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Departments of
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Cell Biology and Genetics and
Immunology, Faculty of Medicine, Erasmus University, Rotterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The xid phenotype, which is present both in CBA/N mice
carrying an Arg28 mutation and in mice with targeted
disruptions of Btk in their germline, is distinct from XLA
(4, 5, 6). Compared with normal mice, Btk-deficient mice have 
50%
fewer B cells in the periphery, with an overrepresentation of immature
IgMhighIgDlow cells. The CD5+ B-1 B
cell population is absent, and the levels of IgM and IgG3 in the serum
are reduced. Although Btk-deficient mice do not make Abs to a subset of
T cell-independent (TI) type II Ags, they are able to respond to most T
cell-dependent (TD) Ags. Btk-deficient B cells do not enter S phase
after BCR triggering (4, 5). Although Xid is characterized by a defect
in peripheral B cell maturation and function, there is no substantial
block in early B cell development, as in XLA. Nevertheless, by analysis
of competition in vivo between wild-type (WT) and Btk-deficient cells,
it was shown that the first selective disadvantage of Btk-deficient
cells in the mouse is also at the transition from pre-B to immature B
cells (6).
Btk is a 659-aa protein that contains a single C-terminal catalytic domain, the Src homology domains 2 and 3, and a unique pleckstrin homology (PH) domain at the amino terminus with an adjacent proline- and cysteine-rich Tec homology domain (1, 2). Btk is expressed throughout B cell development, but not in plasma cells (1, 6, 7).
It has been shown that Btk tyrosine phosphorylation and in vitro kinase
activity increase upon BCR stimulation (8, 9, 10). BCR engagement leads to
activation of phosphatidylinositol 3-kinase, which generates the second
messenger, phosphatidylinositol-(3, 4, 5)triphosphate (PIP3).
In concert with Src family kinases, PIP3
initiates Btk activation by targeting the kinase to the plasma membrane
through interactions with the Btk PH domain (11, 12, 13, 14, 15). These
interactions are critical to the activity of Btk and result in
phospholipase C-
tyrosine phosphorylation, inositol triphosphate
production, and calcium mobilization. This pathway is inhibited by
engagement of the Fc receptor IIB through the activity of the
Src homology 2-containing inositol polyphosphatase SHIP,
which regulates the association of Btk with the membrane by reducing
the level of PIP3 (14, 15). Btk-deficient B cells manifest
a reduced inositol triphosphate peak level and Ca2+ flux
following BCR cross-linking (16). Conversely, a constitutively active
form of Btk, E41K, a Glu-to-Lys mutant at position 41 in the PH domain
(close to the predicted inositol phosphate binding site) (17), is
associated with increased membrane localization and tyrosine
phosphorylation of Btk (18). Expression of the E41K Btk mutant was
shown to stimulate the growth of NIH-3T3 fibroblasts in soft agar (18)
and to enhance sustained Ca2+ increases in Ramos B cells
and blast formation of splenic B cells in response to BCR cross-linking
(16, 19). We have shown previously that transgenic expression of the
E41K Btk mutant as driven by the MHC class II Ea gene locus control
region resulted in an immunodeficient phenotype that was more severe
than Xid (19). Recirculating B cells were lacking, in vivo B cell
responses to both TD and TI type II Ags were essentially absent, and
splenic architecture was disrupted (19). The arrest in development in
the spleen coincided with a marked increase in expression of the
transgene during maturation from IgMhigh to
IgMlow peripheral B cells. In the MHC class
II-hBtkE41K mice, we did not observe any defects
in developing B cells in the BM. However, this finding does not imply
that constitutive activation of Btk does not affect early B cell
development, as the expression level of the MHC class
II-hBtkE41K transgene may not have reached a
critical value.
To study the effect of constitutive Btk activation in early B cell development, we have now placed Btk expression under the control of the human CD19 promoter, which was shown to provide expression throughout the B cell lineage (20, 21). Mice in which expression of the E41K Btk mutant was driven by the CD19 promoter manifested an almost complete arrest in B cell development at the transition of IgMlow to IgMhighIgD- immature B cells in the BM. Crosses onto a Btk- background demonstrated that this developmental block was independent of the presence or absence of the endogenous intact Btk gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cosmid clone containing the human CD19 promoter region
(kindly provided by M. Busslinger, Research Institute of Molecular
Pathology, University of Vienna, Vienna, Austria) was isolated from a
human genomic cosmid library by screening with a homologous murine CD19
cDNA probe (20). A 16-kb XbaI fragment containing 5'
flanking DNA and a 317-bp XbaI-MspI (blunted)
fragment (positions 1072–1389 of the human CD19 gene; 20) were
cloned into cosmid vector pTL5 (22) using a
BglII-MluI-XbaI-SmaI-PvuI-NotI-BglII
polylinker. A unique EagI site in the 16-kb XbaI
fragment was used to isolate a 6.3-kb EagI
(blunted)-PvuI fragment and to ligate it to the 27.1-kb
PvuI-NotI fragment containing 400 bp
(Btk exons 1–3) of the WT or E41K-mutated hBtk
cDNA, a 27-kb genomic DNA fragment (Btk exons 3–19), and
a 109-bp LoxP fragment (19).
The 34-kb MluI-NotI inserts of the
CD19-hBtkWT and
CD19-hBtkE41K constructs were excised from the
vector and gel-purified. DNA (2 ng/µl) was injected into the
pronuclei of FVB x FVB fertilized oocytes, which were
subsequently implanted into pseudopregnant foster mice. To determine
the genotype of the founder mice and the subsequent generations
generated by crosses with Btk-/lacZ
mice of a mixed 129/Sv x C57BL/6 background (6), tail DNA was
analyzed by Southern blotting of BamHI digests and
subsequent hybridization to a partial hBtk cDNA probe
(hBtk position 133-1153). Southern and Western blotting
techniques have been described previously (6).
Flow cytometric analyses
The preparation of single-cell suspensions and three- or
four-color flow cytometry have been described previously (6, 19). The
following mAbs were obtained from PharMingen (San Diego, CA):
FITC-conjugated anti-B220/RA3-6B2, anti-Ig
/R5-240,
anti-heat stable Ag (HSA)/M1/6, anti-CD3, and
anti-BP-1/6C3; PE-conjugated anti-CD43/S7,
anti-CD5/Ly-1, anti-CD4, anti-CD19, and
anti-CD11b/Mac-1; CyChrome-conjugated anti-B220/RA3-6B2 and
anti-CD8; and biotinylated anti-HSA/M1/6,
anti-Ig
/R26-46, and anti-IgM. PE-conjugated anti-IgD was
purchased from Southern Biotechnology Associates (Birmingham, AL).
Affinity-purified polyclonal rabbit-anti-Btk (PharMingen) was used
for intracellular flow cytometric detection of cytoplasmic Btk protein,
as described previously (19). The secondary Abs used were tricolor- or
PE-conjugated streptavidin (SA) (Caltag Laboratories, Burlingame, CA),
SA-APC (PharMingen), or FITC-conjugated goat-anti-rabbit Ig
(Nordic, Capistrano Beach, CA).
Serum Ig detection, in vitro immunizations, and immunohistochemistry
Total or nitrophenyl-specific levels of serum Ig were measured by subclass-specific sandwich ELISA as described previously (19). In these assays, nitrophenyl-specific standards were used for IgG1, IgG2a, and IgG2b, whereas values were calculated as arbitrary units for IgM and IgG3 using a reference serum sample. Immunizations with TD and TI type II Ags were essentially performed as described previously (23). Booster doses were administered after 4 wk.
For immunohistochemical analyses, tissue samples were embedded in optimal cutting temperature compound; frozen 5-µm cryostat sections were acetone-fixed, and single and double labelings were performed using standard procedures (24). The mAbs anti-B220/RA3-6B2, anti-CD3/KT3, anti-CD11c/N418 (25), and MOMA-1 (26) were applied as hybridoma culture supernatants; biotinylated anti-IgM was obtained from PharMingen, biotinylated peanut agglutinin (PNA) was supplied by Sigma (St. Louis, MI), and anti-IgD was obtained from Southern Biotechnology Associates. The second-step reagents used have been described previously (19).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 6.3-kb genomic fragment with the CD19 promoter containing the critical B cell-specific B cell-specific activator protein/pax-5 site (20) and 5' flanking DNA was used to express the hBtkWT and hBtkE41K transgenes. To construct the transgenes, two previously described (19) 27.4-kb hBtk cDNA-genomic DNA fusion segments containing either WT or E41K-mutated human Btk cDNA exons 1–3 together with a genomic DNA fragment with Btk exons 3–19 were used. These segments were ligated to an MspI site in exon 1 of the human CD19 gene, located 18 bp 5' of the ATG translation start. After microinjection into fertilized oocytes, two CD19-hBtkWT and four CD19-hBtkE41K founder mice were obtained. These founders were mated onto a Btk-deficient background, in which the Btk gene was inactivated by a targeted in-frame insertion of a lacZ reporter (6). The offspring of these founders did not exhibit developmental defects or any increased susceptibility to malignancies until they were >9 mo of age.
In the BM and spleens of the transgenic mice, expression of Btk was
detected by Western blotting analyses (data not shown) and by
intracellular flow cytometry using a polyclonal rabbit antiserum
specific for Btk (Fig. 1
). Expression of
transgenic Btk was restricted to B220+ cells; T cells,
granulocytes, monocytes, and macrophages did not express detectable
amounts of transgenic Btk (data not shown). Compared with nontransgenic
mice, the CD19-hBtkE41K transgenic mice
manifested a significant overexpression of Btk protein throughout B
cell development, including early pro-B and pre-B cell stages in the BM
(Fig. 1). This expression pattern differed noticeably from the pattern
observed in the previously reported MHC class
II-hBtkE41K mice (19), for which Btk expression
was not detectable by flow cytometry in the BM but was found to
increase significantly as B cells matured to
IgMlowIgDhigh cells in the spleen (Fig. 1).
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B). In these experiments, we observed a dose
dependency of the effect of hBtkE41K expression
in the four independent CD19-hBtkE41K transgenic
lines, which carried different transgene copy numbers. Transgenic CD19-hBtkE41K expression blocks B cell development at the immature B cell stage
To examine the effect of CD19-hBtkWT and
CD19-hBtkE41K expression on B cell development,
total cell suspensions from various lymphoid tissues from transgenic
and nontransgenic mice, either on the Btk+ or the
Btk- background, were analyzed by flow cytometry (Table I and Fig. 2). As described previously (6), the
Btk- mice had fewer mature B cells (30–50% of normal)
in the spleen, lymph node, peripheral blood, and BM
(B220highIgM+ fraction; Fig. 2A).
They also exhibited a specific deficiency of mature
IgMlowIgDhigh B cells in the spleen (Fig. 2B) and CD5+ B-1 B cells in the peritoneal
cavity (Fig. 2C). In addition, we observed a small decrease
in the size of the pre-B cell population in the BM (Table I). In mice
that expressed the CD19-hBtkWT transgene on the
Btk- background, a complete correction of B cell numbers
was observed in all lymphoid tissues analyzed. On the
Btk+ background, expression of
CD19-hBtkWT did not appear to have any
effect on the sizes of B cell subpopulations (Table I; Fig. 2).
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). Compared with WT or
Btk- mice, the size of the immature B cell population was
reduced by a factor of two to three and lacked IgMhigh
cells (Table I; Fig. 2). In the IgMlowIgD-
population present, the surface IgM heavy (H) chains were associated
with conventional light (L) chains and not with the surrogate L chain
5 and Vpre-B gene products (reviewed in 27), as
assayed by flow cytometry using Ig and Ig L chain-specific mAbs
(data not shown). The almost complete absence of the
B220highIgM+ population of mature recirculating
B cells in the BM and the peripheral lymphoid compartments analyzed
indicated a reduction of the mature B cell pool to <1% of the normal
size (Table I and Fig. 2). In addition to the defect in conventional B
cells, the CD19-hBtkE41K transgenic mice also
lacked CD5+ B-1 B cells in the peritoneal cavity.
The six groups of mice analyzed did not manifest significant
differences in pro-B cell fractions in the BM, as assayed by expression
of the B220, CD43, HSA, and BP-1 surface markers (Ref. 28 and data not
shown). The CD19-hBtkE41K transgenic mice showed
a relative increase of the proportions of T cells in the spleen, lymph
node, and peritoneal cavity, but their absolute numbers were in the
same ranges as those for the nontransgenic or
CD19-hBtkWT transgenic mice (Table I); no
differences in thymocyte subpopulations were observed.
In summary, the expression of constitutively active Btk resulted in an almost complete arrest of B cell development within the immature B cell stage in the BM, irrespective of the presence or absence of endogenous intact murine Btk.
The residual splenic B cell population in CD19-hBtkE41K mice has an aberrant surface phenotype
The residual splenic B cells that were present in the
CD19-hBtkE41K transgenic mice were further
characterized by three- and four-color flow cytometry. As shown in Fig. 3, those B220+ B cells
present manifested close to normal IgM expression on the membrane, but
the level of cell surface IgD was decreased compared with
CD19-hBtkWT transgenic B cells. The B cells
manifested a normal forward scatter profile and did not appear to
belong to the B-1 lineage, as CD5 or Mac-1 were not present on the cell
surface (data not shown). The B cells had a
B220lowHSAhigh phenotype, reminiscent of recent
emigrants from the BM that have not yet differentiated into mature
B220highHSAlow cells of the long-lived B cell
pool (29). The lack of B220highHSAlow cells
(Fig. 3) indicated that in CD19-hBtkE41K
transgenic mice, those few immature B cells that have left the BM
failed to mature in the spleen into long-lived recirculating cells.
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To evaluate the capacities of the residual B cell population
present in the CD19-hBtkE41K mice, serum Ig
levels were determined in 2-mo-old CD19-hBtkWT
and CD19-hBtkE41K transgenic mice, whereas
nontransgenic Btk+ and Btk- mice were used as
controls (Fig. 4A). The levels
of IgM and IgG3 in the sera of Btk- mice were decreased
compared with those in control Btk+ littermates (4, 5, 30).
This defect was corrected by transgenic
CD19-hBtkWT expression: IgM levels were somewhat
elevated and IgG3 levels were in the normal range.
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A).
In the CD19-hBtkE41K transgenic mice, the serum
IgM levels increased with age: when the mice were 6–7 mo old, the IgM
concentration in the serum was elevated compared with normal or
CD19-hBtkWT transgenic mice (Fig. 4B). At this age, IgG1 and IgG2a remained undetectable,
IgG2b and IgG3 were in the range of Btk- mice, and IgA was
variable. The finding of substantial IgM levels in CD19-hBtkE41K transgenic mice implied that, despite the reduction of the mature B cell pool to <1% of the normal size, significant numbers of IgM-secreting plasma cells were present.
Ag responses are defective in CD19-hBtkE41K transgenic mice
To investigate whether functional Abs could be produced when the CD19-hBtkE41K transgene was expressed, we analyzed the immune responses to a TI type II Ag, DNP-Ficoll, and a TD Ag, trinitrophenol (TNP)-keyhole limpet hemocyanin (KLH).
Consistent with the reported findings (4, 5, 30), the Btk-
mice were unresponsive to DNP-Ficoll: the detected NP-specific IgM or
IgG3 levels at day 7 after i.p. injection did not differ from the
values of unimmunized animals. Whereas expression of the
CD19-hBtkWT transgene restored the TI type II
response in Btk- mice, expression of the
CD19-hBtkE41K transgene abolished this response,
irrespective of the Btk+ or Btk- background
(Fig. 5A).
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B). Taken together, these data demonstrate that expression of the CD19-hBtkWT transgene corrected the in vivo B cell response to TI type II Ags in Btk- mice, and that, by contrast, CD19-hBtkE41K B cells did not respond productively to TD or TI type II Ags.
Follicular and marginal zone B cells are absent but plasma cells are present in CD19-hBtkE41K mice
At 7 days after booster injection with the TD Ag TNP-KLH, the
spleens from 3-mo-old nontransgenic mice or
CD19-hBtkWT and
CD19-hBtkE41K transgenic mice, each on the
Btk+ or Btk- background, were examined by
immunohistology. Double labelings of serial spleen sections with mAbs
specific for B cells (anti-IgM, anti-IgD, and anti-B220), T
cells (anti-CD3), metallophilic macrophages (MOMA-1), or
interdigitating dendritic cells (anti-CD11c/N418), as well as
PNA are shown in Fig. 6. The spleens of
nontransgenic or CD19-hBtkWT mice, either on the
Btk+ or Btk- background, demonstrated a
characteristic histological organization in terms of separate T and B
cell areas in the white pulp, with T cells surrounding central
arterioles (Fig. 6, a–c) and B cells in follicles
(Fig. 6, a–c, e–g, and i–k) with
PNA+ germinal centers (Fig. 6, e–g). In these
mice, the marginal zones at the outer boundaries of the white pulp
contained IgDlow B cells as well as MOMA-1+
macrophages interrupted with nests of strongly N418-expressing
dendritic cells that formed bridging channels into the red pulp (24, 25).
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, d, h,
and l). There was no evidence for the presence of a
distinct marginal zone, as the outer boundary of the white pulp did not
contain B220+ cells; MOMA-1+ metallophilic
macrophages were largely absent, except for the incidental presence of
some MOMA-1+ cells close to the small B cell clusters (Fig. 6p). In the red pulp, IgM+ plasma cells were
present in apparently normal numbers (Fig. 6d). Consistent
with the absence of TD immune responses in vivo, PNA+
germinal center B cells were not detectable (Fig. 6h). In summary, these findings show that in CD19-hBtkE41K transgenic mice, those few B cells that emerged from the BM did not develop into follicular or marginal zone B cells, but were present in T cell areas and were efficiently driven into IgM+ plasma cell differentiation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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27 kb of genomic DNA from the Btk gene itself, it is
very possible that Btk endogenous regulatory elements contributed to
the expression pattern of the transgene. When the
CD19-hBtkWT mice were mated onto a
Btk- background, the appropriate expression of transgenic
human Btk resulted in a complete correction of the Xid features. This
observation paralleled our earlier findings in transgenic mice, in
which Btk expression was driven by either the MHC class II Ea gene
locus control region or endogenous regulatory regions in a 340-kb yeast
artificial chromosome (19, 23, 30). In contrast, expression of the E41K Btk mutant under the control of the CD19 promoter resulted in an almost complete arrest of B cell development in the immature B cell stage in the BM. The phenotype of CD19-hBtkE41K transgenic mice differed markedly from our previously reported MHC class II-hBtkE41K transgenic mice, which did not exhibit any detectable defects in developing B cells in the BM, but manifested a deficiency of recirculating B cells (19). From our flow cytometric analyses showing the amount of hBtkE41K expressed in vivo from the two transgene constructs, we conclude that the differences between the two mouse strains most likely reflect the earlier expression during B cell development of the transgene driven by the CD19 promoter region.
Expression of the CD19-hBtkE41K transgene did
not appear to affect the pre-B to immature B cell transition, which is
defective in XLA and partially blocked in Xid (1, 2, 6), because
IgMlow immature B cells that expressed significant levels
of the E41K Btk mutant were still present. Instead, B cell development
was blocked at the progression from IgMlow into
IgMhigh B cells. It was recently shown that this transition
is accompanied by a differential response to autoantigen recognition
(31). With productive L chain gene rearrangement and the assembly
of surface IgM, immature B cells acquire Ag specificity and pass
through a tolerance-susceptible stage (27, 32). Autoantigen binding in
newly generated IgMlowIgD- immature B cells
results in continued Ig L chain rearrangement (i.e., receptor editing)
(31, 33). In contrast, immature B cells that have advanced to the
IgMhigh stage lose this ability and concomitantly acquire
sensitivity to Ag-mediated apoptosis (31, 33). Thus, our findings show
that in CD19-hBtkE41K transgenic mice, B cell
development is arrested at the first immune tolerance checkpoint at
which autoreactive B cells become susceptible to apoptosis. Assuming
that the constitutive activation of Btk mimics B cell occupancy by self
Ags, the finding of developmental arrest would be consistent with a
role for Btk in the transduction of BCR-linked apoptotic signals in
immature B cells. This hypothesis is supported by the observation that
Btk regulates the apoptosis induced by IL-3 withdrawal in cultured mast
cells (34), and that BCR-engagement triggers apoptosis in WT but not in
Btk-deficient DT-40 chicken lymphoma B cells (35). The alternative
explanation, which is that the developmental arrest in the
CD19-hBtkE41K transgenic mice merely resulted
from a direct inhibition of the basal BCR signal that is thought to be
required for the survival of B cells (36, 37), seems less likely. As
inferred from the phenotype of mice that lack essential signaling
components of the BCR complex, such as the CD79
(Ig) cytoplasmic
tail or the tyrosine kinase syk, a basic BCR signaling
defect results in an arrest of B cell development at a slightly later
stage. In contrast to CD19-hBtkE41K transgenic
mice, Ig-deficient or syk-/- mice do have
IgMhigh immature B cells in the BM; however, due to
defective BCR signaling, the recruitment of these cells into the
circulating B cell pool is hampered (38, 39).
The nature of the signals provided by BtkE41K is obviously different from that of the EBV-encoded latent membrane protein-2A (LMP-2A), which is the only other protein that reportedly possesses constitutive BCR-linked signaling activity (40). When expressed in progenitor mouse B lymphocytes in vivo in transgenic mice, LMP-2A can provide signals that mimic those initiating from a functional Ig H chain in the context of the pre-BCR, allowing Ig- cells to colonize peripheral lymphoid organs (40). Apparently, the LMP-2A-derived signals obviate the necessity for basal BCR-mediated survival signals and, as a result, B-lineage cells are maintained in the absence of a competent BCR (40). In contrast to the Btk E41K mutant, LMP-2A signaling does not appear to mimic BCR signals in immature B cells that trigger a checkpoint that physically eliminates autoreactive B cells in the BM (27, 32). This finding may reflect the complicated nature of the constitutive signaling activity of LMP-2A, which forms tyrosine-phosphorylated aggregates in the plasma membrane, has the capacity to associate with the BCR-linked kinases syk and lyn, and prevents normal calcium fluxes and the accumulation of tyrosine-phosphorylated proteins following BCR cross-linking (40, 41).
In CD19-hBtkE41K transgenic mice, the developmental block at the IgMhigh immature B cell stage was leaky, allowing very small numbers of B cells to populate the peripheral immune system. Such cells were almost exclusively found in the spleen as B220lowHSAhigh cells, which normally represent immature B cells that have just left the BM. In parallel with our findings in the spleens of MHC class II-hBtkE41K transgenic mice (19), the expression of BtkE41K further impeded the follicular entry of these cells, as B220highHSAlow cells were absent in the spleen; recirculating B cells in the BM or lymph nodes were completely lacking.
Consistent with the severe reduction of the mature B cell pool to <1% of the normal size and the absence of germinal center formation, serum concentrations of IgA and IgG subclasses were very low, and IgG responses to TD and TI type II Ags were lacking in the CD19-hBtkE41K mice. However, significant numbers of IgM-secreting plasma cells were present in the splenic red pulp. Despite the substantial serum IgM levels, which increased with age to levels that were elevated compared with those in normal mice, specific IgM responses to TD and TI type II Ags were absent. Therefore, we conclude that the constitutive activation of Btk drives the residual B cells that were able to emerge from the BM into IgM+ plasma cell differentiation, apparently without functional selection.
Taken together, the phenotypes of Btk-deficient and MHC class II-hBtkE41K or CD19-hBtkE41K transgenic mice indicate that Btk is essential in the multiple signaling pathways that govern the maturation of peripheral B cells, such as B cell follicular entry, maturation of B cells, and plasma cell differentiation (4, 5, 6, 19). Regarding the role for Btk in early B cell development, evidence for a role for Btk in pre-BCR signaling is lacking. In the absence of Btk, signaling through the pre-BCR complex still mediates its normal checkpoint function (27, 32) by effecting Ig H chain allelic exclusion, IL-7 driven proliferative expansion, and progression to the resting small pre-B cell stage. However, it is obvious that Btk transduces BCR signals at several checkpoints in B cell development in the BM. The phenotype of XLA patients as well as the selective disadvantage of Btk- cells during the pre-B to immature B cell transition (1, 2, 6) indicates that Btk-mediated BCR signaling is required for the survival of immature B cells that have performed a successful Ig L chain locus rearrangement. The present study shows that activation of Btk blocks the progression of IgMlowIgD- immature B cells into the subsequent stage of IgMhighIgD- cells. This finding would be consistent with a role for Btk in a BCR-linked signaling pathway that eliminates autoreactive B cells in the BM. Additional experiments are needed to directly demonstrate the involvement of Btk as a mediator of apoptosis of autoreactive B cells in the BM.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rudolf W. Hendriks, Department of Immunology, Faculty of Medicine, Erasmus University, Dr. Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail address:
3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BCR, B cell receptor; XLA, X-linked agammaglobulinemia; Xid, X-linked immunodeficiency; BM, bone marrow; TI, T cell independent; TD, T cell dependent; WT, wild type; PH, pleckstrin homology; PIP3, phosphatidylinositol-(3,4,5)triphosphate; HSA, heat stable Ag; SA, streptavidin; TNP, trinitrophenol; KLH, keyhole limpet hemocyanin; PNA, peanut agglutinin; H chain, heavy chain; L chain, light chain; LMP-2A, latent membrane protein-2A.
Received for publication November 23, 1998. Accepted for publication March 12, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Middendorp, G. M. Dingjan, A. Maas, K. Dahlenborg, and R. W. Hendriks Function of Bruton's Tyrosine Kinase during B Cell Development Is Partially Independent of Its Catalytic Activity J. Immunol., December 1, 2003; 171(11): 5988 - 5996. [Abstract] [Full Text] [PDF]
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 R. Kersseboom, S. Middendorp, G. M. Dingjan, K. Dahlenborg, M. Reth, H. Jumaa, and R. W. Hendriks Bruton's Tyrosine Kinase Cooperates with the B Cell Linker Protein SLP-65 as a Tumor Suppressor in Pre-B Cells J. Exp. Med., July 7, 2003; 198(1): 91 - 98. [Abstract] [Full Text] [PDF]
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K. Saijo, I. Mecklenbrauker, A. Santana, M. Leitger, C. Schmedt, and A. Tarakhovsky Protein Kinase C {beta} Controls Nuclear Factor {kappa}B Activation in B Cells Through Selective Regulation of the I{kappa}B Kinase {alpha} J. Exp. Med., June 17, 2002; 195(12): 1647 - 1652. [Abstract] [Full Text] [PDF]
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M. C. Nawijn, G. M. Dingjan, R. Ferreira, B. N. Lambrecht, A. Karis, F. Grosveld, H. Savelkoul, and R. W. Hendriks Enforced Expression of GATA-3 in Transgenic Mice Inhibits Th1 Differentiation and Induces the Formation of a T1/ST2-Expressing Th2-Committed T Cell Compartment In Vivo J. Immunol., July 15, 2001; 167(2): 724 - 732. [Abstract] [Full Text] [PDF]
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G. M. Dingjan, S. Middendorp, K. Dahlenborg, A. Maas, F. Grosveld, and R. W. Hendriks Bruton's Tyrosine Kinase Regulates the Activation of Gene Rearrangements at the {lambda} Light Chain Locus in Precursor B Cells in the Mouse J. Exp. Med., May 21, 2001; 193(10): 1169 - 1178. [Abstract] [Full Text] [PDF]
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