for anergy induction.
Although receptor editing in the 3-83 anti-class I MHC Tg system
was associated with increased expression of 
light chains in vivo
(5), we have never observed elevated levels of
(defined as >1% of B220+ cells also positive
for ) in vivo, either on anergic HEL-Ig/sHEL B cells (data not
shown) or previously on anergic
HEL-Ig/bcl-xL double Tg B cells escaping
deletion in mHEL mice (10). Only when HEL-Ig B cells
generated in the in vitro system were incubated with very high
concentrations of HEL (e.g., 50 µg/ml) or received strong
cross-linking from anti-IgM Abs have we observed significant
staining (up to 3 or 4% of B220+ cells, data not
shown). Even under these extreme in vitro conditions, the cell
populations did not show a detectable loss of HEL binding as determined
by an HEL-specific sandwich assay (data not shown). The relative
resistance of HEL-Ig Tg B cells to edit away from HEL specificity is
probably explained by the fact that the HEL-Ig transgenes are
conventional and randomly integrated in the genome and not subject to
normal excision by deletion. In addition, it is possible that the
highly somatically mutated anti-HEL heavy chain either does not
pair well with endogenous or light chains, or that endogenous
light chains do not compete well with the Tg chain for pairing with
the Tg heavy chain.
Our finding of elevated levels of endogenous V-J1 rearrangements in
anergic HEL-Ig/sHEL B cells is supported by gene-expression array
analysis, which documented elevated levels of V-J5 transcripts in
anergic double Tg B cells compared with naive B cells
(27). These results, however, contrast with a recent paper
by Kraus et al. (28), who found no evidence for light
chain editing in anergic B cells compared with HEL-Ig B cells using a
single-cell analysis. It is likely that at least two factors are
contributing to this difference. The first is that Kraus et al.
(28) performed their single-cell analysis on sorted BM
cells, whereas we have consistently found higher levels of
rearrangements in splenic B cells. Furthermore, the PCR strategy they
used detected all of the potential V-J joints, including the Tg
V-J2 rearrangement, using a degenerate V primer and a primer
downstream of J5. In their strategy, V-J1 was the longest of the
PCR products to be amplified, and it had to compete with the Tg V-J2
rearrangement. Since V-J1 is the preferred J segment for de novo
rearrangements (29, 30) and the data shown by Kraus et
al. (28) did not identify a single V- J1 product, we
conclude that their assay may have missed the J1 rearrangements in
HEL-Ig/sHEL mice that we were able to detect using a V-J1-specific
strategy in unfractionated BM and spleen. Definitive resolution of this
issue would likely require a single-cell PCR analysis along with
Southern blot detection of V-J1 joints.
Another question that arises from these data concerns the identity of
the cell population that responds to BCR cross-linking with an editing
response in vitro. Specifically, is this a broad response across the
population of cells, or does this represent a selective response of an
activated subpopulation? Our data at present are most consistent with
the notion that the editing observed represents a broad response across
the population. This is supported by the proliferation data shown in
Fig. 4, where incubation with Ag resulted in lower, not higher, levels
of proliferation than cultures not containing Ag. In addition, in
recent experiments we have labeled cells with CFSE at the end of
first-stage cultures and then followed the fate of these cells in
second-stage cultures. After 2 days, there were no significant
differences detected in CFSE staining between second-stage B cells
cultured in the absence or presence of HEL Ag. Thus, we find no
evidence for the selective outgrowth of a subpopulation in response to
incubation with HEL.
The data presented here are consistent with the hypothesis that the editing response is regulated primarily by BCR signaling strength. In one respect, this is not particularly surprising, given the critical role that BCR signaling has at virtually every stage in B cell development. Currently, there are a number of important and unresolved issues regarding central receptor editing. These include the nature of the signaling pathways downstream of the BCR that regulate editing, the molecular mechanism by which editing is confined to a specific developmental stage in BM B cell development, and the mechanism by which light chain allelic exclusion is maintained during editing. The in vitro system described here, where the degree of BCR signaling and receptor editing can be modulated using soluble ligands, may prove useful in addressing some of these issues.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Timothy W. Behrens, University of Minnesota Medical School, 6-126 BSBE Building, 312 Church Street SE, Minneapolis, MN 55455.
3 Abbreviations used in this paper: BCR, B cell receptor; HEL, hen egg lysozyme; mHEL, membrane-bound HEL; sHEL, soluble HEL; BM, bone marrow; Rag, recombination-activating gene; DEL, duck egg lysozyme; MFI, mean fluorescence intensity.
Received for publication May 2, 2000. Accepted for publication September 18, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
immunoglobulin light-chain locus: NF-B-dependent and -independent pathways of activation. Mol. Cell. Biol. 17:3477.[Abstract]
/ ratio in surface Ig molecules on B lymphocytes differentiating from DHJH-rearranged murine pre-B cell clones in vitro. Eur. J. Immunol. 21:2895.[Medline]
and L chain gene loci. J. Exp. Med. 178:1263. region characterized by the circular excision products in mouse splenocytes. J. Exp. Med. 173:1065.
cytoplasmic truncation renders immature B cells more sensitive to Ag contact. Immunity 11:537.[Medline]
families and V exons: implications for the available B cell repertoire. J. Immunol. 147:3194.[Abstract]
chain expression in early ontogeny: biased utilization of V exons and preferential V-J recombinations. J. Exp. Med. 177:1317.This article has been cited by other articles:
|
|
 |
|
  E. J. Cadera, F. Wan, R. H. Amin, H. Nolla, M. J. Lenardo, and M. S. Schlissel NF-{kappa}B activity marks cells engaged in receptor editing J. Exp. Med., August 3, 2009; 206(8): 1803 - 1816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Taylor, B. S. Cobb, L. Bruno, Z. Webster, A. G. Fisher, and M. Merkenschlager A reappraisal of evidence for probabilistic models of allelic exclusion PNAS, January 13, 2009; 106(2): 516 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Diz, S. K. McCray, and S. H. Clarke B Cell Receptor Affinity and B Cell Subset Identity Integrate to Define the Effectiveness, Affinity Threshold, and Mechanism of Anergy J. Immunol., September 15, 2008; 181(6): 3834 - 3840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kiefer, P. B. Nakajima, J. Oshinsky, S. H. Seeholzer, M. Radic, G. C. Bosma, and M. J. Bosma Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM J. Immunol., May 1, 2008; 180(9): 6094 - 6106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Schram, L. E. Tze, L. B. Ramsey, J. Liu, L. Najera, A. L. Vegoe, R. R. Hardy, K. L. Hippen, M. A. Farrar, and T. W. Behrens B Cell Receptor Basal Signaling Regulates Antigen-Induced Ig Light Chain Rearrangements J. Immunol., April 1, 2008; 180(7): 4728 - 4741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Caucheteux, C. Vernochet, J. Wantyghem, M.-C. Gendron, and C. Kanellopoulos-Langevin Tolerance induction to self-MHC antigens in fetal and neonatal mouse B cells Int. Immunol., January 1, 2008; 20(1): 11 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Bai, Y. Chen, Y. He, X. Dai, X. Lin, R. Wen, and D. Wang Phospholipase C{gamma}2 Contributes to Light-Chain Gene Activation and Receptor Editing Mol. Cell. Biol., September 1, 2007; 27(17): 5957 - 5967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Roy, N.-H. Chang, Y. Cai, G. Bonventi, and J. Wither Aberrant IgM Signaling Promotes Survival of Transitional T1 B Cells and Prevents Tolerance Induction in Lupus-Prone New Zealand Black Mice J. Immunol., December 1, 2005; 175(11): 7363 - 7371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-Y. Ryu, J. K. Emberley, J. J. Schlezinger, L. L. Allan, S. Na, and D. H. Sherr Environmental Chemical-Induced Bone Marrow B Cell Apoptosis: Death Receptor-Independent Activation of a Caspase-3 to Caspase-8 Pathway Mol. Pharmacol., October 1, 2005; 68(4): 1087 - 1096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Hippen, B. R. Schram, L. E. Tze, K. A. Pape, M. K. Jenkins, and T. W. Behrens In Vivo Assessment of the Relative Contributions of Deletion, Anergy, and Editing to B Cell Self-Tolerance J. Immunol., July 15, 2005; 175(2): 909 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Swanson-Mungerson, R. G. Caldwell, R. Bultema, and R. Longnecker Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen J. Virol., June 15, 2005; 79(12): 7355 - 7362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hillion, A. Saraux, P. Youinou, and C. Jamin Expression of RAGs in Peripheral B Cells outside Germinal Centers Is Associated with the Expression of CD5 J. Immunol., May 1, 2005; 174(9): 5553 - 5561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Schlezinger, G. J. Howard, C. H. Hurst, J. K. Emberley, D. J. Waxman, T. Webster, and D. H. Sherr Environmental and Endogenous Peroxisome Proliferator-Activated Receptor {gamma} Agonists Induce Bone Marrow B Cell Growth Arrest and Apoptosis: Interactions between Mono(2-ethylhexyl)phthalate, 9-cis-Retinoic Acid, and 15-Deoxy-{Delta}12,14-prostaglandin J2 J. Immunol., September 1, 2004; 173(5): 3165 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Swanson, S. L. Kuslak, W. Fang, L. Tze, P. Gaffney, S. Selby, K. L. Hippen, G. Nunez, C. L. Sidman, and T. W. Behrens Fatal Acute Lymphoblastic Leukemia in Mice Transgenic for B Cell-Restricted bcl-xL and c-myc J. Immunol., June 1, 2004; 172(11): 6684 - 6691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Feuerstein, D. Shivers, F. Chen, R. A. Eisenberg, and T. H. Finkel Chronic GVH prevents anergy in bone marrow self-reactive B cells: a selective increase in post-endoplasmic reticulum processing and trafficking to the cell surface of autoreactive IgM receptors Int. Immunol., August 1, 2003; 15(8): 975 - 985. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Tze, K. L. Hippen, and T. W. Behrens Late Immature B Cells (IgMhighIgDneg) Undergo a Light Chain Receptor Editing Response to Soluble Self-Antigen J. Immunol., July 15, 2003; 171(2): 678 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mehr, G. Shahaf, A. Sah, and M. Cancro Asynchronous differentiation models explain bone marrow labeling kinetics and predict reflux between the pre- and immature B cell pools Int. Immunol., March 1, 2003; 15(3): 301 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Schlezinger, B. A. Jensen, K. K. Mann, H.-Y. Ryu, and D. H. Sherr Peroxisome Proliferator-Activated Receptor {gamma}-Mediated NF-{kappa}B Activation and Apoptosis in Pre-B Cells J. Immunol., December 15, 2002; 169(12): 6831 - 6841. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
