Role of Bruton's Tyrosine Kinase in B Cell Development

X-linked agammaglobulinemia (XLA) is one of the most frequent inherited immunodeficiency diseases in man and is characterized by an almost complete arrest of B cell differentiation at the pre-B cell stage. The gene defective in XLA encodes the cytoplasmic signaling molecule Bruton's tyrosine kinase (Btk). Next to the CBA/N strain of mice, carrying a single amino acid substitution mutation in the Btk gene, which results in the X-linked immunodeficiency (xid) phenotype, additional mouse models have been developed to study the role of Btk in vivo. This review discusses the analyses of Btk null-mutants, obtained by gene targeting in embryonic stem cells, and transgenic mice that express wild-type or mutated forms of the Btk gene. These studies provided information on the function of Btk at several important checkpoints throughout B cell development. Analyses of the mouse models indicated that Btk is not essential for pre-B cell receptor signaling in the mouse. By contrast, Btk-mediated B cell receptor signaling appears to be required for the survival of immature B cells in the bone marrow, that have performed a successful immunoglobulin (Ig) L chain locus rearrangement, resultirig in the expression of a non-autoreactive Ig on the membrane. Btk is also shown to be involved in signaling pathways that govern the development of peripheral B cells, including follicular entry, follicular maturation and plasma cell differentiation.

X-LINKED AGAMMAGLOBULINEMIA AND X-LINKED IMMUNODEFICIENCY B cell development is regulated by multiple signals derived from stromal cell contact, cytokines, antigens and helper T cells. Biochemical analyses and in vivo gene targeting experiments have implicated tyrosine kinases as key regulators of many of these signaling pathways (Satterthwaite and Witte, 1996). Bruton's tyrosine kinase (Btk) is one of the non-receptor protein tyrosine kinases involved in regulating the B cell development and function , Vetrie et al., 1993Rawlings et al., 1993, Thomas et al., 1993 for review see: Sideras and Smith, 1995, Desiderio, 1997, Conley and Cooper, 1998Mohamed et al. 1999). Btk is a 659 amino acid protein and belongs to a subfamily of tyrosine kinases, which also includes Itk, Tec and Bmx. Members of this family are expressed in haematopoietic cells and are all involved in signal transduction pathways activated by growth or differentiation factors. Btk contains, in addition to the Src homology domains SH2 and SH3 and a single C-terminal catalytic domain, a unique pleckstrin homology (PH) domain at the N-terminus and an adjacent proline-and cysteine-rich Tec homology domain. The biochemistry of Btk activation after B cell receptor signaling has recently been reviewed (Mohamed et al., 1999).
Defects in the Btk protein result in the B cell differentiation defects X-linked agammaglobulinemia (XLA; Bruton's disease) in man and X-linked immunodeficiency (xid) in mice. XLA patients manifest recurrent bacterial infections due to a profound reduction of serum immunoglobulin (Ig) of all classes. They have very low B cell numbers in the peripheral blood, and those few B cells present exhibit an immature IgM high phenotype (Conley 1985;Campana et al., 1990). If stimulated with anti-CD40 in vitro these B cells are able to proliferate and differentiate into specific Ig-producing cells (Nonoyama et al., 1998). Plasma cells are almost completely lacking. Because the numbers of pre-B cells in the bone marrow are not significantly reduced, XLA reflects impaired developmental progression or increased cell death at the transition from pre-B to immature B cells in the bone marrow. Since the discovery of the Btk gene, a large variety of mutations, including single nucleotide substitutions, insertions and deletions, distributed over the entire Btk coding region have been characterized in 471 unrelated XLA families (Vihinen et al., 1999). There is phenotypic heterogeneity among patients, even among patients from single XLA pedigrees (Bykowsky et al., 1996, Holsinki-Feder et al., 1998, Vihinen et al., 1999. The arrest of B cell development in XLA patients is not precisely defined and was shown to vary between patients (Campana et al., 1990). So far, it has not been possible to correlate severity of the phenotypic presentation with the genotype. Recently developed techniques, such as single-cell PCR analysis, and a detailed characterization of cell surface markers in XLA bone marrow samples could help to investigate the role of Btk in human B cell development. However, the large genetic variety, differences in habitat or therapeutic interventions between patients might complicate these studies. Therefore, mouse models could serve as an alternative to study the role of Btk and the effect of Btk mutations in B cell development, since they exhibit a uniformly genetic background and can be kept under comparable conditions. Since the early 1970s, an impressive amount of data has been accumulated concerning the functional defects in the CBA/N strain of mice, carrying the X-linked immunodeficiency (xid) mutation (Wicker and Scher, 1986). Shortly after the identification of the Btk gene, it was shown that these mice have a mutation in the Btk PH domain, of the highly conserved Arg28 residue into cysteine , Thomas et al., 1993. The xid phenotype in the mouse is less severe than XLA in humans (Table I). These mice have -50% fewer B cells in the periphery and the residual cells exhibit an unusual IgM high IgD lw phenotype. They lack the CD5 / B-1 B cell population and the IgM and IgG3 serum levels are severely reduced. Btk-deficient B cells do not enter S phase after anti-IgM stimulation in vitro. Although the immune response of xid mice to T cell-dependent antigens is undisturbed, they fail to make antibodies to T cell-independent type 2 antigens in vivo.
The milder phenotype of murine xid, when compared with human XLA, cannot be explained by the nature of the mutations involved. Mutation of the same Arg28 amino acid has been observed in patients with classic severe XLA phenotypes (de Weers et al., 1994a;Vihinen et al. 1999). Furthermore, the analysis of mice deficient for Btk in their germline, which were generated by gene targeting, showed that the complete absence of Btk protein also results in the mild xid phenotype (Khan et al., 1995;Hendriks et al., 1996). The molecular basis of the differences in phenotype between the two species is not well understood, although it is shown that the severity of the xid mutation is dependent on the genetic background of mice (Bona et al., 1980, Khan et al., 1996. Differences can partially be caused by the relative contributions of alternative pathways of B cell differentiation in a Btk-independent manner. An alternative explanation would be that there are differences in availability or functionality of Btk-relatives that may compensate for the absence of functional Btk, as expression of wild-type Btk or other Tec-family members can restore Ca 2/ fluxes in cell lines derived from XLA patients (Fluckinger et al., 1998). In this context, the B cell receptor (BCR)-induced hydrolysis of phospholipids in XLA cell lines was found to be severely reduced, leading to a strong reduction in Ca 2/ flux, whereas xid B cells demonstrate only a two-fold reduction in phosphatidyl-inositide hydrolysis (Fluckinger et al., 1998, Takata & Kurosaki, 1996.

2.
Peripheral B lymphocyte population: absent or very low in XLA, and only a limited reduction (~50%) in xid.

3.
Ig levels in the serum: very low levels of all isotypes in XLA, reduced levels of IgM and IgG3 and generally normal levels of other isotypes in xid.

4.
In vivo responses to T cell dependent antigens: low but detectable in XLA, and normal in xid.
Intrinsic B cell defect; other haematopoietic lineages are unaffected.

2.
Early B cell development up to the pre-B cell stage in the bone marrow is normal.

3.
Residual peripheral B cells have an immature IgM high surface phenotype.

4.
T cell independent antibody responses in vivo are lacking.

5.
Normal responses to anti-CD40 stimulation in vitro.

6.
Heterozygous female carriers are normal, but have non-random X chromosome inactivation in the mature B cell population.

7.
Phenotypic heterogeneity between patients within single XLA families and between families; expression of the xid phenotype is dependent on the background ofthe mouse strain.
In spite of the obvious differences in severity of the phenotype, Btk appears to be a conserved key factor involved in both murine and human B cell development. Btk has been shown to be involved in the BCR signal transduction pathways in both human (De Weers et al., 1994b, Hinshelwood et al., 1995 and murine (Aoki et al., 1994, Saouf et al., 1994) B cells. The genomic organization of the murine Btk gene is very homologous to-the human Btk gene and the Btk proteins share 99.3% homology . Transgenic human Btk could fully compensate for the absence of murine Btk (Drabek et al., 1997, Dingjan et al., 1998, Maas et al., 1997, indicating that the essential sites for Btk interaction with other signal transduction components are conserved between human and mouse. In addition, large scale comparative sequence analysis of the human and murine Btk loci revealed clusters of sequence conservation in non-coding regions throughout the loci (Oeltjen et al., 1997), which may play an essential role in the complex gene regulation. When human Btk was expressed under the control of the endogenous cis-acfing elements in a 340 kb or a 240 kb transgene construct, the expression pattern of transgenic Btk paralleled that of the endogenous Btk (Maas et al., 1997, A.M., unpublished results). We conclude that also cis-acting elements that regulate Btk expression have been conserved. Finally, in vivo competition experiments between B cells either expressing the wild-type Btk + gene or a targeted disrupted Btk-allele (see below), demonstrated that Btkmurine pre-B cells are also hampered in their progression to the immature B cell stage (Hendriks et al., 1996). Thus, XLA and xid may well involve the same stages of B cell development, but with quantitative differences between the species (Table I).

TARGETED MUTATION OF BTK BY INSERTION OF A LACZ REPORTER
To determine the stage in B cell development at which defects in Btk become apparent, a mouse model was generated in which the Btk gene was inactivated through a targeted in-frame insertion of a l-galactosidase (lacZ) reporter (Hendriks et al., 1996). The xid phenotype in these mice confirmed the earlier findings in Btk null-mutant mice (Khan et al., 1995;Kerner et al., 1995) that the elimination of Btk function does not lead to an almost complete block in B cell development, which is typical for XLA in man.
The presence of the lacZ reporter enabled us to determine the Btk expression profile in vivo. We found that Btk is expressed throughout B cell development, from the pro-B cell stage to the most mature IgMlWlgD high peripheral B cell and activated B lymphoblasts stage. Btk expression is down-regulated in plasma cells, and was also not found in T cells or NK cells. These patterns were consistent with other Btk expression pattern studies, either in cell lines and leukemias (De Weers et al., 1993, Genevier et al., 1994, Smith et al., 1994 or in vivo using intracellular flow cytometric Btk detection (Maas et al., 1999). Expression of the Btk gene was not restricted to the B cell lineage. In the bone marrow, the ER-MP20 high precursor cells of the monocyte lineage showed high lacZ activity, whereas the ER-MP20 medium fraction of granulocyte precursors manifested heterogeneous levels of lacZ expression ( Figure 1). In the erythroid lineage (Ter-ll9/; ER-MP201w), lacZ activity was mainly detected in the most immature population of large erythroid precursors ( Figure 1). This wide expression pattern of Btk suggests a role for Btk in multiple signaling pathways. Indeed, apart from its role in BCR-signaling Btk has been implicated as a mediator of signals from the interleukin 5 receptor (IL-5R), IL-6R and CD38 in B lymphocytes, the FceRI in myeloid cells, as well as the collagen receptor glycoprotein VI in platelets Smith, 1995, Wahl et al., 1997;Quek et al., 1998). It was recently reported that Btk-deficient macrophages produce less nitric oxide than wild-type macrophages in response to a variety of stimuli (Mukhopadhyay et al., 1999). Despite these findings, Btk is only essential in B cell development and cells from other haematopoietic lineages do not appear to be affected in XLA nor in xid (Sideras and Smith, 1995).
Using the Btk-/lacZ mouse model it was also possible to detect selective disabilities of Btk-deficient cells in each successive step of B cell development in an in vivo competition strategy (Hendriks et al.,

CORRECTION OF THE XID PHENOTYPE BY TRANSGENIC EXPRESSION OF BTK
We and others have generated transgenic mice in which expression of Btk is driven by various promoters, in order to correct the xid phenotype in Btk-deficient mice. In these experiments, also the minimal dosage required for Btk function was studied and the effects of Btk overexpression on B cell development and B cell function were analyzed.
Transgenic expression of the human Btk gene was driven by the murine MHC class II Ea gene locus control region (LCR), which was shown to provide position-independent and copy-number dependent expression from the pre-B cell stage onwards (Drabek et al., 1997). When these transgenic mice were mated onto a Btk-deficient background, correction of the xid B cell defects was observed. B cell differentiated to IgMlWlgD high stages in spleen or lymph nodes and peritoneal CD5 + B-1 B cells were present. In the serum the levels of IgM and IgG3 were in the normal ranges, and B cell responses to the T cell independent type II antigen di-nitrophenol-Ficoll were present. A A:. erhmid linea MI" small FSC cells Using the Ig H chain enhancer and promoter to rescue the xid phenotype, it was shown that murine Btk expression that equaled~25% of endogenous levels was sufficient to restore normal numbers of B cells in the spleen, which were phenotypically mature (Satterthwaite et al., 1997). However, serum Ig levels, T cell independent type II responses, CD5+B 1 B cell development and in vitro responses to anti-IgM stimulation remained significantly impaired in these animals. These data indicated that the development of mature conventional B cells, the development of CD5 + B-1 B cells, and B cell responses to antigen in vivo may require higher levels of Btk activity.
In a next series of experiments, we increased the expression levels of transgenic Btk by including genomic DNA from the Btk gene (16 out of 18 introns) and the endogenous 3' untranslated region (Dingjan et al. 1998). Although we observed a significant overexpression (up to~14x in the spleen of one of the transgenic lines generated), this did not appear to result in adverse effects on B cell development or function. Complete correction of all xid features was also observed by transgenic expression of the wild-type human Btk under the control of the B-cell specific CD 19 promoter region or endogenous regulatory sequences present on a 340 kb yeast artificial chromosome Btk construct (Maas et al., 1997(Maas et al., , 1999. Therefore it is concluded that Btk overexpression per se does not lead to significant activation of downstream signaling pathways in the mouse.

EXPRESSION OF THE E41K BTK MUTANT IN TRANSGENIC MICE
It has been shown that Btk tyrosine phosphorylation and the in vitro kinase activity of Btk increase upon BCR stimulation (Saouf et al., 1994;De Weers et al., 1994;Aoki et al., 1994), placing Btk in the BCR signal transduction pathway. BCR engagement leads to activation of phosphatidyl-inositol-triphosphate (PIP3). PIP 3 initiates Btk activation by targeting the kinase to the plasma membrane through interactions with the Btk PH domain, a pathway which is inhibited by the activity of the Src homology 2 containing inositol polyphospatase SHIP (Bolland et al., 1998;Scharenberg et al., 1998;Pearse et al., 1999). In concert with this phosphatidylinositol (PI) 3-kinase and PIP3-dependency, Btk activity is regulated by the (z-subunit of the Gq class of G proteins, and the Src family kinases (Bence et al., 1997, Li et al., 1997. Upon BCR or IL-5R stimulation in B cells and FceRI in mast cells, Src family kinases rapidly induce phosphorylation of Y551 in the Btk kinase domain. This phosphorylation is followed by an autophosphorylation at Y223 in the SH3 domain . These concerted phosphorylation events were shown to be enhanced by an E41K mutation (Glu-to-Lys) in the PH domain of Btk . The E41K Btk mutant, isolated using a retroviral random mutagenisis scheme, was able to induce transformation of NIH 3T3 fibroblast in soft agar cultures and relieved the IL-5 dependence of pro-B cell line Y16 (Li et al., 1995). The nature of E41K transforming activity is associated with an increased membrane localization (Li et al., 1995;Varnai, et al., 1999), thereby positioning Btk in close proximity to other signaling molecules, needed for activation.
To identify B cell signaling pathways activated by Btk in vivo we generated transgenic mice, which express an E41K human Btk mutant. When expression was driven by the CD19 promoter, B cell development was arrested within the immature IgM+IgD B cell stage in the bone marrow, irrespective of the presence or absence of the endogenous intact murine Btk gene (Maas et al., 1999). The arrest occurred at the progression from IgM lw into IgM high B cells, which reflects the first immune tolerance checkpoint at which autoreactive B cells become susceptible to apoptosis. Whereas the numbers of peripheral mature B cells in spleen and lymph nodes were reduced to < 1% of the normal numbers, a significant population of IgM + plasma cells was present in the spleen. Serum levels of IgM were substantial and increased with age (Maas et al., 1999).
A different phenotype was observed when the E41K mutant was expressed under the control of the MHC class II Ea locus control region (Dingjan et al., 1998). These mice did not exhibit any detectable defects in developing B cells in the bone marrow, but manifested a deficiency of recirculating B cells. A marked reduction of the B cell compartment was found in the spleen. Furthermore, the mice manifested a disorganization of the B cell areas and marginal zones in the spleen. In the spleen, B cell areas typically contained unusually high numbers of T cells and the T-cell area-associated CD 1 lc + interdigitating dendritic cells, which normally do not extend into B cell follicles. These findings suggested that the expression of the E41K mutant in peripheral B cells results in follicular exclusion, followed by apoptosis for the majority of peripheral B cells in the spleen. In the lymph nodes, peripheral blood and peritoneal cavity only very few B cells were present. Furthermore, the expression of the E41K mutant was shown to enhance blast formation of purified splenic B cells in vitro in response to anti-IgM or LPS stimulation. The differences between the two Btk E41K expressing mouse strains most likely reflect the earlier expression during B cell development of the transgene driven by the CD19 promoter region. The MCHII-hBtk E41K expressing cells may well escape negative selection i,n the bone marrow because the expression level of the transgene had not reached a critical threshold value. Due to the nature of the MHC class II Ea LCR, transgenic Btk E41K expression is significantly upregulated only after cells have arrived in the spleen (Dingjan et al., 1998;Maas et al., 1999).

ROLE FOR BTK IN THE INDIVIDUAL STEPS OF B CELL DEVELOPMENT
Btk is not required in pro and large pre-B cells Several lines of evidence indicate that Btk is not critical for the assembly of the t H chain and the transition from the pro-B to the small pre-B cell stage. (1) Pre-B cells are generally present at normal numbers in XLA patients (Sideras and Smith, 1995).
(2) In heterozygous Btk +/female mice, the absence of Btk did not result in a selective disadvantage up to the small pre-B cell stage (Hendriks et al., 1996). (3) The xid phenotype can be corrected by transgenic expression of Btk from the pre-B cell stage onwards (Drabek et al., 1997). (4) Pre-B cell receptor-mediated events, such as allelic exclusion and proliferation of B cell precursors that have performed a successful Ig H chain rearrangement, proceed normally in xid or XLA (Sideras and Smith, 1995). (5) Despite high levels of E41K mutated Btk in the pro-B and pre-B stages, the CD19-hBtk E41K transgenic mice showed defects only from the immature B cell stage onwards (Maas et al., 1999). Also when these mice were crossed on a RAG-l-deficient background, activated Btk did not signal developmental progression of pro-B lymphocytes (R.W.H., unpublished results).
Btk is critical for the transition from the pre-B to the immature-B cell stage The first role for Btk is evident at the transition of the small resting pre-B cell stage to the IgMlWlgD immature B cell stage (Figure 2). This transition is affected both in XLA patients (Conley, 1985) and in mice, in which B cells that lack Btk exhibited a selective disadvantage compared to B cells that express Btk (Hendriks et al., 1996). These findings suggest that Btk may be an essential transducer of signals that govern Ig L chain rearrangement events, such as chromatin structure changes that allow the recombinase access to the Ig L chain gene segments. Alternatively, Btk-mediated signals may regulate the re-expression of the RAG gene products, which are absent in the large cycling pre-B cells and reactivated in small resting pre-B cells for Ig L chain rearrangement (Grawunder et al., 1995). Finally, Btk signaling may regulate the survival of immature B cells that have performed a successful Ig L chain rearrangement. This would be supported by the finding that Bcl-2 expression is reduced and surface Ig mediated Bcl-x L induction is absent in xid B cells (Anderson et al., 1996;Woodland et al., 1996;Solvason et al., 1998).  (Goodnow, 1996). Recently, two subpopulations within the immature B cell stage with differences in apoptosis sensitivity were described (Melamed et al., 1998 (Russel et al., 1991, Eibel et al., 1994, Goodnow et al., 1995. It is likely that the activated state of the MHCII-hBtk E41K B cells may result in an inhibition of follicular entry. As T helper cell-derived rescue signals are absent, follicular exclusion will result in apoptosis of most of the B cells. In summary, the findings in the MHCII-hBtk E4]K mice imply that Btk-mediated BCR signals are decisive for the choice between follicular entry and follicular exclusion. This would be consistent with the block of B cell follicular entry in mice with targeted mutations in other BCR signaling components, such as Ig-ot or Syk, (Torres et al., 1996, Turner et al., 1995. After follicular entry, B cells are positively selected to become long-lived IgMlWIgD high recirculating B cells, a process which is probably mediated by low-level BCR signaling (Gu et al., 1991, Lam et al., 1997. Btk-deficient B cells are not excluded from B cell follicles, but fail to develop from the immature IgMhighIgD lw stage into the long-lived recirculating follicular IgMlWIgD higt B cell stage (Wicker and Scher, 1986, Khan et al., 1995, Hendriks et al., 1996. As xid B cells were shown to express only low levels of the anti-apoptotic bcl-2 protein and to undergo spontaneous apoptosis more rapidly than wild-type B cells in vitro (Woodland et al., 1996), we conclude that Btk must play a critical role in survival and maturation into long-lived recirculating B cells.

Btk and plasma cell differentiation
Another role for Btk was apparent from in the CD19-hBtk E41K transgenic mice. As the block at the IgMhighIgD immature B cell stage was leaky, B cells were found in the peripheral organs in very low numbers, almost exclusively beating an IgMhighHSAhighB22(J lw immature phenotype. Despite the very severe reduction of the mature B cell pool, significant numbers of IgM secreting plasma cells were present in the splenic red pulp. Therefore, we conclude that Btk activation quite efficiently induced terminal differentiation of the residual B cells into IgM-producing plasma cells, apparently without functional selection. In the CD19-hBtk E41K transgenic mice, serum levels of the IgG and IgA subclasses were severely decreased, confirming that constitutive BCR signaling in the absence of co-stimulation by CD40-CD40L interactions did not induce B cells to perform IgH chain class switch or germinal center formation (Foy et al., 1996).

CONCLUDING REMARKS
The analyses of the various mouse models generated show that Btk is expressed throughout B cell development and that signaling cascades activated by Btk are critical at several checkpoints throughout B cell differentiation. In all maturation steps the strength of the BCR-mediated signal is critical to guide further development. In the absence of Btk and even more so in the case of constitutive activation, B cell development is impaired. The molecular mechanisms, by which Btk mediates B cell development, cell activation and cell death need to be further elucidated, and await a detailed characterization of downstream signaling targets and pathways. Very likely, the proteins that interact with the different domains of Btk vary between the individual stages of B cell development. The biochemical characterization of downstream signaling targets at these individual stages could be facilitated by the generation of transgenic mice that express tagged forms of the Btk protein. Similar approaches could be used for other components of the BCR signaling pathways and would eventually lead to a better insight in B cell development and function.