Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

Gemma M. Dingjan; Alex Maas; Martijn C. Nawijn; Linda Smit; Jane S.A. Voerman; Frank Grosveld; Rudolf W. Hendriks

doi:10.1093/emboj/17.18.5309

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

Dingjan, Gemma M.; Maas, Alex; Nawijn, Martijn C.; Smit, Linda; Voerman, Jane S.A.; Grosveld, Frank; Hendriks, Rudolf W. 1998-09-15 00:00:00 The EMBO Journal Vol.17 No.18 pp.5309–5320, 1998 Severe B cell deﬁciency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton’s tyrosine kinase 1,2 1 XLA patients displaying a large variety of mutations in Gemma M.Dingjan , Alex Maas , 2 3 the Btk gene (Vihinen et al., 1998) are very susceptible Martijn C.Nawijn , Linda Smit , 2 1 to bacterial infections. XLA is characterized by an almost Jane S.A.Voerman , Frank Grosveld and 1,4 complete block in B cell development at the pre-B cell Rudolf W.Hendriks stage, resulting in a severe decrease of circulating B cells. 1 2 Department of Cell Biology and Genetics and Department of Plasma cells are virtually absent and serum levels of all Immunology, Faculty of Medicine, Erasmus University Rotterdam, Ig classes are very low. The B cell defects in the CBA/N Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, and xid mice which carry an R28C mutation in the Btk PH Division of Cellular Biochemistry, The Netherlands Cancer Institute, domain are less severe (Wicker and Scher, 1986). These Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands mice have ~50% fewer B cells in the periphery and the Corresponding author high low residual cells exhibit an unusual IgM IgD proﬁle. e-mail: [email protected] They lack the CD5 B-1 B cell population and have low levels of IgM and IgG3. Although xid mice are generally To identify B-cell signaling pathways activated by able to respond to T cell-dependent (TD) antigens, they Bruton’s tyrosine kinase (Btk) in vivo, we generated cannot make antibodies to thymus-independent type 2 (TI- transgenic mice in which Btk expression is driven by II) antigens. Detection of a similar PH domain mutation, the MHC class II Ea gene locus control region. Btk R28H, in a patient with classical XLA suggested that the overexpression did not have signiﬁcant adverse effects distinction between the two phenotypes did not result on B cell function, and essentially corrected the X-linked from an allelic difference (DeWeers et al., 1994a). This immunodeﬁciency (xid) phenotype in Btk mice. In was conﬁrmed by the construction of null mutations in contrast, expression of a constitutively activated form the mouse Btk gene, which also resulted in xid phenotypes of Btk carrying the E41K gain-of-function mutation (Kahn et al., 1995; Kerner et al., 1995; Hendriks et al., resulted in a B cell defect that was more severe than 1996). By analysis of competition in vivo between wild- xid. The mice showed a marked reduction of the B cell type and Btk cells, it was shown that the ﬁrst selective compartment in spleen, lymph nodes, peripheral blood disadvantage of Btk-deﬁcient cells in the mouse is also at and peritoneal cavity. The levels in the serum of most the transition from pre-B to immature B cell (Hendriks immunoglobulin subclasses decreased with age, and et al., 1996). B cell responses to both T cell-independent type II Btk is expressed throughout B cell development, from and T cell-dependent antigens were essentially absent. the earliest pro-B cell stage up to mature B cells, and Expression of the E41K Btk mutant enhanced blast expression is downregulated in plasma cells (DeWeers formation of splenic B cells in vitro in response to anti- et al., 1993; Sideras and Smith et al., 1995; Hendriks IgM stimulation. Furthermore, the mice manifested a et al., 1996). Btk is also expressed in cells of the myeloid disorganization of B cell areas and marginal zones in lineage, but not in T cells. Btk has been implicated as a the spleen. Our ﬁndings demonstrate that expression mediator of signals from various receptors, including the of constitutively activated Btk blocks the development of follicular recirculating B cells. antigen receptor, interleukin 5 receptor (IL-5R), IL-6R, Keywords: B cell antigen receptor/B lymphocytes/Btk/ and CD38 in B lymphocytes, and the FcεRI in myeloid xid/XLA cells (reviewed in Desiderio, 1997). Btk activity is regu- lated by Src family kinases, phosphatidylinositol (PI) 3-kinase-γ and the α-subunit of the G class of G proteins (Rawlings et al., 1996; Bence et al., 1997; Li et al., 1997). Introduction After stimulation of the antigen receptor or IL-5R in B cells and the FcεRI in mast cells, Src family kinases Bruton’s tyrosine kinase (Btk) is a non-receptor protein rapidly induce phosphorylation of Y551 in the Btk kinase tyrosine kinase that is crucial for B lymphocyte develop- domain, followed by Btk autophosphorylation at Y223 in ment and function. Mutations in the Btk gene are the the SH3 domain (Wahl et al., 1997). These concerted genetic basis for X-linked agammaglobulinemia (XLA) in phosphorylation events were shown to be enhanced by a man and X-linked immunodeﬁciency disease (xid)inthe Glu-to-Lys mutation, E41K, in the PH domain of Btk mouse (Rawlings et al., 1993; Thomas et al., 1993; (Park et al., 1996). The E41K mutant, which was isolated Tsukada et al., 1993; Vetrie et al., 1993). Btk encodes a using a retroviral random mutagenesis scheme, was shown 659 amino acid protein that contains, in addition to the to induce transformation of NIH 3T3 ﬁbroblasts in soft Src homology domains SH2 and SH3 and a single catalytic agar cultures and factor-independent growth of the IL-5- domain, a unique pleckstrin homology (PH) domain at the N-terminus and an adjacent proline- and cysteine-rich dependent pro-B cell line Y16 (Li et al., 1995). The Tec homology (TH) domain (for review see Sideras and transforming activity of the E41K mutation is associated Smith, 1995; Desiderio, 1997). with increased membrane localization and tyrosine © Oxford University Press 5309 G.M.Dingjan et al. phosphorylation of Btk in transfected NIH 3T3 ﬁbroblast cells. PH domains recruit signaling molecules to the cell surface through speciﬁc interactions with phospholipids and proteins (reviewed in Lemmon et al., 1996). Binding of the Btk PH domain to various (phosphatidyl)-inositol phosphates, βγ-subunits of heterotrimeric G proteins and protein kinase C isoforms has been described (Tsukada et al., 1994; Yao et al., 1994; Fukada et al., 1996; Salim et al., 1996). The activating nature of the E41K mutation might be explained by its close proximity to the predicted inositol-phosphate binding site, as was indicated by X-ray crystallography studies (Hyvo¨nen and Saraste, 1997). In this context, the E41K mutant binds inositol 1,2,3,4,5,6- hexakiphosphate with a 2 higher afﬁnity than wild-type Btk (Fukada et al., 1996). We have previously described the generation of trans- genic mice that express human Btk (hBtk) under the control of the class II major histocompatibility complex (MHCII) Ea gene locus control region, which provides gene expres- sion in myeloid cells and in B-lineage cells from the pre-B cell stage onwards (Drabek et al., 1997). When the MHCII- hBtk mice were mated onto a Btk background, Btk protein expression was restored to apparently normal levels in the spleen and the Btk phenotype was corrected. B cells now low high differentiated to mature IgM IgD stages, peritoneal CD5 B cells were present and serum Ig levels and in vivo responses to TI-II antigens were in the normal ranges (Drabek et al., 1997). These results indicated that in this system the hBtk gene was appropriately targeted to both conventional and CD5 B-1 B cells. The activation of Btk by B cell antigen receptor- mediated phosphorylation (Aoki et al., 1994; DeWeers et al., 1994b; Saouaf et al., 1994; Wahl et al., 1997) raises the question about the nature of speciﬁc events that are controlled by Btk in developing B cells. To be able to identify signaling pathways that are activated by Btk in vivo, we have now modiﬁed the MHCII-hBtk transgene construct and generated two different types of transgenic mice, which either overexpress wild-type hBtk, or express Fig. 1. Structure and protein expression of the hBtk transgenes. various levels of the E41K gain-of-function Btk mutant. (A) Map of the transgene constructs, showing the locations of the ﬁve These transgenic models would indicate whether over- DNase I hypersensitivity sites present in the 10.6 KpnI–PvuI mouse expression or constitutive activation of Btk leads to MHC class II upstream Ea fragment. The WT-hBtk and E41K-hBtk proliferation of cells in the B cell lineage, immunodeﬁci- transgenes contain a 27.4 kb hBtk cDNA–genomic DNA fusion ency caused by elimination of B cells from the circulation, segment with exons 1–19, as well as a loxP sequence. The MHCII- hBtk transgene contains a 2.1 kb hBtk cDNA fragment (hatched box) or induction of B cell anergy. and a 2.8 kb human β-globin fragment with part of exon 2, exon 3 In this report we show that overexpression of hBtk had and the 3 untranslated region. E, EcoRI; K, KpnI; M, MluI; P, PvuI; only minor effects on B cell development and function. S, SwaI. (B) Western blot analysis of Btk protein expression in total In contrast, E41K hBtk mutant mice manifested an spleen cell lysates (210 cells/lane) from the indicated mice. Transgenic mice were on the Btk background. A polyclonal rabbit immunodeﬁcient phenotype that is more severe than xid antiserum was used, which was raised against fusion proteins of and is characterized by very low numbers of circulating gluthatione S-transferase (GST) and amino acids 163–218 of hBtk and B cells, an almost complete absence of B cell responses also recognized E41K hBtk or murine Btk. Ranges of the relative in vivo and a disruption of the cellular architecture of densities of the 77 kDa Btk signals as compared with the control Btk the spleen. mice are given at the bottom. Values were corrected for differences in the proportion of B cells in the spleen, which were determined by ﬂow cytometry. (C) Intracellular Btk expression during B cell maturation in Results the spleen. Single splenic cell suspensions from 3-month-old non- transgenic Btk or Btk mice, as well as WT-hBtk or E41K-hBtk Generation of WT-hBtk and E41K-hBtk transgenic transgenic mice on the Btk background were stained for surface B220 and IgM and subsequently for intracellular Btk. Data are shown as 5% mice probability B220/IgM contour plots of total lymphocytes, which were The constructs used in this study containing either the gated by forward and side scatter characteristics (top). The indicated wild-type (WT-hBtk) or the E41K mutant (E41K-hBtk) high low IgM and IgM B220 B cell populations were gated and Btk gene, as well as the MHCII-hBtk construct previously analyzed for Btk expression (bottom). The results are displayed as histograms of the indicated mice (solid lines), together with the used to obtain transgenic Btk expression (Drabek et al., background staining as determined in Btk mice (broken lines). 1997), are shown in Figure 1A. The E41K mutation was 5310 E41K Btk transgenic mice introduced byaGtoA replacement at position 257, using transgenic mice, the majority of Btk protein was found in in vitro site-directed mutagenesis. Since only high copy the cytosolic fraction. Also, in vivo tyrosine phosphoryl- number MHCII-hBtk transgenic mice (two out of ﬁve ation or in vitro autokinase activity of the hBtk protein in lines) expressed hBtk levels similar to those found in these cells did not appear to be enhanced by the E41K normal mice (Drabek et al., 1997), we attempted to mutation (data not shown). increase hBtk expression levels by including more hBtk genomic DNA, as well as the endogenous 3 untranslated Depletion of peripheral B cells in E41K-hBtk region, in the transgene construct. The transgenes con- transgenic mice tained a 10.6 kb MHC class II genomic DNA fragment, The B cell populations in bone marrow, peripheral blood, a 0.3 kb fragment with the ﬁrst three exons of hBtk as a spleen, mesenteric lymph nodes and peritoneal cavity from cDNA sequence, as well as a 27.1 kb genomic DNA E41K-hBtk and WT-hBtk mice on the Btk or Btk fragment, encompassing the hBtk exons 3–19 (Figure 1A). background were examined by ﬂow cytometry in 6- to The transgene constructs were microinjected into fertilized 8-week-old mice (Table I; Figures 2 and 3). Cells from non- oocytes and four independent E41K-hBtk transgenic lines transgenic Btk and Btk littermates served as controls, (#8, #10, #11 and #14) and three WT-hBtk transgenic lines showing that the Btk-deﬁcient mice had fewer mature B (G5, F2 and A5) were obtained. Founder mice were mated cells (~30–50% of normal) in peripheral blood, spleen, to Btk /lacZ mice, in which the Btk gene is inactivated by mesenteric lymph node (Figure 2A) and bone marrow a targeted in-frame insertion of a lacZ reporter in exon 8 (IgM IgD fraction), and a speciﬁc deﬁciency of mature low high (Hendriks et al., 1996). surface IgM IgD B cells (Figure 3A) as previously described (Hendriks et al., 1996). In the peritoneal cavity Expression levels of the E41K and WT hBtk of Btk mice, the numbers of conventional B cells were proteins reduced and CD5 cells were lacking (Table I; Figure 2B). Btk protein expression was evaluated in transgenic mice Correction of the xid B cell deﬁciency, although not on the Btk background by Western blotting of total spleen complete, was obtained by WT-hBtk transgene expression cell lysates (Figure 1B). The mice exhibited a wide range on the Btk background. In the peripheral blood, spleen, of transgenic Btk expression levels in the spleen, which mesenteric lymph node and bone marrow the B cell were directly correlated with the transgene copy number numbers only reached values similar to those of Btk as estimated by genomic Southern blotting analyses. mice, but the numbers of peritoneal CD5 cells were in To estimate the Btk expression levels of the individual the normal ranges, and the peripheral B cells exhibited a transgenic mouse lines, the densities of the Western blot normal surface IgM/IgD proﬁle (Table I; Figures 2 and Btk protein signals were quantiﬁed and corrected for the 3A). The effect of Btk overexpression on the Btk proportion of B cells in the spleen (which were signiﬁcantly background was limited: the numbers of B220 cells were lower in the E41K-hBtk transgenic mice; see below). In slightly reduced in peripheral blood, but were in the contrast to the MHCII-hBtk transgenic mice which showed normal ranges in the other organs analyzed (Table I). approximately endogenous Btk levels, the WT- and E41K- In contrast, when E41K-hBtk mice on the Btk back- hBtk mice manifested up to 14 overexpression of hBtk in ground were compared with Btk mice, a further depletion their splenocytes (Figure 1B). The experiments described of B cells was observed in all lymphoid tissues analyzed below were mainly performed on WT-hBtk line A5, and (Table I; Figure 2). Also in the mesenteric lymph nodes on E41K-hBtk line #8. Except where speciﬁcally indicated, from the three other independent E41K-hBtk transgenic no differences were detected between independent lines lines, the proportion of B cells was 1–5%. Expression of in the performed analyses, either for the WT-hBtk or the the E41K-hBtk transgene on the Btk background resulted E41K-hBtk transgenic mice. in an analogous reduction in the numbers of circulating Using intracellular ﬂow cytometry, we compared the B cells, although the effect was less severe than on the Btk expression levels of transgenic WT and E41K hBtk with background (Table I). The reduction of the proportions of the endogenous murine Btk during B cell differentiation. circulating B cells in the E41K-hBtk transgenic mice was The individual subpopulations of developing B cells in accompanied by a relative increase of the percentages of the bone marrow or spleen showed equivalent expression CD4 and CD8 T cells (Table I). The six groups of mice levels of the endogenous Btk (shown for spleen in Figure did not manifest signiﬁcant differences in the numbers of low high 1C). In constrast, a signiﬁcant increase in WT or E41K Mac-1 DX5 NK cells or Mac-1 myeloid cells in the transgenic hBtk protein was found as B cells maturated spleen cell suspensions (data not shown). In the bone high low from IgM to IgM cells in the spleen (Figure 1C). In marrow of E41K-hBtk mice, pro-B, pre-B and immature the bone marrow, transgenic Btk was only detected in B cells were present in normal proportions, whereas recirculating IgM IgD cells. Additional ﬂow cytometric mature recirculating IgM IgD B cells were virtually analyses demonstrated that transgenic Btk was also absent (Table I). Additional analysis of the three pro-B expressed in peritoneal B-1 B cells, in 10% of the cell subfractions, as deﬁned by expression of surface Mac-1 myeloid cells in the spleen and peritoneum, but markers B220, heat-stable antigen (HSA) and BP-1 (Hardy not in T cells or NK cells (data not shown). et al., 1991) in E41K-hBtk mice revealed no detectable When transfected into NIH 3T3 ﬁbroblasts, the E41K alterations from the distribution in normal or WT-hBtk Btk mutant manifested enhanced auto-phosphorylation mice (data not shown). and increased membrane targeting, while the in vitro In strong contrast to the restored IgM/IgD expression kinase activity was similar to wild-type Btk (Li et al., proﬁle found on peripheral B cells from WT-hBtk trans- 1995). However, when we analyzed unstimulated spleno- genic Btk mice, B cells in spleen, mesenteric lymph node cytes, whether from normal mice, WT-hBtk or E41K-hBtk and peritoneal cavity from E41K-hBtk transgenic Btk 5311 G.M.Dingjan et al. Table I. Frequencies of lymphocyte populations in WT-hBtk and E41K-hBtk transgenic mice Compartment Cell population Non-transgenic WT-hBtk E41K-hBtk a – – – Btk Btk Btk Btk Btk Btk b –6 Spleen Nucleated cells (10 ) 190 50 84 22 163 34 112 17 194 20 191 38 B220 cells (%) 38 915 534 10 22 520 610 3 CD3 CD4 (%) 20 322 526 423 826 727 5 CD3 CD8 (%) 11 212 312 312 315 415 4 Lymph node B220 cells (%) 24 48 221 57 25 0.4 2 1 CD3 CD4 (%) 46 553 250 456 160 562 3 CD3 CD8 (%) 22 131 225 227 328 429 3 Blood B220 cells (%) 34 912 419 610 47 24 1 low low of which B220 IgD (%) 10 441 16 10 523 725 14 51 14 high high of which B220 IgD (%) 71 336 15 66 546 537 12 17 10 Peritoneum CD5 IgM B cells (%) 16 8 0.4 0.3 28 16 14 73 22 1 CD5 IgM B cells (%) 24 10 10 424 15 8 14 32 1 CD5 IgM T cells (%) 31 346 829 547 11 65 462 8 Bone marrow B220 cells (%) 37 336 139 10 36 737 531 2 – c CD43 IgM pro-B cells (%) 6 0.3 8 16 38 27 36 0.4 – – CD43 IgM pre-B cells (%) 15 216 114 512 613 512 3 IgM IgD B cells (%) 7 28 0.3 9 28 28 0.3 7 1 IgM IgD B cells (%) 6 22 0.5 5 12 0.3 1 0.3 0.4 0.2 a / – – –/– b Btk mice were Btk /Y males or Btk females; Btk mice (Hendriks et al., 1996) were either Btk /Y males or Btk females. Mice were 6–8 weeks old. Data are mean values standard deviations from three mice analyzed, except for spleen where values are from 5–20 mice per group. The phenotype of lymphocyte populations was determined by ﬂow cytometry; dead cells and high side scatter cells were excluded by gating. Classiﬁcation of pro-B and pre-B cells was according to Hardy et al. (1991). high low mice manifested an IgM IgD phenotype, reminiscent of the B cell population found in non-transgenic Btk mice (shown for spleen in Figure 3A). The peripheral blood contained mainly newly-generated B cells that had low low just left the bone marrow (B220 IgD cells), rather than recirculating cells migrating between follicles high high (B220 IgD cells: only 17 10% of B cells in E41K- hBtk transgenic mice and 71 3% in normal Btk mice). As these observations suggested a maturational defect in the peripheral B cell compartment, we investigated the expression levels of B220 and HSA: B cells that are low high B220 HSA have recently left the bone marrow and high low further differentiate into mature B220 HSA cells of the long-lived B cell pool (Allman et al., 1993). While the spleen of non-transgenic Btk or Btk mice contained high low ~60% mature B220 HSA cells, a small reduction in this population was observed in WT-hBtk transgenic mice and a 3- to 4-fold reduction in E41K-hBtk transgenic mice (Figure 3B). These results indicated that recent emigrants from the bone marrow failed to mature in the spleen into high low long-lived B220 HSA B cells. Serum immunoglobulin levels in E41K-hBtk and WT-hBtk mice Serum Ig levels were determined by ELISA in 2-month- old non-transgenic Btk and Btk mice, as well as E41K- hBtk and WT-hBtk transgenic mice (Figure 4). The Btk mice had severely decreased levels of IgM and IgG3, Fig. 2. Depletion of peripheral B cells in E41K-hBtk mice. Flow variable levels of IgG1 and somewhat decreased levels of cytometric analysis of (A) mesenteric lymph node and (B) peritoneal IgG2a as compared with control Btk littermates (Drabek cavity from 7-week-old mice of non-transgenic Btk or Btk mice, and WT-hBtk or E41K-hBtk transgenic mice on the Btk background. et al., 1997). When the WT-hBtk transgene was expressed Single-cell suspensions were stained with biotinylated anti-IgM and on the Btk background, IgM levels were elevated and all streptavidin-TriColor, and either FITC-conjugated anti-B220 or PE- other Ig subclasses were restored to normal levels, similar conjugated anti-CD5. Data are displayed as 5% probability contour to the correction previously observed as a result of MHCII- plots of total lymphocytes, which were gated by forward and side hBtk transgene expression (Drabek et al., 1997). In the scatter characteristics. Percentages of total lymphocytes within the indicated gates are given. Data shown are representative of the mice WT-hBtk transgenic mice on the Btk background, serum examined (Table I). Ig subclass levels were in the same ranges. In the E41K- 5312 E41K Btk transgenic mice Fig. 3. Expression of E41K Btk induces a dominant maturational defect in peripheral B cells. (A) Surface IgM-IgD proﬁles of splenic low high B cells. Percentages of B220 cells that are IgM IgD are given. high low These cells are mature B cells, whereas IgM IgD cells are more immature (Hardy et al., 1982). Data are displayed as dot plots of all gated viable B220 cells from 310 total events. (B) Surface B220- HSA proﬁles of splenic B cells. Percentages of IgM cells that are low high high low immature (B220 HSA ) or mature (B220 HSA ) are indicated. Data are displayed as dot plots of all gated viable IgM cells from 10 total events (or 310 total events for E41K-hBtk transgenic mice on the Btk background). Spleen-cell suspensions from 7-week-old mice of the indicated genotypes were incubated with biotinylated anti-IgM and streptavidin-TriColor, FITC-conjugated anti-B220 and either PE- Fig. 4. Effects of transgenic WT-hBtk and E41K-hBtk expression on conjugated anti-IgD or anti-HSA and analyzed by three-color ﬂow serum Ig levels. Serum concentrations of the indicated Ig subclasses in cytometry. Data shown are representative of the mice examined; non-transgenic mice (Btk , n 11; Btk , n 16), as well as WT- lymphocytes were gated on the basis of forward and side scatter. hBtk (n 7) and E41K-hBtk (n 20) transgenic mice on the Btk background, whereby each symbol indicates an individual animal. Mice were 2 months old and Ig levels were determined by ELISA. hBtk mice serum IgM was restored to normal or elevated levels, IgG1 was similar to the levels in Btk littermates, while serum IgG3 was corrected to normal values for 12 B cells, signiﬁcant numbers of B cells were induced to out of 20 animals analyzed (Figure 4). The concentrations differentiate into Ig-producing plasma cells. However, the of IgG2a and Ig2b were generally in the normal ranges, possibility remained that these B cells could not mount whereas IgA was quite variable but on average reduced speciﬁc antibody responses. Therefore, we tested the compared with the other three groups of mice. No inﬂuence responses of 2-month-old non-transgenic, WT-hBtk and – – of the Btk or Btk background was detected. E41K-hBtk transgenic mice on the Btk or Btk back- Except for IgM and IgG2b, the serum Ig concentrations grounds to TI-II and TD antigenic challenges in vivo. of the E41K-hBtk mice decreased signiﬁcantly with age. The responsiveness to the TI-II antigen dinitrophenol In 6-month-old E41K-hBtk mice, the levels of IgG1, (DNP)-ﬁcoll was measured seven days after intraperitoneal IgG2a, IgG3 were only 37 10, 38 20 and 21 (i.p.) injection by enzyme-linked immunosorbent assay 15 μg/ml, respectively (n 3). In age-matched WT-hBtk (ELISA; Figure 5A). Consistent with previous ﬁndings in mice these levels were 970 170, 470 140 and 280 Btk-deﬁcient mice (Wicker and Scher, 1986; Kahn et al., 100 μg/ml (n 4). 1995; Drabek et al., 1997), DNP-speciﬁc IgM or IgG3 was completely absent in Btk mice, as the absorbence Defective in vivo responses in E41K-hBtk measured did not differ from the values of unimmunized transgenic mice animals. On the Btk background, the TI-II response of The absence of a dramatic decrease of serum Ig in E41K- WT-hBtk mice was comparable with the response of hBtk transgenic mice at the age of 2 months indicated normal mice, while on the Btk background, expression that despite the observed maturation defect of peripheral of the WT-hBtk transgene could only partially restore the 5313 G.M.Dingjan et al. in WT-hBtk mice on the Btk background. In strong contrast to these limited adverse effects of the WT-hBtk transgene, we found that secondary TD responses were very low or lacking in E41K-hBtk mice, irrespective of their Btk or Btk background (Figure 5C). Because Btk- deﬁcient mice show a normal secondary response to TD antigens (Wicker and Scher, 1986; Kahn et al., 1995; Drabek et al., 1997; Figure 5C), the ﬁnding that TD responses are essentially absent in E41K-hBtk transgenic mice was striking. Aberrant in vitro responses of E41K-hBtk transgenic B cells To examine the capacity of B cells to respond to mitogenic signals in vitro, B cell-enriched spleen cell suspensions were stimulated with LPS, anti-CD40 and IL-4, and different concentrations of a goat-antiserum to mouse IgM. Proliferative responses to LPS, determined by quantitat- ing [ H]thymidine incorporation after 60 h of culture, were low in Btk B cells compared with control B cells (Figure 6A). Expression of the WT-hBtk transgene completely restored the proliferative capacity, while expression of E41K-hBtk had a minor effect. These differences in proliferative responses to LPS were paralleled by the in vitro Ig production proﬁles in LPS- stimulated cell cultures as measured by ELISA at day 7 (Figure 6B). The defective IgM, IgG1 and IgG3 production in Btk B cells was corrected by transgenic WT-hBtk expression, but the effect of E41K-hBtk expression was moderate for IgM, and negligible for IgG1 and IgG3. Consistent with previous reports (Kahn et al., 1995; Fig. 5. Defective in vivo responses in E41K-hBtk mice. (A) IgM and Anderson et al., 1996; Ridderstad et al., 1996) Btk B IgG3 responses to the TI-II antigen DNP-ﬁcoll. (B) Primary IgM cells showed normal proliferation induced via CD40 in responses to the TD antigen TNP-KLH, determined 7 days after i.p. the presence of IL-4. Btk overexpression in the WT-hBtk injection. (C) Secondary TD responses to TNP-KLH, determined transgenic mice was accompanied by a small but signiﬁcant 7 days after i.p. injections performed 5–8 weeks after the primary immunization. Serial serum dilutions were analyzed for DNP/TNP- increase in proliferative responses. Transgenic expression speciﬁc antibodies of the speciﬁc subclasses and optical densities are of the E41K mutant did not have a detectable effect on shown as mean values from 4–12 mice in each group. The optical proliferation in response to anti-CD40 and IL-4 (Figure densities in the pre-immune sera did not vary signiﬁcantly between the 6B), nor in response to anti-CD40 alone or to anti-CD40 six groups and are shown in (C). and INF-γ (data not shown). When the production of IgM, IgG1 and IgG3 in anti-CD40- and IL-4-stimulated cell TI-II response. The TI-II antibody response in E41K-hBtk cultures was evaluated, a marginally lower production was mice was very low but detectable, whether on the Btk observed in Btk and E41K-hBtk B cells as compared or the Btk background. with normal Btk or WT-hBtk B cells. To investigate TD antibody responses, mice were injected One of the hallmarks of Btk B cells is that they do i.p. with trinitrophenol-keyhole limpet haemocyanin (TNP- not enter S phase when triggered through their surface KLH). After 7 days, TNP-speciﬁc IgM was measured by IgM receptor (Wicker and Scher, 1986; Kahn et al., 1995; ELISA (Figure 5B). Btk mice mounted a moderate Anderson et al., 1996). We observed that the absence of primary IgM response compared with wild-type litter- proliferative response to anti-IgM antibodies was corrected mates, as previously found in xid mice (Wicker and by transgenic expression of both wild-type and E41K Scher, 1986). Whereas Btk overexpression in the WT- mutated hBtk (Figure 6C). Moreover, when the E41K- hBtk transgenic mice appeared to have a minor inhibitory hBtk transgene was expressed, B cells enlarged more effect on the levels of TNP-speciﬁc IgM and IgG1, the rapidly in response to anti-IgM stimulation (Figure 6D). TD primary response was completely absent in E41K- After 24 h of culture, the E41K-hBtk B220 cells showed hBtk transgenic mice, both on the Btk and on the Btk a signiﬁcantly higher proportion of large blast cells when background (shown for IgM in Figure 5B). compared with non-transgenic Btk and Btk or WT-hBtk When the mice were 3–4 months old, TNP-KLH booster transgenic B220 cells. Blast formation was already injections were given and secondary TD responses were manifest in the absence of anti-IgM, but became more measured at day 7. TNP-speciﬁc levels of IgM, IgG1 and pronounced in the presence of anti-IgM up to 1 μg/ml. IgG2b were not signiﬁcantly different between the non- At high anti-IgM concentrations also WT-hBtk transgenic transgenic or WT-hBtk transgenic Btk or Btk groups of B cells showed enhanced blastogenesis when compared mice (Figure 5C). TNP-speciﬁc IgG2a was decreased in with B cells from non-transgenic mice. Increased blast WT-hBtk transgenic mice and TNP-speciﬁc IgG3 was low formation of E41K-hBtk transgenic B cells was also 5314 E41K Btk transgenic mice observed in response to stimulation with 50 μg/ml LPS (data not shown). The impairment of TD responses in the E41K-hBtk did not appear to be due to defective induction of MHC class II or the co-stimulatory molecule B7.2 (CD86) on activated B cells, as these activation markers were upregulated after stimulation with goat anti-mouse IgM, LPS, or anti-CD40 and IL-4; culture with anti-CD40 and IL-4 also induced upregulation of CD23 on E41K-hBtk B cells (data not shown). Disrupted splenic architecture in E41K-hBtk mice By immunohistochemical analyses, the spleens of 2-month-old unimmunized Btk , Btk or WT-hBtk trans- genic mice demonstrated a characteristic organization, in terms of segregation of T and B cells in the white pulp, with T cells clustered in the periarteriolar lymphocyte sheath (PALS) and surrounded by B cell-rich areas con- taining follicles, and the presence of marginal zones at the outer boundaries of the white pulp. In contrast, E41K- hBtk mice derived from all separate founder lines showed a speciﬁc effect of the E41K Btk mutation on the architec- ture of the spleen. When expressed on the Btk background, a disruption of splenic architecture was observed, charac- terized by a reduction in the number and size of B cell areas (consistent with our ﬂow cytometric analyses), loss of the strict compartmentalization of B and T lymphocytes and loss of a distinct marginal zone. Figure 7 shows double-labeling of serial spleen sections with either anti-B220, anti-CD3 or MOMA-1, a mono- clonal antibody speciﬁc for the metallophilic macrophages, which constitute a major component of the marginal zone (Kraal, 1992), together with the N418 antibody speciﬁc for the CD11c integrin on dendritic cells (Steinman et al., 1997). In the E41K-hBtk mice, B220 cells were present in B-cell areas neighboring the T cell zones (Figure 7A, B and C), but B cell numbers were reduced. These B cell areas contained signiﬁcantly more T cells than the B cell follicles in control Btk or Wt-hBtk transgenic mice (compare Figure 7D, E and F), and contained also CD11c interdigitating dendritic cells, which normally do not extend into B cell follicles (Steinman et al., 1997) (compare Figure 7G, H and I). No distinct marginal zones were present; the outer boundary of the white pulp did not contain B220 cells and metallophilic macrophages were only incidently present as a small rim of MOMA-1 cells Fig. 6. Transgenic E41K-hBtk B cells show aberrant responses to adjacent to the B cell areas (Figure 7H). In this area, an mitogenic signals in vitro.(A) Proliferation, determined by expansion was manifest of CD11c marginal dendritic [ H]thymidine incorporation, of cells cultured in medium alone, in the presence of LPS, or anti-CD40 antibodies and IL-4. (B) Ig levels of cells, which normally form bridging channels into the red supernatants of 7-day cultures in the presence of LPS or anti-CD40 pulp (Steinman et al., 1997) (compare Figure 7G, H and and IL-4. (C) Proliferation, determined by [ H]thymidine I). The numbers of ER-TR9 marginal zone macrophages incorporation, in response to various concentrations of goat anti-mouse – (Kraal, 1992) were severely reduced, and the reticular IgM. (A–C) Enriched splenic B cell fractions Btk , Btk ,WT-hBtk ﬁbroblast network, characteristic for marginal zones, as transgenic and E41K-hBtk transgenic mice were cultured in vitro with various mitogens. The groups of transgenic mice contained animals on revealed by ER-TR7 staining (VanVliet et al., 1986) Btk or Btk background; no differences were detected between mice appeared to be absent (data not shown). We observed a from these two backgrounds. Data are given as mean values SE dose-dependency of the effect of E41K-hBtk expression with 4–9 animals in each group (A), as symbols that indicate the on the disruption of splenic architecture, because in line values in cultures of individual animals (B) or as mean values from 4–9 mice in each group (C). (D) Total splenocytes from Btk , Btk , #14, which showed a higher transgene expression level – – WT-hBtk transgenic Btk and E41K-hBtk transgenic Btk mice were than line #8 (Figure 1B), MOMA-1 cells were essentially cultured with 1 μg/ml goat-anti-mouse IgM for the indicated time absent (Figure 7K and L). Nevertheless, in line #10 with points (left), or in the presence of the indicated αIgM concentrations low-level transgene expression (Figure 1B) the MOMA-1 for 24 h (right). The proportion of blast cells was determined by ﬂow- cytometric analysis of forward light scatter of viable B220 cells. cells were reduced in number, CD11c cells extended into Data are given as mean values from 2–4 mice in each group. the B cell areas and the strict separation of B and T cell 5315 G.M.Dingjan et al. Fig. 7. Disrupted splenic architecture in E41K-hBtk transgenic mice. Immunohistochemical analysis of 5 μm splenic frozen sections from Btk mice, – – WT-hBtk Btk mice and E41K-hBtk transgenic mice [(C), (F), (I) and (K), line #8, and (L), line #14] on the Btk background. Sections were stained with anti-B220 [blue, (A–C)] for B cells, anti-CD3 [blue, (D–F)] for T cells or MOMA-1 [blue, (G–L)] for metallophilic marginal zone macrophages, together with anti-CD11c/N418 (brown) to detect dendritic cells. Arrows [sections (I) and (K)] indicate the presence of small rims of MOMA-1 cells in the marginal zone. a, central arteriole; f, B cell follicle. Original magniﬁcations: 50 (A–I) and 16 (J–L). areas was lost (data not shown). In WT-hBtk mice, Analogous to the disruption of splenic architecture, also MOMA-1 cells were not affected and B cell areas did in mesenteric lymph nodes from E41K-hBtk mice on the not contain CD11c interdigitating cells (Figure 7H and Btk background B cell distribution defects were observed. J). The splenic abnormalities of E41K-hBtk transgenic B220 cells expressing IgM and IgD were almost absent mice on the Btk background were variable, but became in the cortical B cell areas, whereas IgM IgD B cells more severe with age. When spleens from 4- to 5-month- were present in low numbers as clusters in the paracortical old mice were analyzed, the segregation of B and T cell T cell zones (data not shown). areas was lost and the numbers of MOMA-1 and ER- TR9 cells were severely reduced. Discussion Germinal center formation was evaluated by immuno- histochemical analysis of the spleen, both in 4- to 5-month- To date, numerous interactions of the individual domains old unimmunized mice and in mice 7 days after booster of Btk with various protein or lipid molecules have been injection with TNP-KLH adsorbed to alum. In contrast to reported (Fukada et al., 1996; Bence et al., 1997; Li et al., – – the Btk mice, Btk and WT-hBtk transgenic Btk mice, 1997; Wahl et al., 1997; reviewed in Mattsson et al., the E41K-hBtk transgenic Btk mice did not develop 1996). To be able to study the impact of these particular germinal centers, as B220 IgD cells that showed binding interactions on B cell differentiation and function in vivo, of peanut agglutinin (PNA) were completely absent (data we have developed mouse models in which wild-type or not shown). mutated Btk can be appropriately expressed as transgenes. 5316 E41K Btk transgenic mice We previously showed that expression of hBtk driven by presence of intact endogenous murine Btk, substantiated the class II MHC Ea locus LCR resulted in physiological the dominant nature of the E41K mutation. Btk protein levels in the spleen, which completely cor- When expressed in NIH 3T3 cells in vitro, the gain-of- rected the features of the xid phenotype (Drabek et al., function activity of the E41K Btk mutant was associated 1997). with increased membrane localization and was shown to require kinase activity (Li et al., 1995). Our biochemical analyses in unstimulated B cells demonstrated that the Limited effects of overexpression of Btk E41K mutant had in vitro kinase activity, but did not By the introduction of 16 out of 18 intron segments of reveal a dramatic increase in membrane targeting. Never- the Btk gene, together with the endogenous 3 untranslated theless, we cannot exclude that the E41K mutation was region, high-level Btk expression was obtained. It seems associated with relatively small changes in subcellular likely that these modiﬁed transgenes contained endogenous localization of Btk, which are difﬁcult to detect in bio- regulatory elements of the Btk gene, since Btk intron chemical assays. In this context, it was shown that subtle sequences were shown to contain multiple clusters of variations in antigen concentration or expression of CD19, extensive conservation between mouse and man (Oeltjen a co-receptor that lowers the threshold for antigen receptor et al., 1997), one of which, a 229 bp region in the ﬁfth stimulation, resulted in dramatic changes of the fate of intron with 90% identity, showed co-localization with a B cells in vivo (Cook et al., 1997; Tedder et al., 1997). DNase I hypersensitive site present in B cells but not in Nevertheless, two of our ﬁndings indicated that the E41K T cells from human tonsils (G.M.Dingjan, unpublished mutation represented an activated form of Btk. (i) Expres- results). Thus, the transgenic Btk expression pattern may sion of E41K enhanced in vitro blast formation of splenic reﬂect a combination of the effects of MHC class II Ea B cells in culture, either with or without mitogens (Figure gene LCR and endogenous Btk regulatory sequences. 6D). (ii) Splenic E41K-hBtk B cells showed proliferation On the Btk background, overexpression of hBtk cor- upon stimulation with anti-IgM antibodies (Figure 6C), rected most of the xid defects, as indicated by the despite the unusually high proportion of immature low high appearance of normal proportions of mature IgM IgD low high B220 HSA cells in this population. Normally, such circulating B cells, CD5 B cells in the peritoneum, low high B220 HSA virgin B cells are refractory to anti-IgM restored serum IgM and IgG3 levels and in vitro responses stimulation (Allman et al., 1992). to anti-IgM or LPS stimulation. However, in contrast with our ﬁndings in the MHCII-hBtk mice, TI-II responses Expression of E41K-hBtk impedes follicular entry were only partially restored and primary TD IgM responses and disrupts marginal zone microarchitecture were somewhat reduced. Our ﬁndings in the E41K-hBtk transgenic mice indicate Although WT-hBtk overexpression on the Btk back- that constitutive activation of Btk blocks the development ground was found to be associated with partially enhanced of follicular B cells. The numbers of recirculating B cells blastogenesis and proliferation of B cells in response to were severely reduced, speciﬁcally in peripheral blood anti-IgM stimulation in vitro (Figure 6C and D), this did not and lymph nodes. In the spleen, B cell areas typically appear to result in adverse effects on B cell development or contained CD11c interdigitating dendritic cells, which function: B cell numbers were largely in the normal normally do not extend into B cell follicles, as well as ranges, and B cell responses to TI-II or TD antigens unusually high numbers of T cells. Signals that induce in vivo or LPS in vitro were not notably affected. Therefore, B cells to become recirculating follicular B cells are we conclude that Btk overexpression per se does not lead mediated by the B-cell antigen receptor, as most peripheral to signiﬁcant activation of downstream signaling pathways. B cells are ligand-selected (Gu et al., 1991) and mutations of the CD79α/Igα or Syk signaling components of the E41K-hBtk represents an activated form of Btk B cell antigen receptor block the entry of B cells into In contrast, expression of the Btk E41K gain-of-function follicles (Torres et al., 1996; Turner et al., 1997). These mutation on the Btk background blocked maturation of ﬁndings demonstrated that low-level stimulation of the peripheral B cells, leading to a B cell deﬁciency that was B cell antigen receptor provides a signal that is required more severe than the xid phenotype. Although E41K-hBtk for follicular entry. Our results show that in the presence transgenic mice produced normal numbers of immature B of E1K-hBtk expression such signals were not transduced. cells in the bone marrow that began to express IgD, they Together with the reported abnormal maturation into low high failed to become mature recirculating follicular B cells in IgM IgD follicular B cells in Btk-deﬁcient mice the periphery. A severe reduction of B cell numbers was (Wicker and Scher, 1986; Kahn et al., 1995; Hendriks observed in all peripheral tissues and those B cells present et al., 1996), this argues for a crucial role of Btk in the low high in the periphery were mainly B220 and HSA .Itis recruitment of B cells in the long-lived recirculating most probable that these cells represented newly-generated B-cell pool. virgin B cells, which generally have a lifespan of only Although the possibility that constitutive activation of 3 days (Fulcher and Basten, 1997). Total serum Ig levels Btk leads to a general defect that impedes the survival or were not seriously affected at the age of 2 months, but affects the migration of B cells in the periphery cannot they decreased signiﬁcantly with age. The in vivo B cell be excluded, it is attractive to hypothesize that expression response to TI-II antigen was very low and only marginally of the E41K Btk mutant mimics B cell antigen receptor restored compared with Btk mice, while B cell responses engagement. In the E41K-hBtk mice those cells that high high low to TD antigen were lacking. The ﬁnding that E41K-hBtk were present in the spleen were IgM HSA B220 transgene expression resulted in a decrease of circulating immature B cells that have just left the bone marrow. B cell numbers and a loss of B cell functions, even in the Normally, immature B cells in the bone marrow are 5317 G.M.Dingjan et al. susceptible to negative selection (Goodnow et al., 1995) transduction of signals that govern the development of high and immature HSA splenic B cells are refractory to recirculating follicular B cells. The absence of Btk leads to low high stimulation with anti-IgM or phorbol ester and calcium abnormal maturation into IgM IgD follicular B cells ionophore (Allman et al., 1992). Therefore, the immature (Wicker and Scher, 1986; Kahn et al., 1995; Hendriks status of the E41K-hBtk activated B cells may hamper et al., 1996). We have shown that transgenic expression their subsequent expansion or differentiation and induce of the E41K Btk mutant blocks the development of their elimination instead. In this context, E41K-hBtk B cells recirculating follicular B cells, indicating that constitutive may resemble auto-reactive B cells which have received activation of Btk induces the elimination of virgin peri- a stimulatory signal through their antigen receptor, pheral B cells. Further experiments are required to investi- resulting in arrest in the outer PALS where their lifespan gate whether this elimination is caused by the absence of is reduced to 3–4 days in the absence of T cell help a basal antigen receptor signal that is thought to direct (Fulcher and Basten, 1994; Goodnow et al., 1995). Addi- developmental progression of B cells, or by the presence tional parallels with auto-reactive B cells include the rapid of a signal that mimics B cell receptor occupancy by increase in size of auto-reactive B cells upon self-antigen self-antigens. recognition immediately before their disappearance (Fulcher et al., 1996; Rathmell et al., 1996) and their Materials and methods absence in the splenic marginal zones (Mason et al., 1992). In vitro mutagenesis The lack of splenic marginal zone B cells in E41K- The E41K mutation was created byaGtoA replacement at position hBtk mice could be a direct result of the impeded follicular 257 in the hBtk cDNA clone phBtk2.55 in pBlueScript (Drabek et al., entry of B cells, as marginal zone B cells are derived 1997). Double-stranded site-directed mutagenesis (Stratagene, La Jolla, from follicular B cells (Kumararatne and MacLennan, CA) was performed with the E41K mutagenic primer 5-GCACA AACTC TCCTA CTATA AGTAT GACTT TGAAC GTGGG-3 and a 1981). The B cell abnormalities in E41K-hBtk mice may 39 bp KpnI→BglII selection primer, and the obtained mutant plasmids also hamper the normal development of marginal zone were sequenced, using standard methods. From the original wild-type macrophage populations. Further studies will be required hBtk and the E41K mutated cDNA clone, 303 bp PvuI–NlaIV fragments, to clarify whether the B cell or the macrophage population encompassing the ﬁrst two exons and part of exon 3, were used in the construction of the transgenes. of the marginal zone is intrinsically affected in E41K- hBtk mice, or whether the defects in both populations are Construction of WT-hBtk and E41K-hBtk transgenes secondary to accessory cell dysfunction. Since we have The two transgene constructs WT-hBtk and E41K-hBtk are shown in observed a similar disruption of splenic marginal zone Figure 1A. From the MHC class II Ea gene cosmid 32.1 (Carson and Wiles, 1993) a 4.0 kb KpnI fragment, containing DNase I hypersensitivity architecture in mice that express the E41K-hBtk mutant sites (HSS) I and II and a unique PvuI site at position 14 in the Ea under the control of the B cell speciﬁc CD19 promoter gene, was cloned into pBlueScript, using a NotI–SmaI–KpnI–SwaI– (A.Maas, unpublished results), it seems less probable that XhoI–NotI polylinker. The resulting plasmid was partially digested with macrophage development is intrinsically affected by the Asp718 in the presence of ethidium bromide to introduce a 9.4 kb Asp718 fragment from cosmid 32.1, encompassing HSS III to V. expression of the E41K Btk mutant. Clues about the Subsequently, a 13.4 kb NotI fragment from this plasmid was cloned relationship between these cell populations may come into a NotI-digested cosmid vector pTL5 (Lund et al., 1982), in which from experiments in mutant mice that present with closely the same polylinker as described above was introduced at a unique BglII related phenotypes, such as mice deﬁcient in members site. In a next step two fragments were cloned into this plasmid, using the unique PvuI site in the Ea gene and the unique SwaI site within the of the nuclear factor-κB family of transcription factors polylinker: (1) a 303 bp PvuI–NlaIV WT or E41K-mutated hBtk cDNA (Franzoso et al., 1997, 1998; Caaman˜o et al., 1998) or fragment (as described above) and a 97 bp NlaIV–SwaI fragment tumor necrosis factor ligand and receptor family members obtained by PCR ampliﬁcation using primers at positions 46846 and (Matsumoto et al., 1997), which present with defects in 47029 of the hBtk gene (Oeltjen et al., 1997) and subsequent digestion humoral responses, germinal center formation and mar- with NlaIV and SwaI. In the two resulting plasmids with the WT or E41K-mutated ﬁrst three Btk exons, a 109 bp SalI–XhoI fragment ginal zone macrophage subpopulations. containing loxP sequences (obtained by insertion of a loxP oligonucleo- tide, 5-ATAAC TTCGT ATAGC ATACA TTATA CGAAG TTAT-3 The role of Btk in B cell development into the Asp718 site of the pPolyIII vector) was cloned into the XhoI site Studies in transgenic mice carrying rearranged Ig genes within the NotI–SmaI–KpnI–SwaI–XhoI–NotI polylinker. Subsequently, a have established that strong antigen receptor signals (e.g. 23.1 kb SwaI–SalI fragment, encompassing Btk exons 5–19, was intro- duced using unique SwaI and XhoI sites within the polylinker. This transmitted by autoantigens) can result in antigen receptor 23.1 kb SwaI–SalI fragment was from a 32.9 kb cosmid which was editing or elimination of immature B cells in the bone isolated from a mini-library constructed from a 340 kb YAC clone marrow (reviewed in Goodnow et al., 1995; Melamed containing the hBtk gene (DeWeers et al., 1997). Finally, a ~4 kb SwaI et al., 1998). The absence of any defects in developing fragment from the same cosmid clone, which contained Btk exon 4, was introduced into the unique SwaI site. B cells in the bone marrow of E41K-hBtk mice (except in the recirculating mature cells) does not imply that Generation of transgenic mice constitutive activation of Btk would not have any effect The WT-hBtk and E41K-hBtk constructs were digested with NotIto in the bone marrow. Our intracellular ﬂow-cytometric release the ~38.2 kb DNA fragments shown in Figure 1. Gel-puriﬁed DNA was injected into pronuclei of FVBFVB fertilized oocytes at a analyses indicate that the expression level of the E41K- concentration of ~2 ng/μl and implanted into pseudopregnant female hBtk transgene may not have reached a critical threshold mice. Tail DNA was analyzed by Southern blotting using a partial hBtk value in the bone marrow to affect B cell development. cDNA probe (bp 133–1153) to determine the genotype of the founder In fact, the ﬁrst defects in the B cell lineage only became mice and mice generated in subsequent crosses with Btk /lacZ mice of mixed 129/SvC57BL/6 background (Hendriks et al., 1996). apparent in the spleen, where we identiﬁed a signiﬁcant increase in expression of the hBtk transgenes during Flow cytometric analyses high low maturation from IgM to IgM B cells. Preparation of single-cell suspensions and three- or four-color ﬂow In summary, we conclude that Btk is essential for the cytometry have been described (Hendriks et al., 1996). Intracellular ﬂow 5318 E41K Btk transgenic mice cytometric detection of cytoplasmic Btk protein was performed on cells (Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti- that were ﬁrst stained for cell surface markers and subsequently ﬁxed rat Ig alkaline phosphatase (Southern Biotechnology). in 2% paraformaldehyde and permeabilized using 0.5% saponin. Events (3–510 ) were scored using a FACScan or FACSCalibur ﬂow cytometer and analyzed by CellQuest software (Becton Dickinson, Sunnyvale, Acknowledgements CA). The following monoclonal antibodies were obtained from Pharmingen (San Diego, CA): FITC-conjugated anti-B220/RA3-6B2, We thank A.Boonstra, J.Borst, L.Braam, S.Carson, W.van Ewijk, M.Kuit, anti-HSA/M1/6, anti-CD3, anti-DX5, and anti-BP-1/6C3, PE-conjugated P.Leenen and H.Savelkoul for assistance at various stages of the project. anti-CD43/S7, anti-CD11b/Mac1, anti-CD5/Ly-1, anti-CD4, Cy-Chrome R.W.H. was supported by the Royal Academy of Arts and Sciences. conjugated anti-B220/RA3-6B2 and anti-CD8, and biotinylated anti- These studies were supported in part by the Netherlands Organization HSA/M1/6 and anti-IgM. PE-conjugated anti-IgD was purchased from for Scientiﬁc Research NWO (A.M.) and the Dutch Prevention Founda- Southern Biotechnology (Birmingham, AL). Anti-CD23 (B3B4) and tion (A.M. and G.M.D.). anti-CD28/B7.2 (GL1) were puriﬁed monoclonal antibodies conjugated to biotin and FITC, respectively, according to standard procedures. Afﬁnity-puriﬁed polyclonal rabbit anti-Btk was from Pharmingen. Secondary antibodies used were TriColor- or PE-conjugated streptavidin References (Caltag Laboratories, Burlingame, CA), streptavidin-APC (Pharmingen) Allman,D.M., Ferguson,S.E. and Cancro,M.P. (1992) Peripheral B cell or FITC-conjugated goat anti-rabbit Ig (Nordic, Capistrano beach, CA). maturation I. Immature peripheral B cells in adults are heat-stable antigen (hi) and exhibit unique signalling characteristics. J. Immunol., Ig detection and in vitro immunizations 149, 2533–2540. Levels of Ig subclasses in serum or culture supernatants were measured Allman,D.M., Ferguson,S.E., Lentz,V.M. and Cancro,M.P. (1993) by sandwich ELISA, using unlabeled and peroxidase-labeled anti-mouse Peripheral B cell maturation II. Heat-stable antigen (hi) splenic B cells Ig isotype-speciﬁc antibodies (Southern Biotechnology). Serially diluted are an immune developmental intermediate in the production of long- sera were incubated at room temperature for 3 h, and azino-bis-ethylbenz- lived marrow-derived B cells. J. Immunol., 151, 4431–4444. thiazoline sulfonic acid was used as a substrate. Antibody concentrations Anderson,J.S., Teutsch,M., Dong,Z. and Wortis,H.H. (1996) An essential were calculated by using puriﬁed isotype Ig proteins as standards. TD role for Bruton’s tyrosine kinase in the regulation of B-cell apoptosis. and TI-II immunizations and TNP-speciﬁc ELISA were essentially Proc. Natl Acad. Sci. USA, 93, 10966–10971. performed as described previously (Maas et al., 1997). Booster doses Aoki,Y., Isselbacher,K.J. and Pillai,S. (1994) Bruton tyrosine kinase is were given after 5–8 weeks. Serum dilutions were incubated at room tyrosine phosphorylated and activated in pre-B lymphocytes and temperature for 3 h and the biotinylated TNP-KLH step was overnight receptor-ligated B cells. Proc. Natl Acad. Sci. USA, 91, 10606–10609. at 4°C. Bence,K., Ma,W., Kozasa,T. and Huang,X.Y. (1997) Direct stimulation of Bruton’s tyrosine kinase by G -protein α-subunit. Nature, 389, 296–299. In vitro B cell cultures Caaman˜o,J.H., Rizoo,C.A., Durham,S.K., Barton,D.S., Raventos- Whole spleen cell suspensions from 7- to 10-week-old mice were Suarez,C., Snapper,C.M. and Bravo,R. (1998) Nuclear Factor (NF)-κB2 depleted of erythrocytes by standard NH Cl lysis and enriched for B cells (p100/p52) is required for normal splenic microarchitecture and B cell- by incubation with anti-Thy1.2 (30-H12), anti-CD4 (GK1.5) and anti- mediated immune responses. J. Exp. Med., 187, 185–196. CD8 (YTS191) at room temperature for 30 min, and subsequent treatment Carson,S. and Wiles,M.V. (1993) Far upstream regions of class I MHC Ea with rabbit complement (CedarLane Laboratories, Hornby, Ontario, are necessary for position-independent, copy-dependent expression of Canada) at 37°C for 30 min. The fractions of B220 cells remaining Ea transgene. Nucleic Acids Res., 9, 2065–2072. after treatment were determined by FACS analyses. The enriched B Cook,M.C., Basten,A. and Fazekas de St Groth,B. (1997) Outer cells were cultured in round-bottomed microculture plates at a ﬁnal periarteriolar lymphoid sheath arrest and subsequent differentiation of concentration of 10 B cells/well in RPMI 1640 culture medium both naive and tolerant immunoglobulin transgenic B cells is determined –5 supplemented with 5% FCS and 510 M 2-mercaptoethanol, in the by B cell receptor occupancy. J. Exp. Med., 186, 631–643. presence of 50 μg/ml Escherichia coli LPS (serotype 026:B6, Difco DeJong,J.P., Voerman,J.S.A., Leenen,P.J., Van der Sluijs-Gelling,A.J. and Laboratories, Detroit, MI), several concentrations of polyclonal goat- Ploemacher,R.E. (1991) Improved ﬁxation of frozen lympho- anti-mouse IgM (Southern Biotechnology), 10 μg/ml rat anti-mouse haemopoietic tissue sections with hexazotized pararosaniline. CD40 (FGK-45.5, kindly provided by D.Gray), 100 ng/ml rIL-4 (R&D Histochem. J., 23, 392–401. Systems Inc., Minneapolis, MN) or 200 U/ml rINF-γ (R&D Systems Desiderio,S., (1997) Role of Btk in B cell development and signalling. Inc.). To measure DNA synthesis, after 2 days of culture cells were Curr. Opin. Immunol., 9, 534–540. pulsed with [ H]thymidine for 16–18 h, harvested and counted using DeWeers,M., Verschuren,M.C.M., Kraakman,M.E.M., Mensink,R.G.J., standard methods. Culture supernatants were assayed for Ig production Schuurman,R.K.B., Van Dongen,J.J.M. and Hendriks,R.W. (1993) The by ELISA on day 7. 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Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1998
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/17.18.5309
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.17 No.18 pp.5309–5320, 1998 Severe B cell deﬁciency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton’s tyrosine kinase 1,2 1 XLA patients displaying a large variety of mutations in Gemma M.Dingjan , Alex Maas , 2 3 the Btk gene (Vihinen et al., 1998) are very susceptible Martijn C.Nawijn , Linda Smit , 2 1 to bacterial infections. XLA is characterized by an almost Jane S.A.Voerman , Frank Grosveld and 1,4 complete block in B cell development at the pre-B cell Rudolf W.Hendriks stage, resulting in a severe decrease of circulating B cells. 1 2 Department of Cell Biology and Genetics and Department of Plasma cells are virtually absent and serum levels of all Immunology, Faculty of Medicine, Erasmus University Rotterdam, Ig classes are very low. The B cell defects in the CBA/N Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, and xid mice which carry an R28C mutation in the Btk PH Division of Cellular Biochemistry, The Netherlands Cancer Institute, domain are less severe (Wicker and Scher, 1986). These Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands mice have ~50% fewer B cells in the periphery and the Corresponding author high low residual cells exhibit an unusual IgM IgD proﬁle. e-mail: [email protected] They lack the CD5 B-1 B cell population and have low levels of IgM and IgG3. Although xid mice are generally To identify B-cell signaling pathways activated by able to respond to T cell-dependent (TD) antigens, they Bruton’s tyrosine kinase (Btk) in vivo, we generated cannot make antibodies to thymus-independent type 2 (TI- transgenic mice in which Btk expression is driven by II) antigens. Detection of a similar PH domain mutation, the MHC class II Ea gene locus control region. Btk R28H, in a patient with classical XLA suggested that the overexpression did not have signiﬁcant adverse effects distinction between the two phenotypes did not result on B cell function, and essentially corrected the X-linked from an allelic difference (DeWeers et al., 1994a). This immunodeﬁciency (xid) phenotype in Btk mice. In was conﬁrmed by the construction of null mutations in contrast, expression of a constitutively activated form the mouse Btk gene, which also resulted in xid phenotypes of Btk carrying the E41K gain-of-function mutation (Kahn et al., 1995; Kerner et al., 1995; Hendriks et al., resulted in a B cell defect that was more severe than 1996). By analysis of competition in vivo between wild- xid. The mice showed a marked reduction of the B cell type and Btk cells, it was shown that the ﬁrst selective compartment in spleen, lymph nodes, peripheral blood disadvantage of Btk-deﬁcient cells in the mouse is also at and peritoneal cavity. The levels in the serum of most the transition from pre-B to immature B cell (Hendriks immunoglobulin subclasses decreased with age, and et al., 1996). B cell responses to both T cell-independent type II Btk is expressed throughout B cell development, from and T cell-dependent antigens were essentially absent. the earliest pro-B cell stage up to mature B cells, and Expression of the E41K Btk mutant enhanced blast expression is downregulated in plasma cells (DeWeers formation of splenic B cells in vitro in response to anti- et al., 1993; Sideras and Smith et al., 1995; Hendriks IgM stimulation. Furthermore, the mice manifested a et al., 1996). Btk is also expressed in cells of the myeloid disorganization of B cell areas and marginal zones in lineage, but not in T cells. Btk has been implicated as a the spleen. Our ﬁndings demonstrate that expression mediator of signals from various receptors, including the of constitutively activated Btk blocks the development of follicular recirculating B cells. antigen receptor, interleukin 5 receptor (IL-5R), IL-6R, Keywords: B cell antigen receptor/B lymphocytes/Btk/ and CD38 in B lymphocytes, and the FcεRI in myeloid xid/XLA cells (reviewed in Desiderio, 1997). Btk activity is regu- lated by Src family kinases, phosphatidylinositol (PI) 3-kinase-γ and the α-subunit of the G class of G proteins (Rawlings et al., 1996; Bence et al., 1997; Li et al., 1997). Introduction After stimulation of the antigen receptor or IL-5R in B cells and the FcεRI in mast cells, Src family kinases Bruton’s tyrosine kinase (Btk) is a non-receptor protein rapidly induce phosphorylation of Y551 in the Btk kinase tyrosine kinase that is crucial for B lymphocyte develop- domain, followed by Btk autophosphorylation at Y223 in ment and function. Mutations in the Btk gene are the the SH3 domain (Wahl et al., 1997). These concerted genetic basis for X-linked agammaglobulinemia (XLA) in phosphorylation events were shown to be enhanced by a man and X-linked immunodeﬁciency disease (xid)inthe Glu-to-Lys mutation, E41K, in the PH domain of Btk mouse (Rawlings et al., 1993; Thomas et al., 1993; (Park et al., 1996). The E41K mutant, which was isolated Tsukada et al., 1993; Vetrie et al., 1993). Btk encodes a using a retroviral random mutagenesis scheme, was shown 659 amino acid protein that contains, in addition to the to induce transformation of NIH 3T3 ﬁbroblasts in soft Src homology domains SH2 and SH3 and a single catalytic agar cultures and factor-independent growth of the IL-5- domain, a unique pleckstrin homology (PH) domain at the N-terminus and an adjacent proline- and cysteine-rich dependent pro-B cell line Y16 (Li et al., 1995). The Tec homology (TH) domain (for review see Sideras and transforming activity of the E41K mutation is associated Smith, 1995; Desiderio, 1997). with increased membrane localization and tyrosine © Oxford University Press 5309 G.M.Dingjan et al. phosphorylation of Btk in transfected NIH 3T3 ﬁbroblast cells. PH domains recruit signaling molecules to the cell surface through speciﬁc interactions with phospholipids and proteins (reviewed in Lemmon et al., 1996). Binding of the Btk PH domain to various (phosphatidyl)-inositol phosphates, βγ-subunits of heterotrimeric G proteins and protein kinase C isoforms has been described (Tsukada et al., 1994; Yao et al., 1994; Fukada et al., 1996; Salim et al., 1996). The activating nature of the E41K mutation might be explained by its close proximity to the predicted inositol-phosphate binding site, as was indicated by X-ray crystallography studies (Hyvo¨nen and Saraste, 1997). In this context, the E41K mutant binds inositol 1,2,3,4,5,6- hexakiphosphate with a 2 higher afﬁnity than wild-type Btk (Fukada et al., 1996). We have previously described the generation of trans- genic mice that express human Btk (hBtk) under the control of the class II major histocompatibility complex (MHCII) Ea gene locus control region, which provides gene expres- sion in myeloid cells and in B-lineage cells from the pre-B cell stage onwards (Drabek et al., 1997). When the MHCII- hBtk mice were mated onto a Btk background, Btk protein expression was restored to apparently normal levels in the spleen and the Btk phenotype was corrected. B cells now low high differentiated to mature IgM IgD stages, peritoneal CD5 B cells were present and serum Ig levels and in vivo responses to TI-II antigens were in the normal ranges (Drabek et al., 1997). These results indicated that in this system the hBtk gene was appropriately targeted to both conventional and CD5 B-1 B cells. The activation of Btk by B cell antigen receptor- mediated phosphorylation (Aoki et al., 1994; DeWeers et al., 1994b; Saouaf et al., 1994; Wahl et al., 1997) raises the question about the nature of speciﬁc events that are controlled by Btk in developing B cells. To be able to identify signaling pathways that are activated by Btk in vivo, we have now modiﬁed the MHCII-hBtk transgene construct and generated two different types of transgenic mice, which either overexpress wild-type hBtk, or express Fig. 1. Structure and protein expression of the hBtk transgenes. various levels of the E41K gain-of-function Btk mutant. (A) Map of the transgene constructs, showing the locations of the ﬁve These transgenic models would indicate whether over- DNase I hypersensitivity sites present in the 10.6 KpnI–PvuI mouse expression or constitutive activation of Btk leads to MHC class II upstream Ea fragment. The WT-hBtk and E41K-hBtk proliferation of cells in the B cell lineage, immunodeﬁci- transgenes contain a 27.4 kb hBtk cDNA–genomic DNA fusion ency caused by elimination of B cells from the circulation, segment with exons 1–19, as well as a loxP sequence. The MHCII- hBtk transgene contains a 2.1 kb hBtk cDNA fragment (hatched box) or induction of B cell anergy. and a 2.8 kb human β-globin fragment with part of exon 2, exon 3 In this report we show that overexpression of hBtk had and the 3 untranslated region. E, EcoRI; K, KpnI; M, MluI; P, PvuI; only minor effects on B cell development and function. S, SwaI. (B) Western blot analysis of Btk protein expression in total In contrast, E41K hBtk mutant mice manifested an spleen cell lysates (210 cells/lane) from the indicated mice. Transgenic mice were on the Btk background. A polyclonal rabbit immunodeﬁcient phenotype that is more severe than xid antiserum was used, which was raised against fusion proteins of and is characterized by very low numbers of circulating gluthatione S-transferase (GST) and amino acids 163–218 of hBtk and B cells, an almost complete absence of B cell responses also recognized E41K hBtk or murine Btk. Ranges of the relative in vivo and a disruption of the cellular architecture of densities of the 77 kDa Btk signals as compared with the control Btk the spleen. mice are given at the bottom. Values were corrected for differences in the proportion of B cells in the spleen, which were determined by ﬂow cytometry. (C) Intracellular Btk expression during B cell maturation in Results the spleen. Single splenic cell suspensions from 3-month-old non- transgenic Btk or Btk mice, as well as WT-hBtk or E41K-hBtk Generation of WT-hBtk and E41K-hBtk transgenic transgenic mice on the Btk background were stained for surface B220 and IgM and subsequently for intracellular Btk. Data are shown as 5% mice probability B220/IgM contour plots of total lymphocytes, which were The constructs used in this study containing either the gated by forward and side scatter characteristics (top). The indicated wild-type (WT-hBtk) or the E41K mutant (E41K-hBtk) high low IgM and IgM B220 B cell populations were gated and Btk gene, as well as the MHCII-hBtk construct previously analyzed for Btk expression (bottom). The results are displayed as histograms of the indicated mice (solid lines), together with the used to obtain transgenic Btk expression (Drabek et al., background staining as determined in Btk mice (broken lines). 1997), are shown in Figure 1A. The E41K mutation was 5310 E41K Btk transgenic mice introduced byaGtoA replacement at position 257, using transgenic mice, the majority of Btk protein was found in in vitro site-directed mutagenesis. Since only high copy the cytosolic fraction. Also, in vivo tyrosine phosphoryl- number MHCII-hBtk transgenic mice (two out of ﬁve ation or in vitro autokinase activity of the hBtk protein in lines) expressed hBtk levels similar to those found in these cells did not appear to be enhanced by the E41K normal mice (Drabek et al., 1997), we attempted to mutation (data not shown). increase hBtk expression levels by including more hBtk genomic DNA, as well as the endogenous 3 untranslated Depletion of peripheral B cells in E41K-hBtk region, in the transgene construct. The transgenes con- transgenic mice tained a 10.6 kb MHC class II genomic DNA fragment, The B cell populations in bone marrow, peripheral blood, a 0.3 kb fragment with the ﬁrst three exons of hBtk as a spleen, mesenteric lymph nodes and peritoneal cavity from cDNA sequence, as well as a 27.1 kb genomic DNA E41K-hBtk and WT-hBtk mice on the Btk or Btk fragment, encompassing the hBtk exons 3–19 (Figure 1A). background were examined by ﬂow cytometry in 6- to The transgene constructs were microinjected into fertilized 8-week-old mice (Table I; Figures 2 and 3). Cells from non- oocytes and four independent E41K-hBtk transgenic lines transgenic Btk and Btk littermates served as controls, (#8, #10, #11 and #14) and three WT-hBtk transgenic lines showing that the Btk-deﬁcient mice had fewer mature B (G5, F2 and A5) were obtained. Founder mice were mated cells (~30–50% of normal) in peripheral blood, spleen, to Btk /lacZ mice, in which the Btk gene is inactivated by mesenteric lymph node (Figure 2A) and bone marrow a targeted in-frame insertion of a lacZ reporter in exon 8 (IgM IgD fraction), and a speciﬁc deﬁciency of mature low high (Hendriks et al., 1996). surface IgM IgD B cells (Figure 3A) as previously described (Hendriks et al., 1996). In the peritoneal cavity Expression levels of the E41K and WT hBtk of Btk mice, the numbers of conventional B cells were proteins reduced and CD5 cells were lacking (Table I; Figure 2B). Btk protein expression was evaluated in transgenic mice Correction of the xid B cell deﬁciency, although not on the Btk background by Western blotting of total spleen complete, was obtained by WT-hBtk transgene expression cell lysates (Figure 1B). The mice exhibited a wide range on the Btk background. In the peripheral blood, spleen, of transgenic Btk expression levels in the spleen, which mesenteric lymph node and bone marrow the B cell were directly correlated with the transgene copy number numbers only reached values similar to those of Btk as estimated by genomic Southern blotting analyses. mice, but the numbers of peritoneal CD5 cells were in To estimate the Btk expression levels of the individual the normal ranges, and the peripheral B cells exhibited a transgenic mouse lines, the densities of the Western blot normal surface IgM/IgD proﬁle (Table I; Figures 2 and Btk protein signals were quantiﬁed and corrected for the 3A). The effect of Btk overexpression on the Btk proportion of B cells in the spleen (which were signiﬁcantly background was limited: the numbers of B220 cells were lower in the E41K-hBtk transgenic mice; see below). In slightly reduced in peripheral blood, but were in the contrast to the MHCII-hBtk transgenic mice which showed normal ranges in the other organs analyzed (Table I). approximately endogenous Btk levels, the WT- and E41K- In contrast, when E41K-hBtk mice on the Btk back- hBtk mice manifested up to 14 overexpression of hBtk in ground were compared with Btk mice, a further depletion their splenocytes (Figure 1B). The experiments described of B cells was observed in all lymphoid tissues analyzed below were mainly performed on WT-hBtk line A5, and (Table I; Figure 2). Also in the mesenteric lymph nodes on E41K-hBtk line #8. Except where speciﬁcally indicated, from the three other independent E41K-hBtk transgenic no differences were detected between independent lines lines, the proportion of B cells was 1–5%. Expression of in the performed analyses, either for the WT-hBtk or the the E41K-hBtk transgene on the Btk background resulted E41K-hBtk transgenic mice. in an analogous reduction in the numbers of circulating Using intracellular ﬂow cytometry, we compared the B cells, although the effect was less severe than on the Btk expression levels of transgenic WT and E41K hBtk with background (Table I). The reduction of the proportions of the endogenous murine Btk during B cell differentiation. circulating B cells in the E41K-hBtk transgenic mice was The individual subpopulations of developing B cells in accompanied by a relative increase of the percentages of the bone marrow or spleen showed equivalent expression CD4 and CD8 T cells (Table I). The six groups of mice levels of the endogenous Btk (shown for spleen in Figure did not manifest signiﬁcant differences in the numbers of low high 1C). In constrast, a signiﬁcant increase in WT or E41K Mac-1 DX5 NK cells or Mac-1 myeloid cells in the transgenic hBtk protein was found as B cells maturated spleen cell suspensions (data not shown). In the bone high low from IgM to IgM cells in the spleen (Figure 1C). In marrow of E41K-hBtk mice, pro-B, pre-B and immature the bone marrow, transgenic Btk was only detected in B cells were present in normal proportions, whereas recirculating IgM IgD cells. Additional ﬂow cytometric mature recirculating IgM IgD B cells were virtually analyses demonstrated that transgenic Btk was also absent (Table I). Additional analysis of the three pro-B expressed in peritoneal B-1 B cells, in 10% of the cell subfractions, as deﬁned by expression of surface Mac-1 myeloid cells in the spleen and peritoneum, but markers B220, heat-stable antigen (HSA) and BP-1 (Hardy not in T cells or NK cells (data not shown). et al., 1991) in E41K-hBtk mice revealed no detectable When transfected into NIH 3T3 ﬁbroblasts, the E41K alterations from the distribution in normal or WT-hBtk Btk mutant manifested enhanced auto-phosphorylation mice (data not shown). and increased membrane targeting, while the in vitro In strong contrast to the restored IgM/IgD expression kinase activity was similar to wild-type Btk (Li et al., proﬁle found on peripheral B cells from WT-hBtk trans- 1995). However, when we analyzed unstimulated spleno- genic Btk mice, B cells in spleen, mesenteric lymph node cytes, whether from normal mice, WT-hBtk or E41K-hBtk and peritoneal cavity from E41K-hBtk transgenic Btk 5311 G.M.Dingjan et al. Table I. Frequencies of lymphocyte populations in WT-hBtk and E41K-hBtk transgenic mice Compartment Cell population Non-transgenic WT-hBtk E41K-hBtk a – – – Btk Btk Btk Btk Btk Btk b –6 Spleen Nucleated cells (10 ) 190 50 84 22 163 34 112 17 194 20 191 38 B220 cells (%) 38 915 534 10 22 520 610 3 CD3 CD4 (%) 20 322 526 423 826 727 5 CD3 CD8 (%) 11 212 312 312 315 415 4 Lymph node B220 cells (%) 24 48 221 57 25 0.4 2 1 CD3 CD4 (%) 46 553 250 456 160 562 3 CD3 CD8 (%) 22 131 225 227 328 429 3 Blood B220 cells (%) 34 912 419 610 47 24 1 low low of which B220 IgD (%) 10 441 16 10 523 725 14 51 14 high high of which B220 IgD (%) 71 336 15 66 546 537 12 17 10 Peritoneum CD5 IgM B cells (%) 16 8 0.4 0.3 28 16 14 73 22 1 CD5 IgM B cells (%) 24 10 10 424 15 8 14 32 1 CD5 IgM T cells (%) 31 346 829 547 11 65 462 8 Bone marrow B220 cells (%) 37 336 139 10 36 737 531 2 – c CD43 IgM pro-B cells (%) 6 0.3 8 16 38 27 36 0.4 – – CD43 IgM pre-B cells (%) 15 216 114 512 613 512 3 IgM IgD B cells (%) 7 28 0.3 9 28 28 0.3 7 1 IgM IgD B cells (%) 6 22 0.5 5 12 0.3 1 0.3 0.4 0.2 a / – – –/– b Btk mice were Btk /Y males or Btk females; Btk mice (Hendriks et al., 1996) were either Btk /Y males or Btk females. Mice were 6–8 weeks old. Data are mean values standard deviations from three mice analyzed, except for spleen where values are from 5–20 mice per group. The phenotype of lymphocyte populations was determined by ﬂow cytometry; dead cells and high side scatter cells were excluded by gating. Classiﬁcation of pro-B and pre-B cells was according to Hardy et al. (1991). high low mice manifested an IgM IgD phenotype, reminiscent of the B cell population found in non-transgenic Btk mice (shown for spleen in Figure 3A). The peripheral blood contained mainly newly-generated B cells that had low low just left the bone marrow (B220 IgD cells), rather than recirculating cells migrating between follicles high high (B220 IgD cells: only 17 10% of B cells in E41K- hBtk transgenic mice and 71 3% in normal Btk mice). As these observations suggested a maturational defect in the peripheral B cell compartment, we investigated the expression levels of B220 and HSA: B cells that are low high B220 HSA have recently left the bone marrow and high low further differentiate into mature B220 HSA cells of the long-lived B cell pool (Allman et al., 1993). While the spleen of non-transgenic Btk or Btk mice contained high low ~60% mature B220 HSA cells, a small reduction in this population was observed in WT-hBtk transgenic mice and a 3- to 4-fold reduction in E41K-hBtk transgenic mice (Figure 3B). These results indicated that recent emigrants from the bone marrow failed to mature in the spleen into high low long-lived B220 HSA B cells. Serum immunoglobulin levels in E41K-hBtk and WT-hBtk mice Serum Ig levels were determined by ELISA in 2-month- old non-transgenic Btk and Btk mice, as well as E41K- hBtk and WT-hBtk transgenic mice (Figure 4). The Btk mice had severely decreased levels of IgM and IgG3, Fig. 2. Depletion of peripheral B cells in E41K-hBtk mice. Flow variable levels of IgG1 and somewhat decreased levels of cytometric analysis of (A) mesenteric lymph node and (B) peritoneal IgG2a as compared with control Btk littermates (Drabek cavity from 7-week-old mice of non-transgenic Btk or Btk mice, and WT-hBtk or E41K-hBtk transgenic mice on the Btk background. et al., 1997). When the WT-hBtk transgene was expressed Single-cell suspensions were stained with biotinylated anti-IgM and on the Btk background, IgM levels were elevated and all streptavidin-TriColor, and either FITC-conjugated anti-B220 or PE- other Ig subclasses were restored to normal levels, similar conjugated anti-CD5. Data are displayed as 5% probability contour to the correction previously observed as a result of MHCII- plots of total lymphocytes, which were gated by forward and side hBtk transgene expression (Drabek et al., 1997). In the scatter characteristics. Percentages of total lymphocytes within the indicated gates are given. Data shown are representative of the mice WT-hBtk transgenic mice on the Btk background, serum examined (Table I). Ig subclass levels were in the same ranges. In the E41K- 5312 E41K Btk transgenic mice Fig. 3. Expression of E41K Btk induces a dominant maturational defect in peripheral B cells. (A) Surface IgM-IgD proﬁles of splenic low high B cells. Percentages of B220 cells that are IgM IgD are given. high low These cells are mature B cells, whereas IgM IgD cells are more immature (Hardy et al., 1982). Data are displayed as dot plots of all gated viable B220 cells from 310 total events. (B) Surface B220- HSA proﬁles of splenic B cells. Percentages of IgM cells that are low high high low immature (B220 HSA ) or mature (B220 HSA ) are indicated. Data are displayed as dot plots of all gated viable IgM cells from 10 total events (or 310 total events for E41K-hBtk transgenic mice on the Btk background). Spleen-cell suspensions from 7-week-old mice of the indicated genotypes were incubated with biotinylated anti-IgM and streptavidin-TriColor, FITC-conjugated anti-B220 and either PE- Fig. 4. Effects of transgenic WT-hBtk and E41K-hBtk expression on conjugated anti-IgD or anti-HSA and analyzed by three-color ﬂow serum Ig levels. Serum concentrations of the indicated Ig subclasses in cytometry. Data shown are representative of the mice examined; non-transgenic mice (Btk , n 11; Btk , n 16), as well as WT- lymphocytes were gated on the basis of forward and side scatter. hBtk (n 7) and E41K-hBtk (n 20) transgenic mice on the Btk background, whereby each symbol indicates an individual animal. Mice were 2 months old and Ig levels were determined by ELISA. hBtk mice serum IgM was restored to normal or elevated levels, IgG1 was similar to the levels in Btk littermates, while serum IgG3 was corrected to normal values for 12 B cells, signiﬁcant numbers of B cells were induced to out of 20 animals analyzed (Figure 4). The concentrations differentiate into Ig-producing plasma cells. However, the of IgG2a and Ig2b were generally in the normal ranges, possibility remained that these B cells could not mount whereas IgA was quite variable but on average reduced speciﬁc antibody responses. Therefore, we tested the compared with the other three groups of mice. No inﬂuence responses of 2-month-old non-transgenic, WT-hBtk and – – of the Btk or Btk background was detected. E41K-hBtk transgenic mice on the Btk or Btk back- Except for IgM and IgG2b, the serum Ig concentrations grounds to TI-II and TD antigenic challenges in vivo. of the E41K-hBtk mice decreased signiﬁcantly with age. The responsiveness to the TI-II antigen dinitrophenol In 6-month-old E41K-hBtk mice, the levels of IgG1, (DNP)-ﬁcoll was measured seven days after intraperitoneal IgG2a, IgG3 were only 37 10, 38 20 and 21 (i.p.) injection by enzyme-linked immunosorbent assay 15 μg/ml, respectively (n 3). In age-matched WT-hBtk (ELISA; Figure 5A). Consistent with previous ﬁndings in mice these levels were 970 170, 470 140 and 280 Btk-deﬁcient mice (Wicker and Scher, 1986; Kahn et al., 100 μg/ml (n 4). 1995; Drabek et al., 1997), DNP-speciﬁc IgM or IgG3 was completely absent in Btk mice, as the absorbence Defective in vivo responses in E41K-hBtk measured did not differ from the values of unimmunized transgenic mice animals. On the Btk background, the TI-II response of The absence of a dramatic decrease of serum Ig in E41K- WT-hBtk mice was comparable with the response of hBtk transgenic mice at the age of 2 months indicated normal mice, while on the Btk background, expression that despite the observed maturation defect of peripheral of the WT-hBtk transgene could only partially restore the 5313 G.M.Dingjan et al. in WT-hBtk mice on the Btk background. In strong contrast to these limited adverse effects of the WT-hBtk transgene, we found that secondary TD responses were very low or lacking in E41K-hBtk mice, irrespective of their Btk or Btk background (Figure 5C). Because Btk- deﬁcient mice show a normal secondary response to TD antigens (Wicker and Scher, 1986; Kahn et al., 1995; Drabek et al., 1997; Figure 5C), the ﬁnding that TD responses are essentially absent in E41K-hBtk transgenic mice was striking. Aberrant in vitro responses of E41K-hBtk transgenic B cells To examine the capacity of B cells to respond to mitogenic signals in vitro, B cell-enriched spleen cell suspensions were stimulated with LPS, anti-CD40 and IL-4, and different concentrations of a goat-antiserum to mouse IgM. Proliferative responses to LPS, determined by quantitat- ing [ H]thymidine incorporation after 60 h of culture, were low in Btk B cells compared with control B cells (Figure 6A). Expression of the WT-hBtk transgene completely restored the proliferative capacity, while expression of E41K-hBtk had a minor effect. These differences in proliferative responses to LPS were paralleled by the in vitro Ig production proﬁles in LPS- stimulated cell cultures as measured by ELISA at day 7 (Figure 6B). The defective IgM, IgG1 and IgG3 production in Btk B cells was corrected by transgenic WT-hBtk expression, but the effect of E41K-hBtk expression was moderate for IgM, and negligible for IgG1 and IgG3. Consistent with previous reports (Kahn et al., 1995; Fig. 5. Defective in vivo responses in E41K-hBtk mice. (A) IgM and Anderson et al., 1996; Ridderstad et al., 1996) Btk B IgG3 responses to the TI-II antigen DNP-ﬁcoll. (B) Primary IgM cells showed normal proliferation induced via CD40 in responses to the TD antigen TNP-KLH, determined 7 days after i.p. the presence of IL-4. Btk overexpression in the WT-hBtk injection. (C) Secondary TD responses to TNP-KLH, determined transgenic mice was accompanied by a small but signiﬁcant 7 days after i.p. injections performed 5–8 weeks after the primary immunization. Serial serum dilutions were analyzed for DNP/TNP- increase in proliferative responses. Transgenic expression speciﬁc antibodies of the speciﬁc subclasses and optical densities are of the E41K mutant did not have a detectable effect on shown as mean values from 4–12 mice in each group. The optical proliferation in response to anti-CD40 and IL-4 (Figure densities in the pre-immune sera did not vary signiﬁcantly between the 6B), nor in response to anti-CD40 alone or to anti-CD40 six groups and are shown in (C). and INF-γ (data not shown). When the production of IgM, IgG1 and IgG3 in anti-CD40- and IL-4-stimulated cell TI-II response. The TI-II antibody response in E41K-hBtk cultures was evaluated, a marginally lower production was mice was very low but detectable, whether on the Btk observed in Btk and E41K-hBtk B cells as compared or the Btk background. with normal Btk or WT-hBtk B cells. To investigate TD antibody responses, mice were injected One of the hallmarks of Btk B cells is that they do i.p. with trinitrophenol-keyhole limpet haemocyanin (TNP- not enter S phase when triggered through their surface KLH). After 7 days, TNP-speciﬁc IgM was measured by IgM receptor (Wicker and Scher, 1986; Kahn et al., 1995; ELISA (Figure 5B). Btk mice mounted a moderate Anderson et al., 1996). We observed that the absence of primary IgM response compared with wild-type litter- proliferative response to anti-IgM antibodies was corrected mates, as previously found in xid mice (Wicker and by transgenic expression of both wild-type and E41K Scher, 1986). Whereas Btk overexpression in the WT- mutated hBtk (Figure 6C). Moreover, when the E41K- hBtk transgenic mice appeared to have a minor inhibitory hBtk transgene was expressed, B cells enlarged more effect on the levels of TNP-speciﬁc IgM and IgG1, the rapidly in response to anti-IgM stimulation (Figure 6D). TD primary response was completely absent in E41K- After 24 h of culture, the E41K-hBtk B220 cells showed hBtk transgenic mice, both on the Btk and on the Btk a signiﬁcantly higher proportion of large blast cells when background (shown for IgM in Figure 5B). compared with non-transgenic Btk and Btk or WT-hBtk When the mice were 3–4 months old, TNP-KLH booster transgenic B220 cells. Blast formation was already injections were given and secondary TD responses were manifest in the absence of anti-IgM, but became more measured at day 7. TNP-speciﬁc levels of IgM, IgG1 and pronounced in the presence of anti-IgM up to 1 μg/ml. IgG2b were not signiﬁcantly different between the non- At high anti-IgM concentrations also WT-hBtk transgenic transgenic or WT-hBtk transgenic Btk or Btk groups of B cells showed enhanced blastogenesis when compared mice (Figure 5C). TNP-speciﬁc IgG2a was decreased in with B cells from non-transgenic mice. Increased blast WT-hBtk transgenic mice and TNP-speciﬁc IgG3 was low formation of E41K-hBtk transgenic B cells was also 5314 E41K Btk transgenic mice observed in response to stimulation with 50 μg/ml LPS (data not shown). The impairment of TD responses in the E41K-hBtk did not appear to be due to defective induction of MHC class II or the co-stimulatory molecule B7.2 (CD86) on activated B cells, as these activation markers were upregulated after stimulation with goat anti-mouse IgM, LPS, or anti-CD40 and IL-4; culture with anti-CD40 and IL-4 also induced upregulation of CD23 on E41K-hBtk B cells (data not shown). Disrupted splenic architecture in E41K-hBtk mice By immunohistochemical analyses, the spleens of 2-month-old unimmunized Btk , Btk or WT-hBtk trans- genic mice demonstrated a characteristic organization, in terms of segregation of T and B cells in the white pulp, with T cells clustered in the periarteriolar lymphocyte sheath (PALS) and surrounded by B cell-rich areas con- taining follicles, and the presence of marginal zones at the outer boundaries of the white pulp. In contrast, E41K- hBtk mice derived from all separate founder lines showed a speciﬁc effect of the E41K Btk mutation on the architec- ture of the spleen. When expressed on the Btk background, a disruption of splenic architecture was observed, charac- terized by a reduction in the number and size of B cell areas (consistent with our ﬂow cytometric analyses), loss of the strict compartmentalization of B and T lymphocytes and loss of a distinct marginal zone. Figure 7 shows double-labeling of serial spleen sections with either anti-B220, anti-CD3 or MOMA-1, a mono- clonal antibody speciﬁc for the metallophilic macrophages, which constitute a major component of the marginal zone (Kraal, 1992), together with the N418 antibody speciﬁc for the CD11c integrin on dendritic cells (Steinman et al., 1997). In the E41K-hBtk mice, B220 cells were present in B-cell areas neighboring the T cell zones (Figure 7A, B and C), but B cell numbers were reduced. These B cell areas contained signiﬁcantly more T cells than the B cell follicles in control Btk or Wt-hBtk transgenic mice (compare Figure 7D, E and F), and contained also CD11c interdigitating dendritic cells, which normally do not extend into B cell follicles (Steinman et al., 1997) (compare Figure 7G, H and I). No distinct marginal zones were present; the outer boundary of the white pulp did not contain B220 cells and metallophilic macrophages were only incidently present as a small rim of MOMA-1 cells Fig. 6. Transgenic E41K-hBtk B cells show aberrant responses to adjacent to the B cell areas (Figure 7H). In this area, an mitogenic signals in vitro.(A) Proliferation, determined by expansion was manifest of CD11c marginal dendritic [ H]thymidine incorporation, of cells cultured in medium alone, in the presence of LPS, or anti-CD40 antibodies and IL-4. (B) Ig levels of cells, which normally form bridging channels into the red supernatants of 7-day cultures in the presence of LPS or anti-CD40 pulp (Steinman et al., 1997) (compare Figure 7G, H and and IL-4. (C) Proliferation, determined by [ H]thymidine I). The numbers of ER-TR9 marginal zone macrophages incorporation, in response to various concentrations of goat anti-mouse – (Kraal, 1992) were severely reduced, and the reticular IgM. (A–C) Enriched splenic B cell fractions Btk , Btk ,WT-hBtk ﬁbroblast network, characteristic for marginal zones, as transgenic and E41K-hBtk transgenic mice were cultured in vitro with various mitogens. The groups of transgenic mice contained animals on revealed by ER-TR7 staining (VanVliet et al., 1986) Btk or Btk background; no differences were detected between mice appeared to be absent (data not shown). We observed a from these two backgrounds. Data are given as mean values SE dose-dependency of the effect of E41K-hBtk expression with 4–9 animals in each group (A), as symbols that indicate the on the disruption of splenic architecture, because in line values in cultures of individual animals (B) or as mean values from 4–9 mice in each group (C). (D) Total splenocytes from Btk , Btk , #14, which showed a higher transgene expression level – – WT-hBtk transgenic Btk and E41K-hBtk transgenic Btk mice were than line #8 (Figure 1B), MOMA-1 cells were essentially cultured with 1 μg/ml goat-anti-mouse IgM for the indicated time absent (Figure 7K and L). Nevertheless, in line #10 with points (left), or in the presence of the indicated αIgM concentrations low-level transgene expression (Figure 1B) the MOMA-1 for 24 h (right). The proportion of blast cells was determined by ﬂow- cytometric analysis of forward light scatter of viable B220 cells. cells were reduced in number, CD11c cells extended into Data are given as mean values from 2–4 mice in each group. the B cell areas and the strict separation of B and T cell 5315 G.M.Dingjan et al. Fig. 7. Disrupted splenic architecture in E41K-hBtk transgenic mice. Immunohistochemical analysis of 5 μm splenic frozen sections from Btk mice, – – WT-hBtk Btk mice and E41K-hBtk transgenic mice [(C), (F), (I) and (K), line #8, and (L), line #14] on the Btk background. Sections were stained with anti-B220 [blue, (A–C)] for B cells, anti-CD3 [blue, (D–F)] for T cells or MOMA-1 [blue, (G–L)] for metallophilic marginal zone macrophages, together with anti-CD11c/N418 (brown) to detect dendritic cells. Arrows [sections (I) and (K)] indicate the presence of small rims of MOMA-1 cells in the marginal zone. a, central arteriole; f, B cell follicle. Original magniﬁcations: 50 (A–I) and 16 (J–L). areas was lost (data not shown). In WT-hBtk mice, Analogous to the disruption of splenic architecture, also MOMA-1 cells were not affected and B cell areas did in mesenteric lymph nodes from E41K-hBtk mice on the not contain CD11c interdigitating cells (Figure 7H and Btk background B cell distribution defects were observed. J). The splenic abnormalities of E41K-hBtk transgenic B220 cells expressing IgM and IgD were almost absent mice on the Btk background were variable, but became in the cortical B cell areas, whereas IgM IgD B cells more severe with age. When spleens from 4- to 5-month- were present in low numbers as clusters in the paracortical old mice were analyzed, the segregation of B and T cell T cell zones (data not shown). areas was lost and the numbers of MOMA-1 and ER- TR9 cells were severely reduced. Discussion Germinal center formation was evaluated by immuno- histochemical analysis of the spleen, both in 4- to 5-month- To date, numerous interactions of the individual domains old unimmunized mice and in mice 7 days after booster of Btk with various protein or lipid molecules have been injection with TNP-KLH adsorbed to alum. In contrast to reported (Fukada et al., 1996; Bence et al., 1997; Li et al., – – the Btk mice, Btk and WT-hBtk transgenic Btk mice, 1997; Wahl et al., 1997; reviewed in Mattsson et al., the E41K-hBtk transgenic Btk mice did not develop 1996). To be able to study the impact of these particular germinal centers, as B220 IgD cells that showed binding interactions on B cell differentiation and function in vivo, of peanut agglutinin (PNA) were completely absent (data we have developed mouse models in which wild-type or not shown). mutated Btk can be appropriately expressed as transgenes. 5316 E41K Btk transgenic mice We previously showed that expression of hBtk driven by presence of intact endogenous murine Btk, substantiated the class II MHC Ea locus LCR resulted in physiological the dominant nature of the E41K mutation. Btk protein levels in the spleen, which completely cor- When expressed in NIH 3T3 cells in vitro, the gain-of- rected the features of the xid phenotype (Drabek et al., function activity of the E41K Btk mutant was associated 1997). with increased membrane localization and was shown to require kinase activity (Li et al., 1995). Our biochemical analyses in unstimulated B cells demonstrated that the Limited effects of overexpression of Btk E41K mutant had in vitro kinase activity, but did not By the introduction of 16 out of 18 intron segments of reveal a dramatic increase in membrane targeting. Never- the Btk gene, together with the endogenous 3 untranslated theless, we cannot exclude that the E41K mutation was region, high-level Btk expression was obtained. It seems associated with relatively small changes in subcellular likely that these modiﬁed transgenes contained endogenous localization of Btk, which are difﬁcult to detect in bio- regulatory elements of the Btk gene, since Btk intron chemical assays. In this context, it was shown that subtle sequences were shown to contain multiple clusters of variations in antigen concentration or expression of CD19, extensive conservation between mouse and man (Oeltjen a co-receptor that lowers the threshold for antigen receptor et al., 1997), one of which, a 229 bp region in the ﬁfth stimulation, resulted in dramatic changes of the fate of intron with 90% identity, showed co-localization with a B cells in vivo (Cook et al., 1997; Tedder et al., 1997). DNase I hypersensitive site present in B cells but not in Nevertheless, two of our ﬁndings indicated that the E41K T cells from human tonsils (G.M.Dingjan, unpublished mutation represented an activated form of Btk. (i) Expres- results). Thus, the transgenic Btk expression pattern may sion of E41K enhanced in vitro blast formation of splenic reﬂect a combination of the effects of MHC class II Ea B cells in culture, either with or without mitogens (Figure gene LCR and endogenous Btk regulatory sequences. 6D). (ii) Splenic E41K-hBtk B cells showed proliferation On the Btk background, overexpression of hBtk cor- upon stimulation with anti-IgM antibodies (Figure 6C), rected most of the xid defects, as indicated by the despite the unusually high proportion of immature low high appearance of normal proportions of mature IgM IgD low high B220 HSA cells in this population. Normally, such circulating B cells, CD5 B cells in the peritoneum, low high B220 HSA virgin B cells are refractory to anti-IgM restored serum IgM and IgG3 levels and in vitro responses stimulation (Allman et al., 1992). to anti-IgM or LPS stimulation. However, in contrast with our ﬁndings in the MHCII-hBtk mice, TI-II responses Expression of E41K-hBtk impedes follicular entry were only partially restored and primary TD IgM responses and disrupts marginal zone microarchitecture were somewhat reduced. Our ﬁndings in the E41K-hBtk transgenic mice indicate Although WT-hBtk overexpression on the Btk back- that constitutive activation of Btk blocks the development ground was found to be associated with partially enhanced of follicular B cells. The numbers of recirculating B cells blastogenesis and proliferation of B cells in response to were severely reduced, speciﬁcally in peripheral blood anti-IgM stimulation in vitro (Figure 6C and D), this did not and lymph nodes. In the spleen, B cell areas typically appear to result in adverse effects on B cell development or contained CD11c interdigitating dendritic cells, which function: B cell numbers were largely in the normal normally do not extend into B cell follicles, as well as ranges, and B cell responses to TI-II or TD antigens unusually high numbers of T cells. Signals that induce in vivo or LPS in vitro were not notably affected. Therefore, B cells to become recirculating follicular B cells are we conclude that Btk overexpression per se does not lead mediated by the B-cell antigen receptor, as most peripheral to signiﬁcant activation of downstream signaling pathways. B cells are ligand-selected (Gu et al., 1991) and mutations of the CD79α/Igα or Syk signaling components of the E41K-hBtk represents an activated form of Btk B cell antigen receptor block the entry of B cells into In contrast, expression of the Btk E41K gain-of-function follicles (Torres et al., 1996; Turner et al., 1997). These mutation on the Btk background blocked maturation of ﬁndings demonstrated that low-level stimulation of the peripheral B cells, leading to a B cell deﬁciency that was B cell antigen receptor provides a signal that is required more severe than the xid phenotype. Although E41K-hBtk for follicular entry. Our results show that in the presence transgenic mice produced normal numbers of immature B of E1K-hBtk expression such signals were not transduced. cells in the bone marrow that began to express IgD, they Together with the reported abnormal maturation into low high failed to become mature recirculating follicular B cells in IgM IgD follicular B cells in Btk-deﬁcient mice the periphery. A severe reduction of B cell numbers was (Wicker and Scher, 1986; Kahn et al., 1995; Hendriks observed in all peripheral tissues and those B cells present et al., 1996), this argues for a crucial role of Btk in the low high in the periphery were mainly B220 and HSA .Itis recruitment of B cells in the long-lived recirculating most probable that these cells represented newly-generated B-cell pool. virgin B cells, which generally have a lifespan of only Although the possibility that constitutive activation of 3 days (Fulcher and Basten, 1997). Total serum Ig levels Btk leads to a general defect that impedes the survival or were not seriously affected at the age of 2 months, but affects the migration of B cells in the periphery cannot they decreased signiﬁcantly with age. The in vivo B cell be excluded, it is attractive to hypothesize that expression response to TI-II antigen was very low and only marginally of the E41K Btk mutant mimics B cell antigen receptor restored compared with Btk mice, while B cell responses engagement. In the E41K-hBtk mice those cells that high high low to TD antigen were lacking. The ﬁnding that E41K-hBtk were present in the spleen were IgM HSA B220 transgene expression resulted in a decrease of circulating immature B cells that have just left the bone marrow. B cell numbers and a loss of B cell functions, even in the Normally, immature B cells in the bone marrow are 5317 G.M.Dingjan et al. susceptible to negative selection (Goodnow et al., 1995) transduction of signals that govern the development of high and immature HSA splenic B cells are refractory to recirculating follicular B cells. The absence of Btk leads to low high stimulation with anti-IgM or phorbol ester and calcium abnormal maturation into IgM IgD follicular B cells ionophore (Allman et al., 1992). Therefore, the immature (Wicker and Scher, 1986; Kahn et al., 1995; Hendriks status of the E41K-hBtk activated B cells may hamper et al., 1996). We have shown that transgenic expression their subsequent expansion or differentiation and induce of the E41K Btk mutant blocks the development of their elimination instead. In this context, E41K-hBtk B cells recirculating follicular B cells, indicating that constitutive may resemble auto-reactive B cells which have received activation of Btk induces the elimination of virgin peri- a stimulatory signal through their antigen receptor, pheral B cells. Further experiments are required to investi- resulting in arrest in the outer PALS where their lifespan gate whether this elimination is caused by the absence of is reduced to 3–4 days in the absence of T cell help a basal antigen receptor signal that is thought to direct (Fulcher and Basten, 1994; Goodnow et al., 1995). Addi- developmental progression of B cells, or by the presence tional parallels with auto-reactive B cells include the rapid of a signal that mimics B cell receptor occupancy by increase in size of auto-reactive B cells upon self-antigen self-antigens. recognition immediately before their disappearance (Fulcher et al., 1996; Rathmell et al., 1996) and their Materials and methods absence in the splenic marginal zones (Mason et al., 1992). In vitro mutagenesis The lack of splenic marginal zone B cells in E41K- The E41K mutation was created byaGtoA replacement at position hBtk mice could be a direct result of the impeded follicular 257 in the hBtk cDNA clone phBtk2.55 in pBlueScript (Drabek et al., entry of B cells, as marginal zone B cells are derived 1997). Double-stranded site-directed mutagenesis (Stratagene, La Jolla, from follicular B cells (Kumararatne and MacLennan, CA) was performed with the E41K mutagenic primer 5-GCACA AACTC TCCTA CTATA AGTAT GACTT TGAAC GTGGG-3 and a 1981). The B cell abnormalities in E41K-hBtk mice may 39 bp KpnI→BglII selection primer, and the obtained mutant plasmids also hamper the normal development of marginal zone were sequenced, using standard methods. From the original wild-type macrophage populations. Further studies will be required hBtk and the E41K mutated cDNA clone, 303 bp PvuI–NlaIV fragments, to clarify whether the B cell or the macrophage population encompassing the ﬁrst two exons and part of exon 3, were used in the construction of the transgenes. of the marginal zone is intrinsically affected in E41K- hBtk mice, or whether the defects in both populations are Construction of WT-hBtk and E41K-hBtk transgenes secondary to accessory cell dysfunction. Since we have The two transgene constructs WT-hBtk and E41K-hBtk are shown in observed a similar disruption of splenic marginal zone Figure 1A. From the MHC class II Ea gene cosmid 32.1 (Carson and Wiles, 1993) a 4.0 kb KpnI fragment, containing DNase I hypersensitivity architecture in mice that express the E41K-hBtk mutant sites (HSS) I and II and a unique PvuI site at position 14 in the Ea under the control of the B cell speciﬁc CD19 promoter gene, was cloned into pBlueScript, using a NotI–SmaI–KpnI–SwaI– (A.Maas, unpublished results), it seems less probable that XhoI–NotI polylinker. The resulting plasmid was partially digested with macrophage development is intrinsically affected by the Asp718 in the presence of ethidium bromide to introduce a 9.4 kb Asp718 fragment from cosmid 32.1, encompassing HSS III to V. expression of the E41K Btk mutant. Clues about the Subsequently, a 13.4 kb NotI fragment from this plasmid was cloned relationship between these cell populations may come into a NotI-digested cosmid vector pTL5 (Lund et al., 1982), in which from experiments in mutant mice that present with closely the same polylinker as described above was introduced at a unique BglII related phenotypes, such as mice deﬁcient in members site. In a next step two fragments were cloned into this plasmid, using the unique PvuI site in the Ea gene and the unique SwaI site within the of the nuclear factor-κB family of transcription factors polylinker: (1) a 303 bp PvuI–NlaIV WT or E41K-mutated hBtk cDNA (Franzoso et al., 1997, 1998; Caaman˜o et al., 1998) or fragment (as described above) and a 97 bp NlaIV–SwaI fragment tumor necrosis factor ligand and receptor family members obtained by PCR ampliﬁcation using primers at positions 46846 and (Matsumoto et al., 1997), which present with defects in 47029 of the hBtk gene (Oeltjen et al., 1997) and subsequent digestion humoral responses, germinal center formation and mar- with NlaIV and SwaI. In the two resulting plasmids with the WT or E41K-mutated ﬁrst three Btk exons, a 109 bp SalI–XhoI fragment ginal zone macrophage subpopulations. containing loxP sequences (obtained by insertion of a loxP oligonucleo- tide, 5-ATAAC TTCGT ATAGC ATACA TTATA CGAAG TTAT-3 The role of Btk in B cell development into the Asp718 site of the pPolyIII vector) was cloned into the XhoI site Studies in transgenic mice carrying rearranged Ig genes within the NotI–SmaI–KpnI–SwaI–XhoI–NotI polylinker. Subsequently, a have established that strong antigen receptor signals (e.g. 23.1 kb SwaI–SalI fragment, encompassing Btk exons 5–19, was intro- duced using unique SwaI and XhoI sites within the polylinker. This transmitted by autoantigens) can result in antigen receptor 23.1 kb SwaI–SalI fragment was from a 32.9 kb cosmid which was editing or elimination of immature B cells in the bone isolated from a mini-library constructed from a 340 kb YAC clone marrow (reviewed in Goodnow et al., 1995; Melamed containing the hBtk gene (DeWeers et al., 1997). Finally, a ~4 kb SwaI et al., 1998). The absence of any defects in developing fragment from the same cosmid clone, which contained Btk exon 4, was introduced into the unique SwaI site. B cells in the bone marrow of E41K-hBtk mice (except in the recirculating mature cells) does not imply that Generation of transgenic mice constitutive activation of Btk would not have any effect The WT-hBtk and E41K-hBtk constructs were digested with NotIto in the bone marrow. Our intracellular ﬂow-cytometric release the ~38.2 kb DNA fragments shown in Figure 1. Gel-puriﬁed DNA was injected into pronuclei of FVBFVB fertilized oocytes at a analyses indicate that the expression level of the E41K- concentration of ~2 ng/μl and implanted into pseudopregnant female hBtk transgene may not have reached a critical threshold mice. Tail DNA was analyzed by Southern blotting using a partial hBtk value in the bone marrow to affect B cell development. cDNA probe (bp 133–1153) to determine the genotype of the founder In fact, the ﬁrst defects in the B cell lineage only became mice and mice generated in subsequent crosses with Btk /lacZ mice of mixed 129/SvC57BL/6 background (Hendriks et al., 1996). apparent in the spleen, where we identiﬁed a signiﬁcant increase in expression of the hBtk transgenes during Flow cytometric analyses high low maturation from IgM to IgM B cells. Preparation of single-cell suspensions and three- or four-color ﬂow In summary, we conclude that Btk is essential for the cytometry have been described (Hendriks et al., 1996). Intracellular ﬂow 5318 E41K Btk transgenic mice cytometric detection of cytoplasmic Btk protein was performed on cells (Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti- that were ﬁrst stained for cell surface markers and subsequently ﬁxed rat Ig alkaline phosphatase (Southern Biotechnology). in 2% paraformaldehyde and permeabilized using 0.5% saponin. Events (3–510 ) were scored using a FACScan or FACSCalibur ﬂow cytometer and analyzed by CellQuest software (Becton Dickinson, Sunnyvale, Acknowledgements CA). The following monoclonal antibodies were obtained from Pharmingen (San Diego, CA): FITC-conjugated anti-B220/RA3-6B2, We thank A.Boonstra, J.Borst, L.Braam, S.Carson, W.van Ewijk, M.Kuit, anti-HSA/M1/6, anti-CD3, anti-DX5, and anti-BP-1/6C3, PE-conjugated P.Leenen and H.Savelkoul for assistance at various stages of the project. anti-CD43/S7, anti-CD11b/Mac1, anti-CD5/Ly-1, anti-CD4, Cy-Chrome R.W.H. was supported by the Royal Academy of Arts and Sciences. conjugated anti-B220/RA3-6B2 and anti-CD8, and biotinylated anti- These studies were supported in part by the Netherlands Organization HSA/M1/6 and anti-IgM. PE-conjugated anti-IgD was purchased from for Scientiﬁc Research NWO (A.M.) and the Dutch Prevention Founda- Southern Biotechnology (Birmingham, AL). Anti-CD23 (B3B4) and tion (A.M. and G.M.D.). anti-CD28/B7.2 (GL1) were puriﬁed monoclonal antibodies conjugated to biotin and FITC, respectively, according to standard procedures. Afﬁnity-puriﬁed polyclonal rabbit anti-Btk was from Pharmingen. Secondary antibodies used were TriColor- or PE-conjugated streptavidin References (Caltag Laboratories, Burlingame, CA), streptavidin-APC (Pharmingen) Allman,D.M., Ferguson,S.E. and Cancro,M.P. (1992) Peripheral B cell or FITC-conjugated goat anti-rabbit Ig (Nordic, Capistrano beach, CA). maturation I. Immature peripheral B cells in adults are heat-stable antigen (hi) and exhibit unique signalling characteristics. J. Immunol., Ig detection and in vitro immunizations 149, 2533–2540. Levels of Ig subclasses in serum or culture supernatants were measured Allman,D.M., Ferguson,S.E., Lentz,V.M. and Cancro,M.P. (1993) by sandwich ELISA, using unlabeled and peroxidase-labeled anti-mouse Peripheral B cell maturation II. Heat-stable antigen (hi) splenic B cells Ig isotype-speciﬁc antibodies (Southern Biotechnology). Serially diluted are an immune developmental intermediate in the production of long- sera were incubated at room temperature for 3 h, and azino-bis-ethylbenz- lived marrow-derived B cells. J. Immunol., 151, 4431–4444. thiazoline sulfonic acid was used as a substrate. Antibody concentrations Anderson,J.S., Teutsch,M., Dong,Z. and Wortis,H.H. (1996) An essential were calculated by using puriﬁed isotype Ig proteins as standards. TD role for Bruton’s tyrosine kinase in the regulation of B-cell apoptosis. and TI-II immunizations and TNP-speciﬁc ELISA were essentially Proc. Natl Acad. Sci. USA, 93, 10966–10971. performed as described previously (Maas et al., 1997). Booster doses Aoki,Y., Isselbacher,K.J. and Pillai,S. (1994) Bruton tyrosine kinase is were given after 5–8 weeks. Serum dilutions were incubated at room tyrosine phosphorylated and activated in pre-B lymphocytes and temperature for 3 h and the biotinylated TNP-KLH step was overnight receptor-ligated B cells. Proc. Natl Acad. 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The EMBO Journal – Springer Journals

Published: Sep 15, 1998

Keywords: B cell antigen receptor; B lymphocytes; Btk; xid; XLA

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Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

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Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

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