Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40

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International Immunology, Vol. 9, No. 3, pp. 395–405 © 1997 Oxford University Press

Impaired B cell maturation in mice lackingBruton’s tyrosine kinase (Btk) and CD40

Wasif N. Khan, Anna Nilsson 1, Emiko Mizoguchi 2, Emanuella Castigli 3,Johan Forsell 1, Atul K. Bhan 2, Raif Geha 3, Paschalis Sideras 1

and Frederick W. Alt

Howard Hughes Medical Institute and the Department of Genetics and Pediatrics, The Children’sHospital, Boston, MA 02115, USA1Unit of Applied Cell and Molecular Biology, Umeå University, 901 87 Umeå, Sweden2Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston,MA 02114, USA3Division of Immunology, The Children’s Hospital, Boston, MA 02115, USA

Keywords: CD40, CD40L (gp39), germinal center, immune response, immature B cells, mature B cells,surface Ig, thymus independent, thymus dependent, X-linked agammaglobulinemia, X-linkedimmunodeficiency

Abstract

Mutations in Bruton’s tyrosine kinase (Btk) gene, in mice, result in reduced numbers andresponses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells asthey do not proliferate upon sIg cross-linking and lack thymus-independent (TI) type II responses.Signals through sIg and CD40 play a critical role in B cell maturation. To investigate theconsequences of the lack of both Btk and CD40 on B cell development and function, mice weregenerated that were homozygous for targeted mutations in the Btk and the CD40 genes(Btk MCD40M). The CD40 mutation (CD40 M) had a synergistic effect on the Btk M defects. InBtk MCD40M mice the number ofB cells was reduced 3- to 4-fold compared to Btk M mice and mature B cells (IgM low /IgDhigh ) werevirtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I,TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type Btk M andCD40M mice produced germinal centers in response to TI-I antigen, the Btk MCD40M mice did not.Maturational and functional B cell defects in Btk MCD40M mice may result from a combination ofintrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects.These data suggest that signals through Btk and CD40 are necessary for the production andmaintenance of the mature B cell.

Introduction

Development of B lymphocytes from stem cells proceeds The function of the sIg receptor is defective in B cells withdefective Bruton’s tyrosine kinase (Btk) (10,11). Btk along withthrough an antigen-independent phase in the bone marrow

(BM) and an antigen-dependent phase in the secondary Tec, Itk, Txk and BMX comprise a distinct subfamily of src-related cytoplasmic tyrosine kinases (reviewed in 12). Btklymphoid organs. During the first phase ordered rearrange-

ment of Ig heavy and light chain genes (1) leads to the contains the src homology (SH) domains, SH1 (kinase), SH2and SH3, and a unique N-terminal region which is comprisedgeneration of sIgM1 B cells. After their exit from the BM these

immature B cells go through antigen-driven expansion and of a pleckstrin homology domain (13–16) and a Tec homologydomain (17). Btk is expressed in B lymphocytes as well as inselection in the peripheral lymphoid organs, and establish

the peripheral B cell repertoire (2,3). The B lymphocyte bears myeloid and erythroid cells. In B cells, Btk is expressed inmost stages of B cell development, except the terminallytwo major surface receptors, sIg and CD40, which are each

capable of inducing the transition from resting state to cell differentiated plasma cell stage (18–20). X-linked immuno-deficiency in mice (xid, CBA/N) (21–23) is caused by a singlecycle progression in the absence of exogenous lymphokines

as well as modulating B cell maturation (4–9). amino acid substitution of a conserved residue Arg28Cys in

Correspondence to: F. W. Alt

Transmitting editor: H. Ploegh Received 11 October 1996, accepted 21 November 1996

396 B cell deficiency in Btk and CD40 double-deficient mice

the pleckstrin homology domain of the Btk gene (24,25). pared and stained by standard methods (58) using specificComplete elimination of Btk in mice (BtkM mice) also results antibodies to lymphocyte cell surface markers. Cells in thein the xid phenotype (26,27). In humans, mutations in Btk lymphocyte gate by light scatter were analyzed on FACScancause X-linked agammaglobulinaemia (XLA), a more severe (Becton Dickinson, Mountain View, CA) using Lysys software.B cell maturational defect than xid (12,28–30). T cell depletion The antibodies purchased from PharMingen (San Diego, CA.)in CBA/N mice results in severe B cell and serum Ig deficiency were: fluorescein-conjugated RA 3-6B2 (B220/CD45R), AMSthat resembles closely human XLA (31–36). 9.1 (IgD), phycoerythrin (PE)-conjugated DS-1 (IgM), PE-

The xid/BtkM defect (26,37) does not affect B lymphopoiesis conjugated anti-HSA and PE-conjugated anti-Thy-1.2. CD23as pro-B, pre-B and newly generated B cells are present in could not be used for B cell analysis in the spleen ofnormal numbers in the BM (32,38). The overall numbers of BtkMCD40M mice because it was expressed at a lower levelperipheral B cells are reduced to ~50% of normal and show in the CD40M splenic B cells.an immature phenotype (21,37–40). CD51 B-1 cells are not

Immunizations and ELISA assaysdetectable (41). In the xid/BtkM mice, B cells have functionaldefects as they produce reduced levels of serum IgM and To measure the TD immune responses, mice were immunizedIgG3 (42), and are unable to respond to thymus-independent with 100 µg TNP-keyhole limpet hemacyanin precipitated withtype II (TI-II) antigens (37). In vitro, xid/BtkM B cells do not alum and injected i.p. Mice were bled prior to and 9 daysproliferate when triggered through their surface IgM receptor post-immunization to measure the primary antibody responseand show hypoproliferation to bactarial lipopolysaccharide in the serum, and were subsequently sacrificed for immuno-(LPS) stimulation (10,11,26,43.44). Additionally, they have histochemical analysis. To determine TI responses, mice werebeen found to be deficient in their responses to cytokine bled for pre-immune serum and immunized with 10 µg TNP-receptors IL-5, IL-6 and IL-10, and B cell surface molecules Ficoll (kind gift of Dr John Inman, NIH, Bethesda, MD) or 50CD40 and CD38 (45–48). µg of TNP-LPS in PBS i.p. Post-immunization the mice were

CD40 plays an important role in B cell proliferation, isotype bled at day 8 for ELISA assays and sacrificed for immunohisto-switching and differentiation into memory cells (49). CD40 is chemical analysis. For the determination of TNP-specificthe receptor for CD40L (gp39) on B cells and is expressed antibodies (Southern Biotechnology Associates, Birmingham,throughout most stages of B cell development (50). It is also AL) in the serum of pre-immune and immunized mice isotype-expressed on T cells, macrophages, dendritic cells and specific ELISA was performed. The serum was analyzed byeosinophils (49). The CD40L is expressed on the surface of 3-fold serial dilution’s using TNP-BSA (100 µg/ml) coatedactivated CD41 T cells, some CD81 T cells, mast cells and plates and revealed by alkaline phosphatase-labeledbasophils (51). Mutations in CD40L results in hyper-IgM secondary antibodies. Results were plotted as the mean 6 SD.syndrome (52). Inactivation of CD40 or CD40L in mice results For determinations of total IgM, IgG3, IgG1, IgG2a, IgG2bin a phenotype that is similar to the hyper-IgM syndrome. CD40 and IgA in the serum of unimmunized mice, Immulon-1 platesor CD40L mutant mice have defective immune responses (Dynatech, Chantilly, VA) were coated with 5 µg/ml of isotype-and do not form germinal centers in response to thymus- specific rabbit or goat anti-mouse antibodies. Diluted serumdependent (TD) antigens but are apparently normal with samples were incubated in the plates and revealed by alkalineregard to TI immune responses (53–56). phosphatase-labeled secondary antibodies. Purified mouse

Surface Ig and CD40 co-stimulation plays a critical role in Ig of corresponding isotypes were used as standards.antigen-dependent B cell maturation (49). The role of theseinteractions in the establishment of the long lived peripheral Immunohistochemical analysisB cell pool is not known. We asked the question whether lack Tissue samples from spleen, mesenteric lymph nodes andof both Btk and CD40 in vivo will affect B cell maturation small intestine were frozen in OCT compound (Ames, Elkhart,and function, and investigated the consequences of the IN), on dry ice, and stored at –80°C. Frozen tissue sectionsconcomitant inactivation of Btk and CD40 (BtkMCD40M) on B (4 mm thick) were air-dried for 2 h, fixed in cold acetone forcell development, function and immune responses. 7 min, air-dried again and stained by the avidin–biotin complex

method as described previously (59). The sections wereMethods incubated with purified or biotinylated antibodies in pre-

determined optimal dilution. After 1 h incubation at roomGeneration of Btk and CD40 mutant micetemperature, specimens were incubated for 30 min with 0.3%Btk mutant mice were generated as described (26) andhydrogen peroxide in PBS (pH 7.4) to block endogenousreferred here as BtkM. B cells from these mice lack anyperoxidase activity. Endogenous biotin was blocked bydetectable Btk protein. CD40 mutant mice were generatedsequential incubations with avidin (Vector, Burlingame, CA)as described (53,57). Btk homozygous mutant females wereand biotin (Sigma, St Louis, MO). Specimens were thenbred with CD40 homozygous mutant males all of which wereincubated with biotinylated secondary antibodies followed byin 129/SV and C57BL/6 mixed background (26,53,57) toavidin–biotinylated peroxidase complex (Dako, Santa Bar-obtain wild-type, CD40M, BtkM and BtkMCD40M littermates.bara, CA). After washing, the specimens were developed inThus the mice analyzed have a mixed genetic backgrounda solution of 3-amino-9-ethylcarbazole (Aldrich, Milwaukee,of 129/Sv3C57BL/6.WI) and the reaction was stopped by dipping in distilled

Flow cytometry analysis water for 5 min. Specimens were then post-fixed with 2%paraformaldehyde, counter-stained with hematoxylin andSingle-cell suspensions from BM, spleen, thymus and lymph

nodes from normal CD40M, BtkM and BtkMCD40M were pre- mounted with Glycergel (Dako). Each incubation was followed

B cell deficiency in Btk and CD40 double-deficient mice 397

by three washes in PBS. The antibodies used for staining of the Peripheral B cells are severely reduced in BtkMCD40M micecompared to BtkM and these B cells bear an immaturehistological samples were: biotin-labeled peanut agglutinin

(Vector), biotin-labeled IgDa (AMS9.1; PharMingen), biotin- phenotypelabeled IgM (R6-60.2; PharMingen) and biotin-labeled anti- Flow cytometric analysis showed a severe deficiency of BCD45R/B220 (RA3-6B2; PharMingen), and were used by the cells in the spleen and lymph nodes of BtkMCD40M micedirect method and developed by avidin peroxidase. compared to the number of B cells in BtkM mice (Figs 2 and

3) (26). The total numbers of B2201IgM1 B cells in the spleenIn vitro B cell cultures and LPS stimulation of BtkMCD40M mice were 3- to 4-fold reduced relative to theSplenic B cells from wild-type, CD40M, BtkM and BtkMCD40M number of B cells in BtkM mice and 7- to 8-fold reducedmice were purified by positive selection using the Mini- relative to the number of B cells from normal or CD40M miceMACS system and anti-B220 microbead staining following (Fig. 2, BtkMCD40M 3.5 6 1.83106; BtkM 11.6 6 5.33106;the instructions of the manufacturers (Miltenyi Biotech, Bonn, CD40M 22 6 5.83106 and wild-type 25 6 33106). TheGermany). The purity of B2201 cells was verified by immuno- reduction in B cell numbers was mainly due to the reductionfluorescence staining using FACScalibur (Becton Dickinson) in the numbers of mature IgM1IgDhigh B cells that expressand was ~95–98% for wild-type, CD40M and BtkM cells, and lower levels of HSA (Fig. 3B and C) as the number of immature85–90% for BtkMCD40M. Purified B cells were cultured at IgMhighIgD– cells was only 2- to 3-fold lower than in BtkM,106/ml in RPMI 1640 supplemented with 15% FCS, 2 mM CD40M and wild-type mice, and this population represents aL-glutamine, 10 µM sodium pyruvate, 50 µM 2-mercapto- minor fraction of the normal spleen (BtkMCD40M 1 6 0.563106;ethanol, 100 U/ml penicillin and 100 mg/ml streptomycin. BtkM 3.1 6 1.273106; CD40M 2.8 6 13106 and wild-typeDNA synthesis was measured by pulsing the cultures with 2.13 6 1.23106). The B cell deficiency in BtkMCD40M mice1.0 µCi [3H]thymidine/well for 6 h. [3H]thymidine incorporation was more severe in the lymph node as the percentage ofwas determined with a 1218 Rackbeta β scintillator (LKB, cells stained with B220 and IgM was 5-fold less than in BtkM

Stockholm, Sweden). alone and ~10-fold reduced compared to wild-type or CD40M

littermates (Fig. 3D and E, BtkMCD40M 5%; BtkM 25%; CD40M

53% and wild-type 44% B cells). Thus the B lymphocytes inResults doubly mutant mice display the phenotype of immature B

cells and there is a severe deficiency in mature B cellsGeneration of BtkMCD40M mice suggesting a block in the transition from immature to mature

B cells.The BtkM mice were generated from ES cells that replacedexons 12 and 13 of the Btk gene with the neomycin resistance

BtkMCD40M mice have reduced serum levels of IgM, IgG2bgene by homologous recombination (26). The resulting BtkM

and IgA compared to either BtkM or CD40M micemice did not have any detectable Btk protein. ES cells withtargeted deletion of exons coding for transmembrane and Serum levels of all Ig isotypes were determined by ELISAcytoplasmic regions of CD40 were used to generate CD40M in unimmunized BtkM, CD40M and BtkMCD40M mice, andmice (53,57). B cells from CD40M mice did not express any compared to those of normal littermates. Consistent withdetectable CD40 protein on their surface. The BtkMCD40M previous reports, CD40M mice had decreased levels of alldouble mutant mice were generated by breeding BtkM and isotypes except IgM and IgG3 [Fig. 4 (53,54)] and BtkM miceCD40M mice. Littermates carrying the genotypes wild-type, had reduced levels of IgM and IgG3 [Fig. 4 (26,37,42)].BtkM, CD40M and BtkMCD40M were analyzed and compared Levels of all Ig isotypes in double mutant mice were decreasedin the experiments described below. All the mice have a compared to wild-type. Notably, the levels of serum IgM,mixed 129/Sv3C57BL/6 genetic background. IgG2b and IgA were drastically reduced in the BtkMCD40M

mice compared to either mutant alone. The levels of IgG3 andNormal B cell development in the BM of BtkMCD40M mice IgG2a were comparable to BtkM and CD40M mice respectively.

IgM was ~7-fold reduced compared to BtkM mice; IgG2b wasWe investigated the effects of the concomitant inactivation of10-fold reduced relative to CD40M mice and IgA was 40-foldBtk and CD40 genes on B lineage cells in the BM (60). Noreduced relative to CD40M mice. Detection of Ig in the serumsignificant difference in ratios of pro-B (B2201CD431), pre-Bof BtkMCD40M mice indicates that some of the double-(B2201CD43–) and newly generated immature B celldeficient B cells can differentiate into antibody-secreting cells.(B2201IgM1HSAhigh) was discernible in mice of all four geno-A specific reduction in the levels of IgG2b and IgA isotypestypes (wild-type, BtkM, CD40M and BtkMCD40M) analyzedcompared to IgG2a and IgG1 is consistent with either a(Fig. 1A and B, and data not shown). The small reduction indefect in the production of these isotypes per cell or athe B220lowIgM1 immature B cells seen in the BtkMCD40M

reduction in the number of cells producing these isotypes.compared to BtkM mice in this experiment (Fig. 1A, 14 to9.3%) was variable from mouse to mouse and therefore may

Defective humoral immune responses to TD, TI-I and TI-IInot be significant. Mature recirculating B cells which expressantigens in BtkMCD40M micelower levels of HSA and IgM and higher levels of IgD

B220highIgM1, HSAlowIgM1 and B2201IgD1 (60–62) were Both BtkM and CD40M mice mount an antibody response tothe TI-I antigen, TNP-LPS (63,64). We investigated TNP-reduced in BtkM mice as previously reported. Notably, the

size of this mature B cell population was further reduced in specific antibody titers in mice of all four genotypes 8 daysafter immunization with TNP-Ficoll and TNP-LPS. ConsistentBtkMCD40M compared to BtkM mice (Fig. 1A–C).


Fig. 1. Flow cytometric analysis of BM B lineage cells. B cells from 8-week-old littermates of the indicated genotype were stained to revealimmature B and mature B lymphocytes with CyC–anti-B220/CD45R and PE–anti-IgM (A) or FITC–anti-IgM and PE–anti-HSA (B). IgD expressionis shown on all anti-B2201 cells from panel A (C). Cells in the lymphocyte gate were analyzed by light scatter. The results are presented astwo-dimensional dot-plots in which each dot indicates an individual cell or as histogram. The percentage of cells in each region is indicated.These mice are representative of the mice examined (n 5 10).

results suggest that the double mutant mice are defective intheir ability to respond to this TI-I antigen.

Primary immune responses of BtkMCD40M mice to the TDantigen TNP-KLH were evaluated and compared with BtkM,CD40M and normal litter-mates. Wild-type, BtkM and CD40M

responded as expected (Fig. 5B). Notably, in BtkMCD40M

mice, the IgM response was also defective in addition to thelack of an IgG response, compared to CD40M mice (Fig. 5B).

Germinal centers are formed in CD40M but not in BtkMCD40M

mice in response to TI-I antigenThe germinal center reaction in response to TI antigens isless well studied than with TD antigens. However, germinalcenters are formed in response to both TI-I and TI-II antigens(65–67). We investigated the germinal center reaction in micewith all the four genotypes after immunization with the TI-Iantigen TNP-LPS. Immunohistochemical analysis was per-formed on spleens and mesenteric lymph nodes of unimmun-Fig. 2. The numbers of the B2201IgM1 B cells in the spleens of 4-ized and immunized mice using anti-B220, anti-IgD andto 10-week-old littermates. Symbols correspond to individual mice

and are shown under the indicated genotype. The phenotype of B peanut agglutinin. There was no significant difference in thecell populations was assessed by flow cytometry as described in the size of lymphoid follicles in immunized wild-type, CD40M andlegend to Fig. 3. BtkM mice. In contrast, lymphoid follicles were considerably

smaller in BtkMCD40M mice as evidenced by B220 staining(Fig. 6 and IgM staining data not shown), also the B cellsstained weakly for IgD; staining was much less intense thanwith previous studies (53,54), wild-type, BtkM and CD40M

mice responded to TI-I antigen and produced TNP-specific that observed in CD40M or BtkM mice, reflecting the reductionin mature B cells. In wild-type mice prominent germinalIgM and IgG antibodies (Fig. 5A). TNP-specific IgM antibody

titers in BtkMCD40M mice were lower than in either knockout centers were recognized (Fig. 6 and data not shown). How-ever, germinal centers were infrequent and relatively smalleralone and IgG titers were barely detectable (Fig. 5A). These


Fig. 3. Flow cytometric analysis of spleen and lymph node cells. Splenic B cells from 8-week-old littermates of the indicated genotype werestained to reveal immature B and mature B lymphocytes with CyC–anti-B220/CD45R, PE–anti-IgM and FITC–anti-IgD (A). IgM and IgDexpression is shown on B2201 gated cells from panel A (B). To investigate the levels of HSA, cells were stained with CyC–anti-B220/CD45R,FITC–anti-IgM and PE–anti-HSA. HSA expression is shown by a histogram on B2201IgM1 gated cells (C). Lymph node B cells stained withCyC–anti-B220/CD45R, PE–anti-IgM (D) and B and T cell populations in the lymph node are detected by anti-B220 and anti-Thy-1 staining(E). Cells in the lymphocyte gate were analyzed by light scatter. The results are presented as two-dimensional dot-plots in which each dotindicates an individual cell or as a histogram. The percentage of cells in each quadrant/region is indicated. Note that CD40M B cells alsoexpressed higher levels of HSA compared to wild-type B cells; however, the significance of this finding is unclear at this time. Variability wasobserved between mice. These mice are representative of the mice examined (n 5 10).

in BtkM and CD40M mice and were not apparent in BtkMCD40M previous reports, BtkM B cells showed a reduced level ofproliferation compared to wild-type (Fig. 7). The proliferativemice immunized with TNP-LPS (Fig. 6).response of CD40M B cells was similar to wild-type (data not

In vitro responses of purified BtkM and BtkMCD40M B cells shown). We investigated the LPS response of purified B cellsare reduced compared to wild-type and CD40M B cells from BtkMCD40M as compared to that of BtkM B cells. The

proliferation level of BtkMCD40M B cells was found to beWe and others have previously shown that Xid or BtkM Bcells show reduced proliferation in response to LPS in vitro, variable between mice such that in half of the experiments

BtkMCD40M B cells proliferated at a lower level than BtkM Bparticularly at lower concentrations (26,43). Consistent with


Fig. 4. Severely reduced serum Ig levels in BtkMCD40M mice. Serum Ig levels of 6- to 10-week-old mice were determined by ELISA for theindicated Ig isotypes.

cells (Fig. 7A, two representative experiments are shown). revealed in BtkMCD40M mice. Immature sIgM1 B cells areshort lived and are recruited into the long-lived peripheral BHowever, both BtkM and BtkMCD40M B cells consistently

responded at a reduced level than wild-type or CD40M B cells. cell pool, possibly by positive selection by internal and/orexternal antigens or unknown ligand(s) (2,69). The reductionof mature B cells in BtkMCD40M mice suggests that CD40

Discussion and Btk signaling may synergize in the development of thelong-lived mature B cell repertoire by regulating signals toSevere B cell deficiency in mice carrying concomitant Btkthe threshold level required for positive selection.and CD40 mutations

Our studies demonstrated that the CD40 mutation has aPotential mechanisms by which Btk and CD40 may synergizesynergistic effect on some aspects of the Btk phenotype asto produce mature B cell deficiencyBtkMCD40M mice had a 4- to 5-fold reduction in the numberThe B lymphocyte bears two surface receptors, sIg andof B cells in the spleen and an even greater reduction in theCD40, which are each capable of inducing the transition fromnumber of B cells in the lymph nodes compared to BtkM

resting state to cell-cycle progression in the absence ofmice. Mature IgMlowIgDhigh B cells were virtually absent whileexogenous lymphokines (reviewed by in 9,70). However, thethe numbers of immature IgMhigh/IgD– B cells were onlytwo signals seem to modulate B cell fate differentially inmodestly reduced compared to BtkM mice. Serum Ig levelsimportant ways so that sIg cross-linking results in programmedof all isotypes were reduced compared to either mutant alone,cell death, while CD40 co-stimulation with sIg cross-linkingparticularly of IgM, IgG2b and IgA. The in vivo response to asuppresses programmed cell death. This effect is seen bothTI-I antigen (TNP-LPS) was drastically reduced compared toin phenotypically mature and immature B cells, and is mostthe BtkM mice and they failed to produce germinal centers.likely regulated by anti-apoptotic proteins of the Bcl-2 family;In vitro LPS-induced proliferation of double mutant B cellsBcLx in immature and Bcl-2 in mature B cells (4–9). Severewas similarly reduced as BtkM B cells (Fig. 7), and there wasdeficiency of mature B cells in BtkMCD40M mice suggestsno apparent difference in the sIg induced protein tyrosinethat transition from immature to mature B cells, for the mostphosphorylation in BtkMCD40M, BtkM and the wild-type controlpart, relies on the outcome of the signals generated from sIg(data not shown). In some aspects the phenotype ofand CD40 receptors, and that these signals synergize toBtkMCD40M mice is similar to thymectomized CBA/N miceproduce the co-stimulation necessary for B cell entry into the(36) suggesting that CD40–CD40L interaction might be onemature B cell compartment. That Btk and CD40 mutationsof the factors involved in providing T cell help to xid B cells.synergize in B cells is further supported by the findings thatThese findings have revealed the potential importance ofthe signaling pathways utilized by sIg and CD40 differ initiallyCD40 signaling during the transition from immature to maturewith the former being protein kinase C (PKC)-dependent andB cell stage. This function of CD40 is probably masked by

the compensatory activity of Btk in CD40M mice and is the latter PKC-independent, but they potentially converge at


Fig. 5. Defective serum antibody responses to TI-I antigen TNP-LPS (A) and TD antigen TNP-KLH (B) in BtkMCD40M mice. (A) Serial (3-fold)dilutions of serum were analyzed for TNP-specific IgM and IgG by ELISA at day 8 after immunization with TNP-LPS. Results are expressed asOD405 versus dilutions of anti-IgM- and anti-IgG-specific ELISA using TNP-BSA as capture reagent. Two CD40M mice and three each of wild-type, BtkM and BtkMCD40M mice were analyzed. Data are mean 6 SD (B) Serial dilutions of serum were analyzed for TNP-specific IgM byELISA at day 8 after immunization with TNP-KLH. Results are expressed as OD405 of anti-IgM- and anti-IgG-specific ELISA using TNP-BSA ascapture reagent. Two CD40M and three each of wild-type, BtkM and BtkMCD40M mice were analyzed. Data are mean 6 SD. TNP-specificantibody titers for IgG3, IgG1, IgG2a, IgG2b and IgA were also determined, and were undetectable in CD40M as well as BtkMCD40M mice(not shown). Lower primary IgM response to the TD antigen TNP-KLH in CD40M and BtkM mice is in agreement with earlier reports. BtkM andBtkMCD40M mice did not produce antibodies in response to the TI-II antigen TNP-Ficoll (data not shown).

the level of transcription factor regulation. For example, both double mutant mice. However, the deficiency of IgM, IgG2band IgA in BtkMCD40M mice was much more pronouncedCD40 and sIg receptor-mediated signaling lead to the induc-

tion of the same set of transcription factors; NF-κB, AP-1 and than that of IgG1 and IgG2a compared to either mutant alone.This selective deficiency may result from the reduction inNF-AT (69,70). The kinetics of induction of NF-κB and AP-1

is different in some aspects between sIg and CD40 signaling, numbers of mature B cells in BtkMCD40M mice. Alternatively,the selective Ig deficiency may result from defective signalingand this difference may determine the biological outcome

(70), e.g. positive or negative selection of B cells. through CD40 together with receptors utilizing Btk in theirsignaling pathways. Signaling pathways through sIg, IL-5 andAn alternative but not mutually exclusive possibility is that

the B cell phenotype in BtkMCD40M mice is due to defective IL-10 receptors are defective in Btk mutant B cells (45,46),and have been implicated in switching to IgG2b and IgAcytokine(s) secretion by T cells and/or an altered micro-

environment of the primary follicles in the spleen and lymph (73,74). In addition both CD40 and Btk are required for theproliferation necessary for switching to take place.nodes. This is a consideration as B–T cell interactions are

essential for the development of follicular dendritic cells (72– TI-I antigens such as TNP-LPS, provide two independentsignals, polyclonal B cell activation and the hapten-specific74) and may be defective in the BtkMCD40M mice. Thus,

defective primary follicles, in BtkMCD40M mice, may comprom- (TNP) stimulation through sIg. The defective antibody produc-tion and germinal center formation in the BtkMCD40M mice inise B cell maturation or survival of mature B cells as they may

not provide the necessary trophic factors (3). response to TNP-LPS compared to BtkM mice (Figs 5 and 6)may suggest that the double mutant B cells have not matured

Potential reasons for the severe Ig deficiency and impaired to achieve the competence necessary to respond to TI-Iimmune responses in BtkMCD40M mice antigens. The presence of germinal centers in BtkM and

CD40M mice suggests that this process is not absolutelyAs anticipated from the known defects of Btk and CD40mutations, serum levels of all Ig isotypes were decreased in CD40 dependent. Thus CD40-dependent interactions are


Fig. 6. Immunoperoxidase staining of spleens from mice, at day 7 after immunization with TNP-LPS, for B220 (objective 310), IgD (objective310) and peanut agglutinin (objective 320). There is no discernible difference in the B220 and IgD staining pattern between the wild-type,CD40M and BtkM. The BtkMCD40M double mutant mice had significantly smaller follicles as revealed by staining for B220 and they stainedvery weakly for IgD. Germinal centers were smaller in CD40M and BtkM compared to wild-type as revealed by staining for peanut agglutininand were absent in BtkMCD40M mice. Germinal centers were not recognized in unimmunized mice (data not shown).

Fig. 7. LPS response of purified B cells from wild-type, BtkM, CD40M and BtkMCD40M mice at the indicated concentrations. Triplicate cultureswere analyzed and the mean 6 SD of two independent experiments is shown.


8 Wang, Z., Karras, J. G., Howard, R. G. and Rothstein, T. L. 1995.not responsible for the modification of TI-I responses by TInduction of bcl-x by CD40 engagement rescues sIg-inducedcells (67,75).apoptosis in murine B cells. J. Immunol. 155:3722.

The B cell functional defects in BtkMCD40M mice may be 9 Rothstein, T. L. 1996. Signals and susceptibility to programmedthe direct result of signaling defects in BtkM and CD40M

cell death in B cells. Curr. Opin. Immunol. 8:362.10 Mond, J. J., Schaefer, M., Smith, J. and Finkleman, F. D. 1983.signaling pathways combined or the double mutant B cells

Lyb-5- B cells of CBA/N mice can be induced to synthesizehave not matured to achieve the competence necessary toDNA by culture with insolubilized but not soluble anti-Ig. J.respond to various signals compared to Btk mutation alone.Immunol. 131:2107.

Importantly, the generation of BtkMCD40M mice and analysis 11 Rigley, K. P., Harnett, M. M., Phillips, R. J. and Klaus, G. G. 1989.of the B cell compartment has revealed a requirement for T– Analysis of signaling via surface immunoglobulin receptors on B

cells from CBA/N mice. Eur. J. Immunol. 19:2081.B cell interaction in the transition from immature to mature B12 Sideras, P. and Smith, C. I. E. 1995. Molecular and cellularcells and their maintenance.

aspects of X-linked agammaglobulinemia. Adv. Immunol. 59:135.13 Haslam, R. J., Kolde, H. B. and Hemmings, B. A. 1993. Pleckstrin

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modular domain present in diverse signaling proteins. Cell 73:629.We thank Drs Henry Wortis and Christopher C. Goodnow for helpful 15 Musacchio, A., Gibson, T., Rice, P., Thompson, J. and Saraste,discussions, Dr John Inmann for TNP-Ficoll, and Dr Katherine J. Seidl M. 1993. the PH domain: a common piece in the structuraland Dr Abul Abbas for critical reading of the manuscript. This work patchwork of signalling proteins. Trends Biol. Sci. 18:343.was supported by the Howard Hughes Medical Institute and by 19 Mustelin, T. A. B. P. 1993. Regulation of src family tyrosine kinasesNational Institute of Health grants AI20047, PO1AI35714, DK 47677 in lymphocytes. Trends Biol. Sci. 18:215.and DK43551, the Swedish Medical Research Council, the Swedish 17 Vihinen, M., Nilsson, L. and Smith, C. I. E. 1994. Tec homologyCancer Society (Cancerfonden) grants B94-16X-10845-01A and domain (TH) adjacent to PH domain. FEBS Lett. 350:263.1819-B94-06XAC, The Biotechnology Fund of Umeå University and 18 Smith, C. I. E., Baskin, B., Humire-Greiff, P., Zhou, J., Olsson,Baxter Healthcare, Alpha Therapeutics and Quantum Corp. W. N. K. P. G., Maniar, H. S., Kjellen, P., Lambris, J. D., Christiansen, B.,is an Associate of Howard Hughes Medical Institute. Hammarstrom, L., Bently, D., Vetrie, D., Islam, K. B., Vorechovsky,

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Impaired B cell maturation in mice lacking Bruton's tyrosine kinase (Btk) and CD40

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