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/ß1
*
Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and
Division of Pediatric Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß heterodimer. To further characterize the
role of Ig heavy chain-mediated signaling in vivo, as well as in B cell
development and allelic exclusion, we have created transgenic mice in
which the B cells express these signal-capable and signal-incapable
mutant µ chains. Failure of µ to signal via Ig/ß results in a
block in B cell development in mice expressing the signal-incapable
µ. A small number of B cells in these animals do escape the
developmental block and are expressed in the spleen and the periphery
as B220+ transgenic IgM+ cells. These
cells respond to LPS by proliferating but show no response to
T-independent-specific Ag. In contrast, B cells expressing the
signal-capable B cell receptor show a strong signaling response to
Ag-specific stimulus. There is no Ig seen in association with
signal-deficient IgM. Thus, the B cell receptor complex is not
assembled, and no signal can be delivered. Despite the block in
developmental signaling, allelic exclusion is complete. There is no
detectable coexpression of transgenic IgM and endogenous murine IgM,
nor is there rearrangement of the endogenous heavy chain genes. This
suggests that differing signaling mechanisms are responsible for the
developmental transition and allelic exclusion and thus allows for
separate examination of these signaling mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(the product of the mb-1 gene) and Igß (the product of the
B29 gene) (1, 2, 3). The Ig/ß heterodimer (4, 5) has
structural and functional homologies to the CD3 complex (6, 7). Ig
and Igß are noncovalently associated with mIg via polar interactions
in the transmembrane region of the mIg molecule (8). Ig/ß are
required for release of the BCR from calnexin (9) and subsequent
expression of the BCR complex on the cell surface (10) and for
Ag-induced cellular activation (8, 11). The heterodimer is not required
for binding of the BCR to the cytoskeleton and internalization of mIg
after cross-linking (12, 13), while its role in Ag presentation remains
uncertain (14, 15, 16).
The analysis of BCR complex signaling in mature B cells has been
paralleled by similar work in earlier B cell stages. The stages of B
cell development have been well characterized, especially in the mouse.
Recombination of the heavy chain gene begins at the CD43+
pro-B stage (17), while at the pre-B stage, CD43 expression is lost and
µ protein is detected in the cytoplasm. A variety of studies have
shown that µ heavy chain participates in an analogous signaling
complex in pre-B cells, a complex that signals the successful, in-frame
recombination and expression of the µ gene and, in turn, is critical
for the developmental transition through the pre-B stage. This pre-B
cell receptor complex consists of heavy chain and surrogate light chain
(
5 and VpreB) as well as other molecules such as the Ig/ß
heterodimer (18, 19). The complex is involved in several key events in
pre-B cell maturation, including developmental transition (20) as well
as allelic exclusion (21), but the roles of the individual molecules as
well as those the downstream signaling mechanisms involved remain
unclear. The importance of µ and 5 surrogate light chain has been
demonstrated by targeted deletion experiments. Deletion of 5 (22, 23) or the JH or transmembrane domain of the µ gene (24, 25) results in a block in B cell maturation at the pre-B stage. This
observation has now been extended to humans in some cases of
non-X-linked agammaglobulinemia, where various deletions in the µ
gene have been detected (26). The importance of µ chain-mediated
signaling in the maturation of pre-B cells has been further highlighted
by the identification of Btk as the target of mutations that
cause the classic, X-linked form of agammaglobulinemia, in which
maturation of B cells though the pre-B stage is blocked (27, 28). The
same mutation in mice causes a less profound maturational abnormality,
suggesting differences in signaling mechanisms between murine and human
B cell development (29). The importance of the Ig/ß heterodimer in
development has been directly demonstrated by disruption of the
assembly or function of this portion of the pre-BCR complex
(30, 31, 32).
To define the importance of an intact, signaling-competent Ig in B cell
development, we have examined the ability of various µ mutants
expressed as transgenes to support B cell maturation. In previous
experiments, we have transfected into mature B cell lines a number of
human µ constructs with nonconservative mutations in the
transmembrane and cytoplasmic domains (14) to define the functions of
the BCR complex. One such mutant, called YS:VV, in which two polar
transmembrane residues, Tyr587 and Ser588, are
replaced with valines, fails to associate with Ig/ß and fails to
signal upon Ag binding or to efficiently target bound Ag for
intracellular processing and presentation. This transmembrane mutant
µ is expressed on the surface of transfectants as an integral
membrane protein despite its failure to assemble the BCR complex as a
result of differential processing and release from calnexin in the
endoplasmic reticulum (9).
By expressing mutant µ molecules as transgenes, we sought to
extend these studies of BCR complex-mediated signaling to the pre-B
cell developmental transition. Based on the prior work of the
Nussenzweig group (21, 30), we anticipated that this mutant µ would
recapitulate the signaling failure in the context of the pre-B
transition. In designing these experiments, we took advantage of the
known specificity of the complete IgM (phosphorylcholine (PC))
available to us, with the intent of being able to study Ag-specific
signaling through the transgenic receptor. Thus, we provided both the
heavy and the light chain genes to allow for study of signaling via
both the pre-BCR and the complete BCR in mature B cells. Our data show
that disruption of the binding of µ protein to the Ig/ß
heterodimer causes a block in B cell development. Despite this block in
developmental signaling, allelic exclusion is complete. This suggests
the existence of dual signaling pathways directing allelic exclusion
and maturation through the pre-B cell stage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ig constructs used in these studies have been described in
detail previously (14). The wild-type (WT) µ consists of a rearranged
V-D-J from the mouse plasmacytoma S107 and human Cµ regions. The
first poly(A) addition site has been deleted, ensuring the expression
of this protein solely in the membrane-bound form (33). The two mutants
are Tyr587/Ser588 to Val/Val (YS:VV) and
Tyr587 to Phe (Y:F). The functional analysis of these
mutants transfected into A20 cell lines has been described elsewhere
(14). The purified PvuI/ClaI fragment of the µ
construct and the PvuI/BamHI fragment of the 
construct were comicroinjected into fertilized FVB mouse oocytes using
standard techniques (34). Microinjected eggs were transferred into the
oviducts of pseudopregnant Swiss Webster foster females. Potential
founders were screened for the presence of the µ heavy chain and
light chain DNA and protein by Southern analysis with a heavy chain
probe, PCR utilizing a primer pair recognizing the rearranged S107
light chain, and Ab detection of human µ on the surface of
lymphocytes (see below). RAG-2-deficient mice were the kind
gift of Dr. F. W. Alt, Boston, MA. Progeny of WT, YS:VV, and Y:F
transgenic founders were studied at 8 to 12 wk of age except where
noted.
Abs and Ag
Affinity-purified rabbit anti-human µ (RAHµ) absorbed
against mouse Ig and goat anti-mouse µ (GAMµ) adsorbed against
human Ig (Jackson Immunoresearch, West Grove, PA) were use for
cytometric analysis and cross-linking experiments. These Abs and normal
rabbit IgG (Sigma, St. Louis, MO) were coupled with CNBr-activated
Sepharose CL4B (Pharmacia, Piscataway, NJ) for use in preclearing or
immunoprecipitation. Phycoerythrin (PE)- and FITC-labeled Abs
recognizing murine B220, CD3, and CD43 were obtained from PharMingen
(San Diego, CA). For the three-color analysis of bone marrow, PE-B220,
FITC-CD43, and Cy5-RAHµ were utilized. Anti-Ig antiserum was the
kind gift of Dr. J. Jongstra, Toronto Western Hospital, Toronto
Ontario, Canada. PC-dextran was obtained from Dr. Jim Kenny, National
Cancer Institute, Frederick, MD (35). AB1-2 hybridoma specific for the
S107 Id was obtained from American Type Culture Collection, Manassas,
VA. Ab was purified from ascites and biotinylated for labeling
experiments.
B cell purification and culture
B cells for culture experiments were purified by panning on rabbit anti-mouse Ig- and goat anti-human µ-coated plates (both from Cappel, Malvern, PA) at 4°C as described (36). Typically, this protocol yields >95% pure B cells. In proliferation assays, B cells were cultured at 105 cells per well in the presence of LPS (Sigma), dextran, or PC-dextran and pulsed with [3H]TdR for the final 6 h of a 72-h culture.
Western blotting was performed as previously described (9). Briefly, 10
to 25 x 106 cell equivalents were lysed with 1%
dodecyl maltoside (Anatrace, Maumee, OH) or 1% Triton X-100 (Sigma) in
lysis buffer. The lysates were precleared with goat IgG-Sepharose,
immunoprecipitated with Sepharose-coupled goat anti-human µ,
washed, electrophoresed through polyacrylamide SDS gels, and blotted
onto polyvinylidene difluoride membranes. The blots were then probed
with either horseradish peroxidase-anti-human µ or
anti-Ig. To examine the assembly of the BCR complex, the
anti-µ immunoprecipitated proteins were divided into two aliquots
and electrophoresed separately. One aliquot was probed for µ heavy
chain and the other for Ig.
Analysis of intracellular Ca2+ flux
Cells (2 x 106/ml) were loaded for 30 min at 37°C with 4 µg/ml Indo-1/AM (Molecular Probes, Junction City, OR) in DMSO. After washing, the cells were resuspended at 106/ml in RPMI + 2% FCS on ice, shielded from light. Cell were warmed to 37°C and maintained at that temperature for the signaling assay. Cells (106) were assayed for Ca2+ response before and after addition of cross-linking Ab or Ag. The ratio of violet:blue fluorescence (405:485 nm) emitted from Indo-1/AM was measured on an Epics V cytometer (Coulter, Hialeah, FL) as previously described (37). This assay depends on the shift in fluorescence emission of Indo-1/AM from blue to violet (485 to 405 nm) upon binding of Ca2+ and enabled us to examine Ca2+ flux on a cell-by-cell basis. Fluorescence was measured in arbitrary units (405/485 nm), and unstimulated cells were used to establish a baseline for each measurement. The number of responding cells was also expressed as a percentage of the total cell population.
Analysis of VDJ recombination
We used a modification of the procedure of Marshall et al. (38). Splenocytes were labeled with PE-anti-mouse IgM and FITC-anti-human IgM, followed by sorting on a Coulter Epics Elite cell sorter. Sort windows were drawn to collect PE-positive or FITC-positive cells. Then, 20,000 sorted cells were lysed and subjected to PCR using primers designed to recognize 80% of VDJ recombination events: a degenerate VH consensus primer for the 5' end of the VH framework I region (VHall; 5'-AGGTC/GA/CAA/G CTGCAGC/GAGTCA/TGG-3') and a primer recognizing a sequence just 3' to JH4 (JH4; 5'-AAAGACCTGCAGAGGCCATTCTTACC-3') (38). A Perkin-Elmer GeneAmp 2400 PCR machine (Norwalk, CT) was used for lysis and proteinase K inactivation as well as for the PCR reaction. After agarose gel electrophoresis, the PCR products were transferred via vacuum blotting to nylon membranes, followed by Southern analysis using a radiolabeled JH4 probe (JH4IN, which recognizes the JH4 coding sequence: 5'-GAGGAGACGGTGGACTGAGGTTCCAATCCAATGAATACGAATTCCCTTCCCATG-3').
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice were created to study the role of µ-mediated
signaling in B cell development. The hypothesis driving these
experiments was that µ heavy chain participates in a signaling
complex of molecules in the pre-B cell, and that disruption of this
pre-BCR complex would disturb the developmental signal that allows the
pre-B to B cell transition. To test this assumption, we coinjected into
oocytes constructs directing the synthesis of the signal-competent WT
and Y:F heavy chains, as well as the signal-impaired YS:VV heavy chain
(Table I
), each together with the S107
light chain. Founders were screened for expression of both the
transgenic heavy and light chain. In each positive founder, heavy and
light chain were expressed and, based on analysis of subsequent
offspring (data not shown), were cointegrated into the same site in the
genome. Initially, founders were screened by Southern analysis of
genomic DNA obtained from tail biopsies for the presence of the
transgenic heavy chain. Subsequently, we screened offspring utilizing a
combination of PCR detection of the rearranged light chain sequence
present in tail DNA combined with cytometric analysis of peripheral
blood to detect B cells expressing the human IgM. It was also possible
to confirm the expression of the S107 Id by staining with the mAb
AB1-2, as well as to detect PC Ag binding directly using biotinylated
PC-dextran (not shown).
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As shown in Figure 1 and Table II, expression of the YS:VV Ig transgene
induced a significant, although incomplete, block in B cell
development. Figure 1, A and B, shows cytometric
analysis of peripheral blood and spleen in animals expressing the WT
IgM transgene, the signal-capable IgM mutant Y:F, and the
signal-defective mutant YS:VV, as well as a nontransgenic littermate.
There were significantly fewer B220+ cells in both the
blood and spleens of YS:VV transgenics, which in turn resulted in
reduced spleen size (Fig. 1C). Specifically, spleens
from YS:VV animals had 50% or fewer of the normal number of total
splenocytes and, as can be seen in Table II, <10% of the splenic
B220+ µ+ B cells seen in WT or nontransgenic
animals.
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D, there was almost complete absence of
the B220+ CD43- IgM- pre-B cell
population in the bone marrow of these animals (3–7% of
B220+ cells vs 24–32% of B220+ cells in WT or
nontransgenic animals; range of four experiments). These results show
that the YS:VV µ chain, which is incapable of signaling in mature B
cells as a result of failure to assemble with the Ig/ß heterodimer
(8), is also signaling deficient in the context of B cell development.
This in turn provides evidence to support the role of µ-mediated
signaling via the pre-BCR as an important event in the pre-B
transition. However, these data also show that the signaling block was
not complete, because small numbers of B cells expressing human µ
were detectable in the YS:VV transgenic mice.
Interestingly, the signaling failure responsible for the block in B
cell development becomes more apparent over time. Figure 1E
shows cytometric analysis of spleen and peripheral blood from older
animals expressing the YS:VV transgene (YS:VV 195, 5 mo old, and YS:VV
162, 7 mo old). By this time, B cells expressing the YS:VV µ on the
surface have almost completely disappeared from the periphery (Table II), with animal 195 having 1.5% and animal 162 0.7% YS:VV
µ+ cells. A greater proportion of the splenic B cells
were B220+ YS:VV µ- (36% µ-
and 2% µ+ in animal 195; and 21% µ-, 4%
µ+ in animal 162). Thus, 85 to 95% of the splenic
B220+ cells were transgenic µ- in the older
YS:VV animals. This compares with 5 to 20% in WT and Y:F transgenic
splenocytes and 33 to 80% in younger YS:VV transgenics (range of eight
experiments). These results are most easily explained by a relative
growth advantage possessed by YS:VV µ- B cells, a
population that can preferentially expand over time over
growth-impaired YS:VV µ+ B cells. This would further
indicate lack of signaling through the impaired B cell receptor, as the
few YS:VV B cells that escape the block are not expanding in the
periphery. However, these data do not exclude the possibility that the
life span of YS:VV µ+ B cells may also be shortened.
Analysis of allelic exclusion
In addition to driving the pre-B to B transition, the presence of rearranged µ heavy chain also indicates to the recombination machinery of the B cell that the alternate heavy chain locus should not be rearranged. This phenomenon of allelic exclusion ensures that no B cell emerges from the developmental process capable of recognizing more than one Ag. It was our expectation that a failure of developmental signaling would be accompanied by a failure to induce allelic exclusion and that those YS:VV IgM+ B cells that escaped the developmental block would do so as a result of rearrangement and expression of the endogenous heavy chain gene. Thus, the endogenous heavy chain would provide the developmental signal and not the YS:VV µ, and the development of the small number of YS:VV IgM+ B cells would be dependent on the rearrangement of one of the endogenous heavy chain genes. This proved not to be the case.
We initially looked for coexpression of mouse IgM with the transgenic
human IgM on transgenic B cells. As seen in Figure 2A, regardless of the
transgenic line tested, there was no appreciable coexpression of mouse
IgM and human IgM on the surface of splenic B cells. This was true of
peripheral blood as well (data not shown). Although we could not detect
mouse IgM expression on the surface of YS:VV transgenic B cells, this
did not rule out the possibility of endogenous gene rearrangement that
did not result in surface expression of the murine BCR. To explore this
further, we relied on molecular analysis of µ gene rearrangement. We
used a modification of a PCR detection procedure for VDJ recombination
that had been published previously (38), using a degenerate primer that
recognizes most VH exons and a primer that recognizes a
sequence immediately 3' of the JH4 exon, a primer pair that
detects the majority of VDJ recombination events (38). We labeled cells
with the non-cross-reactive PE- and FITC-conjugated Abs used in the
studies depicted in Figure 2A. These cells were then sorted
into PE (mouse IgM)- and FITC (human IgM)-positive fractions. Cells
(20,000–40,000) were lysed and the DNA in the lysate subjected
directly to PCR; the assay has a detection threshold of 10,000 or fewer
templates (data not shown; and 39 . The PCR products were
separated by gel electrophoresis and vacuum blotted onto nylon
membranes. The Southern blot was then probed with the JH4IN
probe complementary to a sequence in the coding region of
JH4 to confirm the identity of the rearranged bands. The
postsort cytometric analysis and the results of the PCR and Southern
analysis are shown in Figure 2B. These data show no evidence
for µ gene rearrangement in any of the human IgM+ B cells
from any transgenic line, including mice expressing the
signal-defective YS:VV transgene. This also provided evidence that,
even in the face of defective developmental signaling through the YS:VV
µ, the signal driving allelic exclusion seems to function
efficiently.
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A, this was observed with the
expression of some B220+ YS:VV µ+ B cells in
the spleens of these animals. The lack of T cells (Fig. 3, B
and C) demonstrates RAG-2 deficiency.
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These data indicate that recombination of the endogenous µ gene
and subsequent expression of endogenous µ protein did not provide the
mechanism by which the small number of YS:VV IgM+ B
cells were able to traverse the developmental block imposed by this
signal-incapable transgenic Ig. Given this finding, it was important to
exclude signaling through the BCR and the Ig/ß heterodimer.
Although our previous work has shown that no assembly of the BCR
complex or signaling is seen in mature B lymphoma cells transfected
with YS:VV IgM, it may be that signaling and/or complex assembly is
possible in this in vivo system.
To explore signaling through the impaired BCR complex in YS:VV
transgenics, we examined both early and late consequences of receptor
cross-linking. As shown in Figure 4A, B cells isolated by
panning from WT transgenic mice demonstrated a proliferative response
to specific, T cell-independent Ag (PC-dextran) as well as to LPS. The
B cells from mice expressing the signal-capable Y:F transgene also
responded to Ag, while YS:VV transgenic B cells showed no proliferative
response to PC-Ag and responded only to LPS. This lack of signaling
response was also seen with the early event of Ca2+
mobilization (Fig. 4B). YS:VV B cells treated with
anti-IgM or PC-Ag showed no calcium response, while this response
was intact in the case of WT or Y:F B cells, with a significant
percentage of splenocytes showing an increase in Ca2+ flux
after receptor cross-linking. The data in Figure 4 demonstrate a
complete lack of detectable signaling through the YS:VV BCR. We also
analyzed B cells from the Ig transgenics for assembly for the BCR
complex. As can be seen in Figure 5,
Ig is found in association with WT and Y:F IgM in dodecyl maltoside
lysates of B cells of these transgenic mice but not in YS:VV B cells.
Taken together, these data indicate that the BCR in B cells expressing
a signal-deficient IgM transgene neither associates with nor is capable
of signaling through the Ig/ß heterodimer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß heterodimer, nor could we
detect any signaling through this complex in mature YS:VV B cells. We
further anticipated that a lack of developmental signaling would be
paralleled by a failure to force allelic exclusion of the endogenous Ig
heavy chain genes, but this result was not seen. Despite impaired
developmental signaling, allelic exclusion is intact in YS:VV animals.
Development of "escape" YS:VV B cells is not dependent on
endogenous gene rearrangement, as shown in the
RAG-2-/- background. These data suggest that
while signaling through Ig/ß is required for development of most B
cells, it is not required for ensuring allelic exclusion.
The contribution of transgenic µ+ B cells to the mature
circulating compartment in YS:VV animals is small to begin with and
diminishes over time. This may be the result of preferential growth of
B cells over the life of the animal that do not express the YS:VV µ
protein and can thus rearrange their own Ag receptor genes. Similarly,
investigators studying the more subtle phenotype of Btk
inactivity in mice have suggested that this phenotype is caused by
impaired expansion of B cell progenitors (42). When allowed to grow in
a RAG-deficient background, these Btk-/- cells
can expand into peripheral compartments, a phenomenon that we observe
here as well. This points to a possible signaling redundancy in the
developmental system, a redundancy that may be more apparent in mice
but may also be detected in humans as the phenotypic spectrum of
Btk inactivation in man becomes more fully appreciated (43, 44). Our data suggest that if there is such redundancy, it may involve
transducers in a pre-BCR complex other than Ig/ß.
The role of Ig/ß in B cell development has also been carefully
studied by the Nussenzweig group, using a construct with a different
VDJ region but an identical alteration in the transmembrane region
(21). Despite the similarity in approaches, our conclusions differ in
that we see the YS:VV µ protein as mediating allelic exclusion. One
possible explanation for the difference would involve greater binding
of surrogate light chain to our µ protein. Surrogate light
chain-mediated signaling may act to screen for µ chains that are
capable of appropriate assembly with light chain (45, 46). In this
regard, we note that our signal-impaired YS:VV µ heavy chain
assembles with 5 (S. R. Rheingold et al., manuscript in
preparation). The developmentally ectopic expression of light chain in
our transgenic system could also play a role, as the ability of any
given light chain to substitute for surrogate light chain in the
pre-BCR is unknown. Additionally, we note that our YS:VV IgM is capable
of surface expression despite failure to assemble with Ig/ß, both
in our transgenic system and in cell lines (9). Whether or not (or to
what extent) surface expression of the pre-BCR is necessary for its
signaling function is controversial. Finally, we paid particular
attention to analyzing only those B cells that expressed the transgenic
µ to focus on the role of YS:VV µ in delivering the signal for
allelic exclusion. PCR analysis of unsorted bone marrow in our
transgenics could easily have detected rearrangements in B cells
expressing endogenous µ in the absence of transgenic µ and led us
to a conclusion opposite to the one we reached: that YS:VV µ does
mediate allelic exclusion.
Using a transgenic system to study the effect of a signaling-deficient
Ig on B cell development, Torres et al. (32) demonstrated results
more consistent with those seen here. They detected a block in B cell
development, with the most significant depletion seen in the peripheral
B cell compartment. The block in development, although profound, was
not complete, and allelic exclusion was intact. These latter two
findings were attributed to signaling via Igß, which is intact in
these animals and which may assemble with the BCR in the presence of
the signal-impaired Ig. This and other work (30) point to redundancy
between Ig and Igß. In this view, each may provide partial or
complete coupling to the downstream signaling machinery. This
redundancy is brought into question by observations in Igß-deficient
mice in which there is a complete block in B cell development (31).
However, the block is at the pro-B stage, not at the pre-B cell stage
during which µ is expressed and the pre-BCR complex can form, and
points to an entirely separate and earlier role for Igß.
The transmembrane mutation (YS:VV) used in the studies reported here
abrogates assembly with both Ig and Igß (8), suggesting that the
Ig/ß heterodimer may not be the only µ-associated structure
through which signaling via the pre-BCR takes place. Both our
observations and those of Torres et al. are consistent with a model in
which separate or parallel signaling mechanisms control the
developmental process and allelic exclusion. Stringent controls
avoiding B cell dual specificity are required, since the result of such
dual specificity could either be autoimmunity or excessive deletion at
the immature B cell stage. Given this result, it is not surprising that
the mechanism controlling allelic exclusion might be separate from or,
more likely, redundant with the developmental signal. Thus, allelic
exclusion may be seen in the context of impaired development as we
report here, while intact B cell development in the absence of allelic
exclusion would not be observed.
The existence of separable signaling pathways raises the possibility
that the pre-BCR may involve signaling transduction molecules other
than Ig/ß. The role of the surrogate light chain has yet to be
clearly delineated in pre-B cell development, but data from the studies
of 5 knock-out mice (22, 23) show clearly that 5 is involved.
Although µ chains, which do no bind surrogate light chains, are
poorly represented in the B cell repertoire, this does not indicate the
mechanistic link between 5, Ig/ß, and any downstream signaling
pathways. Further support for a role for surrogate light chain in
pre-BCR signaling comes from the Nussenzweig group, which has
demonstrated a requirement for surrogate light chain or conventional
light chain for pre-BCR signaling, even when Ig/ß are present
(47).
In summary, our data add to the growing body of evidence implicating µ heavy chain in a pre-BCR complex that controls development and allelic exclusion. Further, we provide evidence that these two pathways may be distinct. The transgenic and knock-out systems that focus on µ heavy chain and the molecules of the pre-BCR complex offer the best opportunity to study the in vivo phenomenon of pre-B cell death or development.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Stephan Grupp, Division of Pediatric Oncology, ARC 902, Children’s Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104.
3 Abbreviations used in this paper: BCR, B cell Ag receptor; mIg, membrane-bound immunoglobulin; GAMµ, goat anti-mouse µ Ab; PC, phosphorylcholine; RAHµ, rabbit anti-human µ Ab; WT, wild-type mIgM; YS:VV, Tyr587/Ser588 to Val/Val transmembrane mutant; Y:F, Tyr587 to Phe transmembrane mutant; OE, phycoerythrin; Btk, Bruton’s tyrosine kinase.
Received for publication April 30, 1997. Accepted for publication March 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Igß are functionally homologous to the signaling proteins of the T-cell receptor. Mol. Cell. Biol. 14:1095. and Ig-ß/
. J. Biol. Chem. 268:25776./ß heterodimer. J. Immunol. 155:3769.[Abstract]
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 T. Kouro, K. Nagata, S. Takaki, S. Nisitani, M. Hirano, M. I. Wahl, O. N. Witte, H. Karasuyama, and K. Takatsu Bruton's tyrosine kinase is required for signaling the CD79b-mediated pro-B to pre-B cell transition Int. Immunol., April 1, 2001; 13(4): 485 - 493. [Abstract] [Full Text] [PDF]
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 J. W. Stern, J. Fang, S. Shusterman, G. Pierson, R. Barr, B. Pawel, L. Diller, and S. A. Grupp Angiogenesis Inhibitor TNP-470 during Bone Marrow Transplant: Safety in a Preclinical Model Clin. Cancer Res., April 1, 2001; 7(4): 1026 - 1032. [Abstract] [Full Text]
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 Y. Chang, M. J. Bosma, and G. C. Bosma Extended Duration of DH-JH Rearrangement in Immunoglobulin Heavy Chain Transgenic Mice: Implications for Regulation of Allelic Exclusion J. Exp. Med., April 19, 1999; 189(8): 1295 - 1305. [Abstract] [Full Text] [PDF]
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