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• W2130963085 abstract "Article15 September 1998free access Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase Gemma M. Dingjan Gemma M. Dingjan Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Alex Maas Alex Maas Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Martijn C. Nawijn Martijn C. Nawijn Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Linda Smit Linda Smit Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Jane S.A. Voerman Jane S.A. Voerman Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Frank Grosveld Frank Grosveld Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Rudolf W. Hendriks Corresponding Author Rudolf W. Hendriks Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Gemma M. Dingjan Gemma M. Dingjan Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Alex Maas Alex Maas Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Martijn C. Nawijn Martijn C. Nawijn Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Linda Smit Linda Smit Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Jane S.A. Voerman Jane S.A. Voerman Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Frank Grosveld Frank Grosveld Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Rudolf W. Hendriks Corresponding Author Rudolf W. Hendriks Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Author Information Gemma M. Dingjan1,2, Alex Maas1, Martijn C. Nawijn2, Linda Smit3, Jane S.A. Voerman2, Frank Grosveld1 and Rudolf W. Hendriks 1 1Department of Cell Biology and Genetics Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands 2Department of Immunology, Faculty of Medicine, Erasmus University Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands 3Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5309-5320https://doi.org/10.1093/emboj/17.18.5309 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially corrected the X-linked immunodeficiency (xid) phenotype in Btk− mice. In contrast, expression of a constitutively activated form of Btk carrying the E41K gain-of-function mutation resulted in a B cell defect that was more severe than xid. The mice showed a marked reduction of the B cell compartment in spleen, lymph nodes, peripheral blood and peritoneal cavity. The levels in the serum of most immunoglobulin subclasses decreased with age, and B cell responses to both T cell-independent type II and T cell-dependent antigens were essentially absent. Expression of the E41K Btk mutant enhanced blast formation of splenic B cells in vitro in response to anti-IgM stimulation. Furthermore, the mice manifested a disorganization of B cell areas and marginal zones in the spleen. Our findings demonstrate that expression of constitutively activated Btk blocks the development of follicular recirculating B cells. Introduction Bruton's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase that is crucial for B lymphocyte development and function. Mutations in the Btk gene are the genetic basis for X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency disease (xid) in the mouse (Rawlings et al., 1993; Thomas et al., 1993; Tsukada et al., 1993; Vetrie et al., 1993). Btk encodes a 659 amino acid protein that contains, in addition to the Src homology domains SH2 and SH3 and a single catalytic domain, a unique pleckstrin homology (PH) domain at the N-terminus and an adjacent proline- and cysteine-rich Tec homology (TH) domain (for review see Sideras and Smith, 1995; Desiderio, 1997). XLA patients displaying a large variety of mutations in the Btk gene (Vihinen et al., 1998) are very susceptible to bacterial infections. XLA is characterized by an almost complete block in B cell development at the pre-B cell stage, resulting in a severe decrease of circulating B cells. Plasma cells are virtually absent and serum levels of all Ig classes are very low. The B cell defects in the CBA/N xid mice which carry an R28C mutation in the Btk PH domain are less severe (Wicker and Scher, 1986). These mice have ∼50% fewer B cells in the periphery and the residual cells exhibit an unusual IgMhighIgDlow profile. They lack the CD5+ B-1 B cell population and have low levels of IgM and IgG3. Although xid mice are generally able to respond to T cell-dependent (TD) antigens, they cannot make antibodies to thymus-independent type 2 (TI-II) antigens. Detection of a similar PH domain mutation, R28H, in a patient with classical XLA suggested that the distinction between the two phenotypes did not result from an allelic difference (DeWeers et al., 1994a). This was confirmed by the construction of null mutations in the mouse Btk gene, which also resulted in xid phenotypes (Kahn et al., 1995; Kerner et al., 1995; Hendriks et al., 1996). By analysis of competition in vivo between wild-type and Btk− cells, it was shown that the first selective disadvantage of Btk-deficient cells in the mouse is also at the transition from pre-B to immature B cell (Hendriks et al., 1996). Btk is expressed throughout B cell development, from the earliest pro-B cell stage up to mature B cells, and expression is downregulated in plasma cells (DeWeers et al., 1993; Sideras and Smith et al., 1995; Hendriks et al., 1996). Btk is also expressed in cells of the myeloid lineage, but not in T cells. Btk has been implicated as a mediator of signals from various receptors, including the antigen receptor, interleukin 5 receptor (IL-5R), IL-6R, and CD38 in B lymphocytes, and the FcϵRI in myeloid cells (reviewed in Desiderio, 1997). Btk activity is regulated by Src family kinases, phosphatidylinositol (PI) 3-kinase-γ and the α-subunit of the Gq class of G proteins (Rawlings et al., 1996; Bence et al., 1997; Li et al., 1997). After stimulation of the antigen receptor or IL-5R in B cells and the FcϵRI in mast cells, Src family kinases rapidly induce phosphorylation of Y551 in the Btk kinase domain, followed by Btk autophosphorylation at Y223 in the SH3 domain (Wahl et al., 1997). These concerted phosphorylation events were shown to be enhanced by a Glu-to-Lys mutation, E41K, in the PH domain of Btk (Park et al., 1996). The E41K mutant, which was isolated using a retroviral random mutagenesis scheme, was shown to induce transformation of NIH 3T3 fibroblasts in soft agar cultures and factor-independent growth of the IL-5-dependent pro-B cell line Y16 (Li et al., 1995). The transforming activity of the E41K mutation is associated with increased membrane localization and tyrosine 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; Fukuda 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 (Hyvönen 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 (Fukuda et al., 1996). We have previously described the generation of transgenic 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 expression 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 differentiated to mature IgMlowIgDhigh 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 various levels of the E41K gain-of-function Btk mutant. These transgenic models would indicate whether overexpression or constitutive activation of Btk leads to proliferation of cells in the B cell lineage, immunodeficiency caused by elimination of B cells from the circulation, or induction of B cell anergy. In this report we show that overexpression of hBtk had only minor effects on B cell development and function. In contrast, E41K hBtk mutant mice manifested an immunodeficient phenotype that is more severe than xid and is characterized by very low numbers of circulating B cells, an almost complete absence of B cell responses in vivo and a disruption of the cellular architecture of the spleen. Results Generation of WT-hBtk and E41K-hBtk transgenic mice The constructs used in this study containing either the wild-type (WT-hBtk) or the E41K mutant (E41K-hBtk) Btk gene, as well as the MHCII-hBtk construct previously used to obtain transgenic Btk expression (Drabek et al., 1997), are shown in Figure 1A. The E41K mutation was introduced by a G to A replacement at position 257, using in vitro site-directed mutagenesis. Since only high copy number MHCII-hBtk transgenic mice (two out of five lines) expressed hBtk levels similar to those found in normal mice (Drabek et al., 1997), we attempted to increase hBtk expression levels by including more hBtk genomic DNA, as well as the endogenous 3′ untranslated region, in the transgene construct. The transgenes contained a 10.6 kb MHC class II genomic DNA fragment, a 0.3 kb fragment with the first three exons of hBtk as a cDNA sequence, as well as a 27.1 kb genomic DNA fragment, encompassing the hBtk exons 3–19 (Figure 1A). The transgene constructs were microinjected into fertilized oocytes and four independent E41K-hBtk transgenic lines (#8, #10, #11 and #14) and three WT-hBtk transgenic lines (G5, F2 and A5) were obtained. Founder mice were mated to Btk−/lacZ mice, in which the Btk gene is inactivated by a targeted in-frame insertion of a lacZ reporter in exon 8 (Hendriks et al., 1996). Figure 1.Structure and protein expression of the hBtk transgenes. (A) Map of the transgene constructs, showing the locations of the five DNase I hypersensitivity sites present in the 10.6 KpnI–PvuI mouse MHC class II upstream Ea fragment. The WT-hBtk and E41K-hBtk transgenes contain a 27.4 kb hBtk cDNA–genomic DNA fusion 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) and a 2.8 kb human β-globin fragment with part of exon 2, exon 3 and the 3′ untranslated region. E, EcoRI; K, KpnI; M, MluI; P, PvuI; S, SwaI. (B) Western blot analysis of Btk protein expression in total spleen cell lysates (2×105 cells/lane) from the indicated mice. Transgenic mice were on the Btk− background. A polyclonal rabbit antiserum was used, which was raised against fusion proteins of gluthatione S-transferase (GST) and amino acids 163–218 of hBtk and also recognized E41K hBtk or murine Btk. Ranges of the relative densities of the 77 kDa Btk signals as compared with the control Btk+ 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 the spleen. Single splenic cell suspensions from 3-month-old non-transgenic Btk+ or Btk− mice, as well as WT-hBtk or E41K-hBtk transgenic mice on the Btk− background were stained for surface B220 and IgM and subsequently for intracellular Btk. Data are shown as 5% probability B220/IgM contour plots of total lymphocytes, which were gated by forward and side scatter characteristics (top). The indicated IgMhigh and IgMlow B220+ B cell populations were gated and analyzed for Btk expression (bottom). The results are displayed as histograms of the indicated mice (solid lines), together with the background staining as determined in Btk− mice (broken lines). Download figure Download PowerPoint Expression levels of the E41K and WT hBtk proteins Btk protein expression was evaluated in transgenic mice on the Btk− background by Western blotting of total spleen cell lysates (Figure 1B). The mice exhibited a wide range of transgenic Btk expression levels in the spleen, which were directly correlated with the transgene copy number as estimated by genomic Southern blotting analyses. To estimate the Btk expression levels of the individual transgenic mouse lines, the densities of the Western blot Btk protein signals were quantified and corrected for the proportion of B cells in the spleen (which were significantly lower in the E41K-hBtk transgenic mice; see below). In contrast to the MHCII-hBtk transgenic mice which showed approximately endogenous Btk levels, the WT- and E41K-hBtk mice manifested up to 14× overexpression of hBtk in their splenocytes (Figure 1B). The experiments described below were mainly performed on WT-hBtk line A5, and on E41K-hBtk line #8. Except where specifically indicated, no differences were detected between independent lines in the performed analyses, either for the WT-hBtk or the E41K-hBtk transgenic mice. Using intracellular flow cytometry, we compared the expression levels of transgenic WT and E41K hBtk with the endogenous murine Btk during B cell differentiation. The individual subpopulations of developing B cells in the bone marrow or spleen showed equivalent expression levels of the endogenous Btk (shown for spleen in Figure 1C). In constrast, a significant increase in WT or E41K transgenic hBtk protein was found as B cells maturated from IgMhigh to IgMlow cells in the spleen (Figure 1C). In the bone marrow, transgenic Btk was only detected in recirculating IgM+IgD+ cells. Additional flow cytometric analyses demonstrated that transgenic Btk was also expressed in peritoneal B-1 B cells, in <10% of the Mac-1+ myeloid cells in the spleen and peritoneum, but not in T cells or NK cells (data not shown). When transfected into NIH 3T3 fibroblasts, the E41K Btk mutant manifested enhanced auto-phosphorylation and increased membrane targeting, while the in vitro kinase activity was similar to wild-type Btk (Li et al., 1995). However, when we analyzed unstimulated splenocytes, whether from normal mice, WT-hBtk or E41K-hBtk transgenic mice, the majority of Btk protein was found in the cytosolic fraction. Also, in vivo tyrosine phosphorylation or in vitro autokinase activity of the hBtk protein in these cells did not appear to be enhanced by the E41K mutation (data not shown). Depletion of peripheral B cells in E41K-hBtk transgenic mice The B cell populations in bone marrow, peripheral blood, spleen, mesenteric lymph nodes and peritoneal cavity from E41K-hBtk and WT-hBtk mice on the Btk+ or Btk− background were examined by flow cytometry in 6- to 8-week-old mice (Table I; Figures 2 and 3). Cells from non-transgenic Btk+ and Btk− littermates served as controls, showing that the Btk-deficient mice had fewer mature B cells (∼30–50% of normal) in peripheral blood, spleen, mesenteric lymph node (Figure 2A) and bone marrow (IgM+IgD+ fraction), and a specific deficiency of mature surface IgMlowIgDhigh B cells (Figure 3A) as previously described (Hendriks et al., 1996). In the peritoneal cavity of Btk− mice, the numbers of conventional B cells were reduced and CD5+ cells were lacking (Table I; Figure 2B). Figure 2.Depletion of peripheral B cells in E41K-hBtk mice. Flow cytometric analysis of (A) mesenteric lymph node and (B) peritoneal 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. Single-cell suspensions were stained with biotinylated anti-IgM and streptavidin-TriColor, and either FITC-conjugated anti-B220 or PE-conjugated anti-CD5. Data are displayed as 5% probability contour plots of total lymphocytes, which were gated by forward and side scatter characteristics. Percentages of total lymphocytes within the indicated gates are given. Data shown are representative of the mice examined (Table I). Download figure Download PowerPoint Figure 3.Expression of E41K Btk induces a dominant maturational defect in peripheral B cells. (A) Surface IgM-IgD profiles of splenic B cells. Percentages of B220+ cells that are IgMlowIgDhigh are given. These cells are mature B cells, whereas IgMhighIgDlow cells are more immature (Hardy et al., 1982). Data are displayed as dot plots of all gated viable B220+ cells from 3×104 total events. (B) Surface B220-HSA profiles of splenic B cells. Percentages of IgM+ cells that are immature (B220lowHSAhigh) or mature (B220highHSAlow) are indicated. Data are displayed as dot plots of all gated viable IgM+ cells from 104 total events (or 3×104 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-conjugated anti-IgD or anti-HSA and analyzed by three-color flow cytometry. Data shown are representative of the mice examined; lymphocytes were gated on the basis of forward and side scatter. Download figure Download PowerPoint Table 1. Frequencies of lymphocyte populations in WT-hBtk and E41K-hBtk transgenic mice Compartment Cell population Non-transgenic WT-hBtk E41K-hBtk aBtk+ Btk− Btk+ Btk− Btk+ Btk− Spleenb Nucleated cells (×10−6) 190 ± 50 84 ± 22 163 ± 34 112 ± 17 194 ± 20 191 ± 38 B220+ cells (%) 38 ± 9 15 ± 5 34 ± 10 22 ± 5 20 ± 6 10 ± 3 CD3+CD4+ (%) 20 ± 3 22 ± 5 26 ± 4 23 ± 8 26 ± 7 27 ± 5 CD3+CD8+ (%) 11 ± 2 12 ± 3 12 ± 3 12 ± 3 15 ± 4 15 ± 4 Lymph node B220+ cells (%) 24 ± 4 8 ± 2 21 ± 5 7 ± 2 5 ± 0.4 2 ± 1 CD3+CD4+ (%) 46 ± 5 53 ± 2 50 ± 4 56 ± 1 60 ± 5 62 ± 3 CD3+CD8+ (%) 22 ± 1 31 ± 2 25 ± 2 27 ± 3 28 ± 4 29 ± 3 Blood B220+ cells (%) 34 ± 9 12 ± 4 19 ± 6 10 ± 4 7 ± 2 4 ± 1 of which B220lowIgDlow (%) 10 ± 4 41 ± 16 10 ± 5 23 ± 7 25 ± 14 51 ± 14 of which B220highIgDhigh (%) 71 ± 3 36 ± 15 66 ± 5 46 ± 5 37 ± 12 17 ± 10 Peritoneum CD5+IgM+ B cells (%) 16 ± 8 0.4 ± 0.3 28 ± 16 14 ± 7 3 ± 2 2 ± 1 CD5−IgM+ B cells (%) 24 ± 10 10 ± 4 24 ± 15 8 ± 1 4 ± 3 2 ± 1 CD5+IgM− T cells (%) 31 ± 3 46 ± 8 29 ± 5 47 ± 11 65 ± 4 62 ± 8 Bone marrow B220+ cells (%) 37 ± 3 36 ± 1 39 ± 10 36 ± 7 37 ± 5 31 ± 2 CD43+IgM− pro-B cellsc (%) 6 ± 0.3 8 ± 1 6 ± 3 8 ± 2 7 ± 3 6 ± 0.4 CD43−IgM− pre-B cells (%) 15 ± 2 16 ± 1 14 ± 5 12 ± 6 13 ± 5 12 ± 3 IgM+IgD− B cells (%) 7 ± 2 8 ± 0.3 9 ± 2 8 ± 2 8 ± 0.3 7 ± 1 IgM+IgD+ B cells (%) 6 ± 2 2 ± 0.5 5 ± 1 2 ± 0.3 1 ± 0.3 0.4 ± 0.2 a Btk mice were Btk+/Y males or Btk+/+ females; Btk− mice (Hendriks et al., 1996) were either Btk−/Y males or Btk−/− females. b 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. c Classification of pro-B and pre-B cells was according to Hardy et al. (1991). Correction of the xid B cell deficiency, although not complete, was obtained by WT-hBtk transgene expression on the Btk− background. In the peripheral blood, spleen, mesenteric lymph node and bone marrow the B cell numbers only reached values similar to those of Btk− mice, but the numbers of peritoneal CD5+ cells were in the normal ranges, and the peripheral B cells exhibited a normal surface IgM/IgD profile (Table I; Figures 2 and 3A). The effect of Btk overexpression on the Btk+ background was limited: the numbers of B220+ cells were slightly reduced in peripheral blood, but were in the normal ranges in the other organs analyzed (Table I). In contrast, when E41K-hBtk mice on the Btk− background were compared with Btk− mice, a further depletion of B cells was observed in all lymphoid tissues analyzed (Table I; Figure 2). Also in the mesenteric lymph nodes from the three other independent E41K-hBtk transgenic lines, the proportion of B cells was 1–5%. Expression of the E41K-hBtk transgene on the Btk+ background resulted in an analogous reduction in the numbers of circulating B cells, although the effect was less severe than on the Btk− background (Table I). The reduction of the proportions of circulating B cells in the E41K-hBtk transgenic mice was accompanied by a relative increase of the percentages of CD4+ and CD8+ T cells (Table I). The six groups of mice did not manifest significant differences in the numbers of Mac-1lowDX5+ NK cells or Mac-1high myeloid cells in the spleen cell suspensions (data not shown). In the bone marrow of E41K-hBtk mice, pro-B, pre-B and immature B cells were present in normal proportions, whereas mature recirculating IgM+IgD+ B cells were virtually absent (Table I). Additional analysis of the three pro-B cell subfractions, as defined by expression of surface markers B220, heat-stable antigen (HSA) and BP-1 (Hardy et al., 1991) in E41K-hBtk mice revealed no detectable alterations from the distribution in normal or WT-hBtk mice (data not shown). In strong contrast to the restored IgM/IgD expression profile found on peripheral B cells from WT-hBtk transgenic Btk− mice, B cells in spleen, mesenteric lymph node and peritoneal cavity from E41K-hBtk transgenic Btk− mice manifested an IgMhighIgDlow 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 just left the bone marrow (B220lowIgDlow cells), rather than recirculating cells migrating between follicles (B220highIgDhigh 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 B220lowHSAhigh have recently left the bone marrow and further differentiate into mature B220highHSAlow cells of the long-lived B cell pool (Allman et al., 1993). While the spleen of non-transgenic Btk+ or Btk− mice contained ∼60% mature B220highHSAlow 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 long-lived B220highHSAlow 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, variable levels of IgG1 and somewhat decreased levels of IgG2a as compared with control Btk+ littermates (Drabek et al., 1997). When the WT-hBtk transgene was expressed on the Btk− background, IgM levels were elevated and all other Ig subclasses were restored to normal levels, similar to the correction previously observed as a result of MHCII-hBtk transgene expression (Drabek et al., 1997). In the WT-hBtk transgenic mice on the Btk+ background, serum Ig subclass levels were in the same ranges. In the E41K-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 out of 20 animals analyzed (Figure 4). The concentrations of IgG2a and Ig2b were generally in the normal ranges, whereas IgA was quite variable but on average reduced compared with the other three groups of mice. No influence of the Btk+ or Btk− background was detected. Figure 4.Effects of transgenic WT-hBtk and E41K-hBtk expression on serum Ig levels. Serum concentrations of the indicated Ig subclasses in non-transgenic mice (Btk+, n = 11; Btk−, n = 16), as well as WT-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. Download figure Download PowerPoint Except for IgM and IgG2b, the serum Ig concentrations of the E41K-hBtk mice decreased significantly with age. In 6-month-old E41K-hBtk mice, the levels of IgG1, IgG2a, IgG3 were only 37 ± 10, 38 ± 20 and 21 ± 15 μg/ml, respectively (n = 3). In age-matched WT-hBtk mice these levels were 970 ± 170, 470 ± 140 and 280 ± 100 μg/ml (n = 4). Defective in vivo responses in E41K-hBtk transgenic mice The absence of a dramatic decrease of serum Ig in E41K-hBtk transgenic mice at the age of 2 months indicated that despite the observed maturation defect of peripheral B cells, significant numbers of B cells were induced to differentiate into Ig-producing plasma cells. However, the possibility remained that these B cells could not mount specific antibody responses. Therefore, we tested the responses of 2-month-old non-transgenic, WT-hBtk and E41K-hBtk transgenic mice on the Btk+ or Btk− backgrounds to TI-II and TD antigenic challenges in vivo. The responsiveness to the TI-II antigen dinitrophenol (DNP)-ficoll was measured seven days after intraperitoneal (i.p.) injection by enzyme-linked immunosorbent assay (ELISA; Figure 5A). Consistent with previous findings in Btk-deficient mice (Wicker and Scher, 1986; Kahn et al., 1995; Drabek et al., 1997), DNP-specific IgM or IgG3 was completely absent in Btk− mice, as the absorbence measured did not differ from the values of unimmunized animals. On the Btk+ background, the TI-II response of WT-hBtk mice was comparable with the response of normal mice, while on the Btk− background, expression of the WT-hBtk transgene could only partially restore the TI-II response. The TI-II antibody response in E41K-hBtk mice was very low but detectable, whether on the Btk+ or the Btk− background. Figure 5.Defective in vivo responses in E41K-hBtk mice. (A) IgM and IgG3 responses to the TI-II antigen DNP-ficoll. (B) Primary IgM" @default.
• W2130963085 created "2016-06-24" @default.
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• W2130963085 date "1998-09-15" @default.
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• W2130963085 title "Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase" @default.
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