Essential, Nonredundant Role for the Phosphoinositide 3-Kinase p110{delta} in Signaling by the B-Cell Receptor Complex

Many receptor and nonreceptor tyrosine kinases activate phosphoinositide3-kinases (PI3Ks). To assess the role of the ${delta}$ isoform of thep110 catalytic subunit of PI3Ks, we derived enzyme-deficientmice. The mice are viable but have decreased numbers of matureB cells, a block in pro-B-cell differentiation, and a B1 B-celldeficiency. Both immunoglobulin M receptor-induced Ca²⁺ fluxand proliferation in response to B-cell mitogens are attenuated.Immunoglobulin levels are decreased substantially. The abilityto respond to T-cell-independent antigens is markedly reduced,and the ability to respond to T-cell-dependent antigens is completelyeliminated. Germinal center formation in the spleen in responseto antigen stimulation is disrupted. These results define anonredundant signaling pathway(s) utilizing the ${delta}$ isoform ofp110 PI3K for the development and function of B cells.

INTRODUCTION

Top

Introduction

Lymphocyte development and function are regulated through thecoordinated action of receptors of the cytokine receptor superfamilyand the B-cell antigen-specific receptor (BCR) or T-cell antigen-specificreceptors (TCR). Engagement of either receptor complex initiatestyrosine phosphorylation of a variety of intracellular substrates,including receptor chains, resulting in the initiation of cellularresponses. Members of the cytokine receptor superfamily utilizeJAK family cytoplasmic kinases (14), while the BCR and TCR complexesutilize members of the Src, Tec, and Zap70/Syk families of tyrosinekinases. In BCR signal transduction, the Tec family kinase Btkplays a critical role as evidenced by the loss of a proliferativeresponse to BCR engagement in Btk-deficient B cells (15, 16).Among the substrates typically phosphorylated and recruitedto hematopoietic receptor complexes are the regulatory subunitsfor phosphoinositide 3-kinases (PI3Ks) (9, 10, 30). In mammalsthere are three genes that encode adapter proteins for PI3Kcatalytic subunits, including p85 ${alpha}$ , p85ß, and p55 ${gamma}$ .The adapter proteins facilitate association of the catalyticsubunits with the receptor complex and are proposed to regulateenzyme activity. The disruption of the p85 ${alpha}$ gene, in a mannerthat deletes the p85 isoform as well as two splice variantsof p55 and p50, results in defective BCR signaling comparableto that seen with Btk deficiency (11, 26). This strongly suggeststhat PI3K activity is critical in BCR signal transduction.

Three of the known PI3Ks, i.e., PI3K ${alpha}$ , PI3Kß, and PI3K ${delta}$ ,are regulated through their interaction with regulatory subunits(30). The fourth PI3K, PI3K ${gamma}$ , functions in the context of heterotrimericG-protein-coupled receptors and is essential for leukocyte function(12). The critical, nonredundant role that PI3K ${alpha}$ plays in cellularresponses has been demonstrated through the derivation of micelacking the gene. This deficiency results in an embryonic lethalityat E9.5 to E10.5 due to a severe proliferative defect in manytissues (2). Similarly, deletion of the PI3Kß genealone results in a very early embryonic lethality (1, 2). Incontrast to PI3K ${alpha}$ and PI3Kß, PI3K ${delta}$ is expressed primarilyin hematopoietic cells (32). To identify proteins that are recruitedto cytokine receptor complexes, we have used affinity columnscontaining phosphopeptides corresponding to the major sitesof tyrosine phosphorylation of JAK2. Two complexes which boundto a phosphopeptide affinity column derived from the amino acidsequence surrounding Jak2 Y⁹⁶⁶ were p85 ${alpha}$ /p110 ${delta}$ and p85ß/p110 ${delta}$ .In order to determine the potential role of PI3K ${delta}$ in signaling,we derived a strain of mice in which the gene was disruptedin a manner to yield a protein null mutant. As demonstratedhere, a deficiency in PI3K ${delta}$ results in a very specific loss offunction of the BCR complex, while signaling through the cytokinereceptor complexes is unaffected.

MATERIALS AND METHODS

Top

Materials and Methods

Construction of p110 ${delta}$ targeting vector. Approximately 9 kb of the p110d gene was isolated from a 129/Jmouse genomic library in pBeloBacII (Incyte Genomics, St. Louis,Mo.) and subcloned into pBluescript. A neomycin resistance cassettewas inserted into the StuI fragment encompassing exons 2 through6. A diphtheria toxin A cassette mediating negative selectionwas inserted in the 5' end of the p110 ${delta}$ -neo construct.

Culture of ES cells and generation of p110 ${delta}$ -deficient mice. E14 (129/Ola mouse strain) ES cells were cultured in Dulbecco'smodified Eagle medium containing 15% fetal calf serum, 1 mMsodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential aminoacids, 55 mM ß-mercaptoethanol, 10 ng of gentamicinper ml, and 1,000 U of leukemia inhibitory factor/ml (all fromGIBCO-BRL, Rockville, Md.). Irradiated SNLH9 fibroblasts wereused as feeders. Twenty-five micrograms of SalI-linearized p110 ${delta}$ targeting construct was electroporated into the ES cells byusing a gene pulser (Bio-Rad, Hercules, Calif.) set at 0.23kV and 500 µF. Selection at 24 h after electroporationwas in 350 µg of G418 (GIBCO-BRL) per ml. Drug-resistantES clones were picked and expanded 7 to 10 days after electroporation.Four correctly targeted and karyotypically normal ES cloneswere injected into C57BL/6 blastocysts, of which two gave germline transmission. The mice were maintained under specific-pathogen-freeconditions according to institutional guidelines. Genotypingof mice was performed by Southern blot analysis or by PCR.

Flow cytometry. Single-cell suspensions were depleted of red blood cells byusing a hypotonic lysis buffer (pH 7.3) containing 150 mM NH₄Cl,1 mM KHCO₃, and 0.1 mM Na₂EDTA as described previously (19).Monoclonal antibodies (MAbs) conjugated to either fluoresceinisothiocyanate (FITC), phycoerythrin (PE), or CyChrome and specificto murine B220/CD45R, immunoglobulin M (IgM), IgD, CD5, CD43,Thy1.2, interleukin-2 (IL-2) receptor (CD25), CD44, CD4, CD8,Mac-1 (CD11b), and Gr-1 (all from PharMingen) were used to label10⁶ cells for 30 min on ice. Fc receptors were blocked withanti-Fc ${gamma}$ III/II R antibody (Ab) (PharMingen). Cells were washedtwice and analyzed on a FACSCalibur with CellQuest software(Becton Dickinson).

Immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis. Immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, and Western blot analysis were performed asdescribed previously (36). The polyclonal anti-p110 ${delta}$ antiserumwas generated by immunizing rabbits with a keyhole limpet hemocyanin(KLH)-conjugated peptide derived from residues 1028 to 1043of murine p110 ${delta}$ . Anti-phospholipase C ${gamma}$ 2 (anti-PLC ${gamma}$ 2) was from SantaCruz Biotechnologies, anti-Bruton's tyrosine kinase (BTK) wasfrom PharMingen, and anti-phosphotyrosine 4G10 was from UpstateBiotechnology.

Immunizations. To measure the thymus-dependent immune response, wild-type andmutant mice (five per group) were immunized intraperitoneallywith 100 µg of trinitrophenyl (TNP)-KLH (Biosearch Technologies)in a 1:1 emulsion with complete Freund's adjuvant (Sigma), followedby a second immunization 15 days later with 100 µg ofTNP-KLH in a 1:1 emulsion with incomplete Freund's adjuvant(Sigma). Sera were collected 21 days after the first immunizationand analyzed for TNP-specific Abs by enzyme-linked immunosorbentassay (ELISA). To measure the TI immune response, p110 ${delta}$ ^-/- miceand wild-type littermates (five per group) were immunized intraperitoneallywith 50 µg of TNP-lipopolysaccharide (LPS) (TI-I antigen)(Sigma) or 25 µg of TNP-Ficoll (TI-II antigen) (BiosearchTechnologies) in phosphate-buffered saline (PBS). Sera collected12 days after immunization were analyzed for TNP-specific Absby ELISA.

ELISAs. Specific concentrations of IgM, IgG1, IgG2a, IgG2b, IgG3, andIgA in serum were measured by coating Nunc Maxisorp 96-wellplates (Thomas Scientific, Swedesboro, N.J.) with isotype-specificgoat anti-mouse Abs (Southern Biotechnology Associates, Birmingham,Ala.). Bound immunoglobulin from test sera was detected by usingalkaline phosphatase-conjugated goat anti-mouse isotype-specificAbs (Southern Biotechnology), followed by alkaline phosphatasesubstrate (p-nitrophenyl phosphate; Sigma). The absorbance ofthe reaction mixture at 405 nm was quantitated with an ELISAplate reader (model 3550; Bio-Rad Laboratories). Each assayincluded the titration of affinity-purified mouse IgM, IgG1,IgG2a, IgG2b, IgG3, and IgA Abs (Southern Biotechnology), whichwas used to construct a standard curve. The concentrations ofserum immunoglobulin were calculated by comparing the opticaldensity obtained to a standard curve of titrated Abs by usinglinear regression analysis. Anti-TNP specific immunoglobulinisotype levels were measured by coating plates with TNP-bovineserum albumin (Biosearch Technologies).

In vitro B- and T-cell proliferative responses. For B-cell proliferation assays, B cells purified by fluorescence-activatedcell sorting (FACS) (10⁵/well; >95% pure) were cultured intriplicate in 96-well U-bottom plates. Purified goat anti-mouseIgM (µ chain) (Jackson ImmunoResearch), anti-CD40 MAb(PharMingen), LPS (Sigma), or phorbol myristate acetate (PMA)and ionomycin (Sigma) were added as mitogens at the indicatedconcentrations. Cells were cultured for 48 h, after which theywere labeled with 1 µCi of [³H]thymidine per well for18 h and harvested with a TOMTECH Harvester 96 MachII to measureincorporated radioactivity. T-cell proliferation assays wereconducted similarly. Percentages of Thy1.2⁺ cells within splenocytesuspensions were identified by FACS analysis, and splenocyteswere cultured in triplicate U-bottom 96-well plates such thata constant number of T cells (1.5 x 10⁵/well) were used perassay. Anti-CD3 MAb (145-2C11; Pharmingen), phytohemagglutinin(PHA), concanavalin A (ConA), and PMA plus ionomycin (Sigma)were added at the indicated concentrations.

Ca⁺² measurement and phosphotyrosine blots. Splenocytes were suspended in Dulbecco's modified Eagle mediumplus 10% fetal calf serum, loaded with Indo-1 (10 µM;Molecular Probes, Eugene, Oreg.) for 30 min at room temperature,and stained with either PE-anti-B220 or PE-anti-CD4 and FITC-anti-CD8for an additional 20 min at 4°C. Cells were stimulated withanti-mouse IgM (Jackson ImmunoResearch) for B cells or rat anti-mouseCD3 Ab (145-2C11) followed by anti-hamster IgG Ab (Jackson ImmunoResearch)for T cells. Ionomycin (Calbiochem) was used as a positive control.To induce tyrosine phosphorylation, splenic B cells were purifiedby complement lysis with anti-Thy1.2 Ab (AT83) and rabbit andguinea pig complement (Cedarlane Laboratory). Purified B cells(90%) were stimulated with anti-IgM (20 µg/ml) for 3 minand then lysed as indicated above.

Immunohistochemistry. For detection of a germinal center formation response, p110 ${delta}$ ^-/-mice and their wild-type littermates were immunized intraperitoneallywith TNP-KLH (Biosearch Technologies) as described above orwith 150 µl of a 10% suspension of sheep red blood cells(Colorado Serum, Denver, Colo.). Spleens were snap frozen inliquid nitrogen and sectioned. Frozen sections 8 nm thick werefixed with acetone for 15 min at -20°C and rehydrated withPBS. Sections were double stained with biotinylated B220 (1:200)and FITC-Thy1.2 (1:50) or FITC-B220 (1:200) (all from PharMingen)or biotinylated peanut agglutinin (PNA) (10 µg/ml; Vector,Burlingame, Calif.) in PBS with 3% bovine serum albumin for2 h at room temperature. Sections were washed and subsequentlycounterstained with streptavidin-tetramethyl rhodamine isocyanate(1:1,000) (Southern Biotechnology) for 1 h at room temperature.After being washed in PBS, the sections were mounted in FluoromountG (Southern Biotechnology). All slides were evaluated and photographedwith a Leica TCS confocal laser scanning microscope.

Bone marrow colony assays. Cells were prepared from bone marrow in ${alpha}$ -MEM medium (Life Technologies)containing 2% fetal bovine serum (StemCell Technologies) andcounted in the presence of 3% acetic acid to lyse red bloodcells. Cells were cultured in 0.9% MethoCult M3230 (StemCellTechnologies) with recombinant cytokines at the following concentrations:BFU-E, 3 U of recombinant human erythropoietin (rhEpo) (Amgen)per ml plus 10 ng of recombinant murine IL-3 (rmIL-3) (R&DSystems) per ml; CFU-Mix, 10 ng of rmIL-3 (R&D) per ml;CFU-Eos, 20 ng of rmIL-5 (R&D) per ml; CFU-Meg, 50 ng ofrcombinant human thrombopoietin (Genzyme) per ml; CFU-G, 10ng of recombinant human granulocyte colony-stimulating factor(rhG-CSF) (Amgen) per ml; CFU-GM, 10 ng of recombinant murinegranulocyte-macrophage CSF (rmGM-CSF) (R&D) per ml; andCFU-M, 10 ng of rmCSF-1 (R&D) per ml. Pre-B-cell assayswere conducted with MethoCult M3630 by using 10 ng of rhIL-7(StemCell Technologies) per ml. Assay mixtures were plated in35-mm-diameter culture dishes in duplicate and scored as previouslydescribed (28).

RESULTS

Top

Results

Generation of p110 ${delta}$ mice. The p110 ${delta}$ gene was disrupted in murine E14 ES cells by the replacementof a 2.6-kb fragment containing exons 2 through 6 with a neogene expression cassette (Fig. 1). The design of the targetingconstruct relied on the finding that deletion of >150 aminoacids from the N-terminal region of p110 ${delta}$ eliminated kinase activity(32). Four correctly targeted ES clones were injected into C57BL/6blastocysts, two of which were transmitted to the germ line.The two mouse lines had comparable phenotypes. Southern blotanalysis confirmed the presence of the targeted locus (Fig.1B). Heterozygous mice were bred to obtain mice homozygous forthe targeted allele. Homozygous offspring were viable, wereobtained with the expected Mendelian ratio, and lacked p110 ${delta}$ protein as assessed by immunoprecipitation and Western blotanalysis of splenic extracts (Fig. 1C). The absence of p110 ${delta}$ had no effect on the levels of p110 ${alpha}$ or p110ß in splenicextracts (data not shown).

View larger version (23K):
[in this window]
[in a new window]
FIG. 1. Targeted disruption of murine p110 ${delta}$ gene. (A) A targeting construct that would replace the indicated exons with a neomycin resistance cassette was developed. The locations of the 3' external probe and PCR primers (P1, P2, and P3) for genotyping are indicated. Restriction enzyme sites: Sa, SacI; K, KpnI; St, StuI. DTA, diphtheria toxin A; Wt, wild type. (B) Southern blot analysis of KpnI-digested genomic DNA from p110 ${delta}$ heterozygous mice probed with the indicated probe. The positions of the mutant 5.8-kb and wild-type 4.3-kb KpnI fragments are indicated. (C) Absence of p110 ${delta}$ protein expression in p110 ${delta}$ ^-/- mice. Total splenic lysate (2 mg) was used for immunoprecipitation and Western blot analysis with rabbit polyclonal anti-p110 ${delta}$ antibodies (see Materials and Methods).

Lack of an effect of p110 ${delta}$ deficiency on hematopoietic tissues. In contrast to the widely distributed p110 ${alpha}$ and p110ßproteins, p110 ${delta}$ is expressed primarily in circulating neutrophils,monocytes, and lymphocytes as well as in lymphoid tissues, includingspleen, lymph nodes, and thymus (32). Therefore, we examineda number of hematopoietic parameters in the mutant mice. Therewere no significant differences between wild-type and mutantmice in the number of red cells (Table 1) or in hemoglobin levelsor hematocrits (data not shown). Similarly, there were no significanteffects of p110 ${delta}$ deficiency on the numbers and morphology ofperipheral blood leukocytes, including neutrophils, eosinophils,basophils, monocytes, and lymphocytes (Table 1). The recoveryof bone marrow cells was normal, and in colony assays of bonemarrow progenitors, there were no significant alterations inthe frequency and morphology of colonies in response to a varietyof cytokines (Table 2).

View this table:
[in this window]
[in a new window]
TABLE 1. Peripheral blood cell numbers in wild-type and p110 ${delta}$ -deficient mice (n = 11)^a

View this table:
[in this window]
[in a new window]
TABLE 2. In vitro colony formation by bone marrow hematopoietic progenitors from wild-type and p110 ${delta}$ -deficient mice in response to various cytokines

Defective B-cell development in p110 ${delta}$ ^-/- mice. Analysis of B-cell populations indicated that the deficiencyof p110 ${delta}$ had a significant effect on the numbers and the varioussubpopulations of differentiating B cells. Although the totalnumbers of bone marrow cells were comparable to those in wild-typemice (54.1 ± 5.3 [x 10⁶] versus 54.3 ± 2.3 [x10⁶]; n = 8), there was an approximately 25% decrease in thepercentage of bone marrow B-lineage cells (21.1 ± 5.2versus 15.6 ± 5.8). Among the bone marrow B-cell subpopulations,the percentages of pro-B cells (CD43⁺ B220⁺) were similar forwild-type and p110 ${delta}$ -deficient bone marrow (Fig. 2A). However,the more mature B-cell subpopulation of B220⁺ CD43^- cells wasreduced (Fig. 2). Of the more mature populations, there weresignificant reductions in the percentages of both the B220⁺IgM⁺ cells and the more mature B220⁺⁺ IgM^low cells.

View larger version (41K):
[in this window]
[in a new window]
FIG. 2. Flow cytometric analysis of B- and T-cell development in the primary and secondary lymphoid organs. Total cells isolated from 3- to 4-month-old mice were stained with the indicated antibodies and analyzed by FACS. The numbers in each region indicate the percentage of the total cells plotted. The data shown are representative of those from a minimum of six pairs of mice examined. (A) Early and late stages (left and right, respectively) of B-cell maturation in the bone marrow. Left panel, B220 versus CD43 profiles of IgM^--gated cells; right panels, B220 versus IgM profiles of CD43^--gated cells (fractions D, E, and F). (B) Analysis of splenic B and T cells, showing profile of B220 versus Thy1.2 gated lymphocytes. (C) Analysis of peritoneal B1a cells, displayed as the profile of IgM⁺ CD5⁺ cells within the lymphocyte gate. (D) Maturation stages of splenic B cells. Left panels, B220 versus IgM profiles of gated lymphocytes; right panels, IgD versus IgM profiles of gated lymphocytes. (E) Analysis of lymph node B cells, showing B220 IgM profiles of gated lymphocytes. (F) Maturation of thymocytes is unimpaired (CD4 versus CD8 profile of gated thymocytes).

Gross macroscopic examination of lymphoid organs revealed aconsistent reduction in the sizes of spleens and lymph nodesisolated from p110 ${delta}$ -deficient animals compared to wild-type littermatecontrols. In preparations of splenocytes, the percentages oftotal B220⁺ lymphocytes was reduced from 42.3% ± 5.0%to 32.1% ± 11.4% (Table 3 and Fig. 2B). As shown in Fig.2D, there was a marked reduction in the IgM^low IgD^high populationand in the most mature B220⁺ IgM^- population. Similar to thecase for the spleen populations, the percentage of B220⁺ lymphocytesin lymph nodes was reduced from 31.6% ± 4.5% to 19.0%± 3.2% (n = 6) (Table 3).

View this table:
[in this window]
[in a new window]
TABLE 3. Lymphocyte populations in wild-type and p110 ${delta}$ -deficient mice^a

Both strains had comparable proportions of CD4⁺ and CD8⁺ peripheralT cells in their spleen and lymph node T-cell compartments (Table3), and the T-cell subpopulations in the thymus were normal(Fig. 2F). Lastly, there was a complete absence of CD5⁺ B1 cellsin peritoneal lymphocytes (Fig. 2C). Collectively, the phenotypicchanges in the B-cell subpopulations are similar to those observedin mice in which the BCR signaling pathway is disrupted by deficienciesin Btk (15, 16), BLNK (21), protein kinase C (PKC) ß(17), PLC ${gamma}$ 2 (35), 85 ${alpha}$ (26), Vav-1/Vav-2 (11, 27), and CD19 (7,23).

Decreased serum immunoglobulin and defective humoral response in p110 ${delta}$ ^-/- mice. The reduction in the number of peripheral B cells in p110 ${delta}$ knockoutmice suggested that B-cell function might be disrupted in theabsence of p110 ${delta}$ . To further assess this, serum immunoglobulinlevels were measured by ELISA. As illustrated in Fig. 3, unimmunizedp110 ${delta}$ -deficient mice had lower levels of serum immunoglobulinof all isotypes examined, with IgM and IgG1 levels being themost dramatically lowered. The requirement for p110 ${delta}$ in the humoralresponse was examined by immunizing mice with T-independentand T-dependent antigens. Administration of TNP-LPS (a T-independentantigen) resulted in hapten-specific IgM and IgG1 responsesin the p110 ${delta}$ -deficient mice that were 30 to 50% lower than thosein wild-type mice (Fig. 4A). In contrast, the levels of hapten-specificIgG2a, IgG2b, and IgG3 in the two strains were equivalent. p110 ${delta}$ -deficientmice immunized with the type II antigen TNP-Ficoll had significantlydecreased production of hapten-specific IgM, IgG1, IgG2b, andIgG3, whereas IgG2a responses were low in all mice (Fig. 4B).Lastly, p110 ${delta}$ ^-/- mice immunized with the T-cell-dependent antigenTNP-KLH failed to mount any detectable response (Fig. 4C).

View larger version (15K):
[in this window]
[in a new window]
FIG. 3. Decreased serum immunoglobulin concentrations in p110 ${delta}$ ^-/- mice. Levels of immunoglobulin isotypes in serum in unimmunized wild type or p110 ${delta}$ -deficient mice were determined by ELISA. The values for each individual mouse tested are plotted on a logarithmic graph. Mean values are indicated by boldface lines. P values as determined by Student's t test are indicated.

View larger version (25K):
[in this window]
[in a new window]
FIG. 4. Defective humoral response to thymus-independent antigens and lack of a humoral response to a thymus-dependent antigen in p110 ${delta}$ -deficient mice. Wild-type (filled circles) or p110 ${delta}$ -deficient (open circles) mice were immunized with either TNP-LPS, a TI-I antigen (A); TNP-Ficoll, a TI-II antigen (B); or TNP-KLH, a T-dependent antigen (C). The serum TNP-specific immunoglobulin isotype response was measured by ELISA. The relative absorbance values at 405 nm for the indicated dilution are plotted for each individual animal. Mean values are indicated by boldface lines. Statistically significant differences (P < 0.05) are indicated (**).

The lack of a response to TNP-KLH suggested that the deficiencyof p110 ${delta}$ might also be associated with a defect in germinal centerformation. As illustrated in Fig. 5, immunocytochemical stainingof spleen cryosections demonstrated that whereas wild-type micedeveloped germinal centers, detected by PNA staining, mutantmice failed to generate germinal centers. To further confirmthat p110 ${delta}$ is required for the process of germinal center formation,wild-type and mutant mice were immunized with sheep red bloodcells. Ten days later, at the peak of germinal center formation,spleens were analyzed for germinal centers by PNA staining.In all p110 ${delta}$ ^-/- mice examined, there was a complete absence ofgerminal center formation (data not shown). In contrast to theeffect of p110 ${delta}$ deficiency on peripheral B cells, we detectedno difference between wild-type and mutant mice in the T-cellpopulations (Thy 1-positive cells) surrounding the splenic follicles(data not shown).

View larger version (52K):
[in this window]
[in a new window]
FIG. 5. Germinal center formation in TNP-KLH-stimulated 4-month-old mice. Germinal centers (arrowheads) were stained with biotinylated PNA followed by streptavidin-tetramethyl rhodamine isocyanate (red). B cells were stained with FITC-B220 (green).

Impairment of in vitro B-cell proliferation with p110 ${delta}$ deficiency. To establish whether the observed in vivo defects in p110 ${delta}$ deficientB cells were due to an intrinsic defect in signal transduction,in vitro B-cell proliferation was examined. The abilities ofsplenocytes to respond to various concentrations of known activatorsare shown in Fig. 6. As members of the PI3K family have beendemonstrated to lie in the B-cell receptor signaling pathwayupstream of PLC ${gamma}$ 2, we compared the mitogenic response of p110 ${delta}$ -deficientB cells with that of B cells isolated from PLC ${gamma}$ 2^-/- mice (35).As illustrated, the proliferation of p110 ${delta}$ -deficient B cellsin response to anti-IgM was markedly reduced. However, thisreduction was not as severe as that associated with a deficiencyof PLC ${gamma}$ 2. There was also a decrease in the proliferative responseof p110 ${delta}$ -deficient B cells relative to wild-type B cells afterstimulation with anti-CD40, similar to the reductions observedwith PLC ${gamma}$ 2-deficient B cells. In addition, the response of p110 ${delta}$ ^-/-B cells to LPS was reduced relative to that in the wild type,while the response to phorbol ester and ionomycin was not significantlyaffected (Fig. 6). Curiously, the response of p110 ${delta}$ ^-/- B cellsto CD40 plus IL-4 was unaffected (data not shown). The responseof splenic T lymphocytes was also examined (Fig. 6B). The proliferativeresponse to stimulation through the TCR complex with anti-CD3was not significantly affected. There was some reduction inthe response to ConA and a more striking reduction in the responseof lymphocytes to PHA. It should be noted, however, that theresponse to PHA is only approximately 1/10 that seen in responseto anti-CD3. Lastly, the response of T cells to PMA and ionomycinwas not altered (Fig. 6), as was the response to stimulationwith anti-CD3 plus anti-CD28, with or without addition of exogenousIL-2 (data not shown).

View larger version (33K):
[in this window]
[in a new window]
FIG. 6. Impaired in vitro proliferative response of p110 ${delta}$ -deficient lymphocytes. (A). Purified splenic B cells from 3- to 4-month-old wild-type mice (WT), p110 ${delta}$ ^-/- mice, and PLC ${gamma}$ 2^-/- mice were incubated in medium alone or in medium containing the indicated concentrations of various stimuli. Proliferative responses are expressed as mean counts of triplicates from a single [³H]thymidine incorporation assay; error bars indicate standard deviations. Results are representative of those from four to six experiments. Ion, ionomycin. (B) Splenic T cells were incubated in medium alone or in medium containing the indicated concentrations of various stimuli. Proliferative responses are expressed as mean counts of triplicates from a single [³H]thymidine incorporation assay; error bars indicate standard deviations. Results are representative of those from four to six experiments.

The induction of an intracellular calcium flux is an early signaltransduction event that regulates many downstream cellular responsesand is a sensitive marker for activation of PLC ${gamma}$ 2 and its upstreamactivators (35). Therefore, we analyzed the ability of anti-IgMto induce an increase in intracellular Ca²⁺ in p110 ${delta}$ ^-/- B cells.In contrast to the absence of detectable Ca²⁺ mobilization inPLC ${gamma}$ 2^-/- B cells, p110 ${delta}$ ^-/- B cells induced an increase in intracellularCa²⁺ that was approximately 25% of that observed in wild-typecells (Fig. 7A). Lastly, mobilization of intracellular Ca²⁺upon anti-CD3 stimulation was unimpaired in p110 ${delta}$ ^-/- T cells(Fig. 7A).

View larger version (49K):
[in this window]
[in a new window]
FIG. 7. Induction of Ca²⁺ mobilization and tyrosine phosphorylation in p110 ${delta}$ -deficient mice. (A) Induction of Ca²⁺ was measured by flow cytometry following stimulation of Indo-1-labeled splenic B cells with anti-IgM or of splenic T cells with anti-CD3 followed by anti-IgG. Arrow 1, time point of specific stimulation; arrow 2, time point of ionomycin addition. The results are representative of those from four independent experiments. WT, wild type. (B) Purified splenic B cells from wild-type and p110 ${delta}$ ^-/- mice were stimulated with anti-IgM (20 µg/ml) for 3 min and analyzed for the induction of tyrosine phosphorylation (pTyr) of Btk and PLC ${gamma}$ 2. IP, immunoprecipitation.

A deficiency in p110 ${delta}$ could potentially affect the recruitmentand activation of components of the BCR complex that utilizepleckstrin homology domains to be localized in the complex,including Btk and PLC ${gamma}$ 2. We therefore assessed the recruitmentof these proteins into the complex by examining their extentof tyrosine phosphorylation following receptor engagement. Asillustrated in Fig. 7B, both Btk and PLC ${gamma}$ 2 were inducibly tyrosinephosphorylated in p110 ${delta}$ -deficient cells at levels comparableto those in wild-type cells.

DISCUSSION

Top

Discussion

The results demonstrate the essential, nonredundant role thatp110 ${delta}$ plays in BCR signal transduction. The remarkable specificityof function was unexpected, since p110 ${delta}$ is expressed in a varietyof hematopoietic lineages. Indeed, our interest in p110 ${delta}$ arosefrom studies of cytokine signaling in myeloid-lineage cells.In particular, the cytoplasmic protein tyrosine kinase Jak2is associated with many cytokine receptors (13), and with receptorengagement it is activated, resulting in phosphorylation ofat least 10 major autophosphorylation sites (unpublished data).When one of these sites, Y⁹⁶⁶, was used to develop phosphopeptideaffinity columns, it was found to bind a number of signal-transducingproteins, including a complex of p85 ${alpha}$ /p85ß with p110 ${delta}$ .Importantly, in the myeloid cell line used, complexes of p85 ${alpha}$ /p85ßwith p110 ${alpha}$ or p110ß were not detected, although bothare expressed in these cells. The basis for this specificityis not known but could reside in the characteristics of p85 ${alpha}$ /p85ßwhen bound to the Y⁹⁶⁶ phosphopeptide. In spite of the abilityof Y⁹⁶⁶ to uniquely recruit the p85 ${alpha}$ /p110 ${delta}$ or p85ß/p110 ${delta}$ complex, the absence of p110 ${delta}$ has no detectable affect on theresponses of a number of cytokines that utilize Jak2, includingIL-3 and Epo. The possibility exists that the recruitment ofPI3K activity to these receptor complexes is not critical fortheir physiological function. Consistent with this, mutant micethat contain a truncated Epo receptor that lacks receptor domainsrequired for optimal activation of PI3K activity (38) have normalerythropoiesis. Alternatively, in the absence of p110 ${delta}$ , p110 ${alpha}$ or p110ß may replace the requirement for p110 ${delta}$ . Itshould be noted, however, that there was no obvious evidencefor altered Epo signaling in mice that are deficient in p85 ${alpha}$ ,and thus a different regulatory subunit would also be involved.

Perhaps one of the most striking features is the similarityof the effect of p110 ${delta}$ deficiency to those of deficiencies ofa number of other genes on BCR signaling. First, the similaritiesseen with deficiencies of p85 ${alpha}$ and p110 ${delta}$ would indicate that p110 ${delta}$ uniquely associates with p85 ${alpha}$ in the context of the BCR. Thisis again surprising, since both p110 ${alpha}$ and p110ß associatewith p85 ${alpha}$ and have previously been shown to be present in B cells(32). Moreover, deficiency of p110 ${delta}$ does not detectably alterthe levels of p110 ${alpha}$ or p110ß in splenic lymphocytes(data not shown). The specificity may reside in the BCR complexand its ability to recruit specific p85 ${alpha}$ /p110 complexes. Forexample, CD19 is uniquely required for B-cell responses to mitogens,generation of phosphatidylinositol triphosphate, PLC activation,and Ca²⁺ mobilization following BCR engagement, and the deficiencyof CD19 results in a very similar in vivo phenotype to thatseen with a deficiency of p110 ${delta}$ (7, 23). Given the lack of asignificant effect of p110 ${delta}$ deficiency on TCR signaling pathways(see below), it can by hypothesized that the lack of T-dependentantibody responses in p110 ${delta}$ ^-/- mice is directly related to impairedCD19 signaling. Interestingly, the properties of CD19-deficientB cells have led to the proposal that tyrosine phosphorylationof CD19 on Y⁴⁸⁴ and Y⁵¹⁵ is uniquely required for activationof PI3K activity, which, in turn, is required for phosphoinositidehydrolysis and Ca²⁺ mobilization (3).

The deficiency in p110 ${delta}$ also has a consequence very similar tothat of a deficiency in PLC ${gamma}$ 2 (35). It should be noted, however,that the deficiency in PLC ${gamma}$ 2 affects a number of receptors ofthe Fc family of receptors that are not detectably affectedby the p110 ${delta}$ deficiency. Thus, only within the context of theBCR is p110 ${delta}$ required for PLC ${gamma}$ 2 function. It is possible thatin the context of other receptor complexes, which do not requireCD19, the p85/p110 complexes may function redundantly or havedifferent specificities. The results demonstrate that in theabsence of p110 ${delta}$ , PLC ${gamma}$ 2 is recruited to the receptor complex basedon its induced tyrosine phosphorylation. However, functionallyit is not fully activated, as evidenced by the greatly reducedCa²⁺ mobilization. It can be proposed that the engagement ofthe pleckstrin homology domain of PLC ${gamma}$ 2 may be required for itsappropriate localization or activation. In this regard it shouldalso be noted that a deficiency in the PIP₃ phosphatase PTENresults in B-cell hyperactivity (6). In addition to potentiallyaffecting PLC ${gamma}$ 2 activity in the complex, p110 ${delta}$ may also affectBtk function. As shown here, Btk is recruited to the complexand is inducibly tyrosine phosphorylated in the absence of p110 ${delta}$ .However, the naturally occurring Xid mutation in mice involvesa single amino acid change in the pleckstrin homology domain(22, 29) and creates a phenotype that is similar to that seenwith Btk deficiency (15, 16, 31, 33) and deficiencies in othercomponents of the complex.

Based on the above observations, it can be proposed that thep85 ${alpha}$ /p110 ${delta}$ is uniquely recruited to the BCR complex, most likelythrough a specificity involving CD19. Once recruited, PIP₃ generationis required to form binding sites for both Btk and PLC ${gamma}$ 2 thatmay stabilize the complex or contribute to the overall activationof the complex. The essential function of the complex is thehydrolysis of membrane phosphoinositides and the generationof IP₃ and diacylglyerol. These mediators, in turn, are essentialfor Ca²⁺ mobilization and the activation of PKC ß.Again, the critical role for PKC ß is evident in thephenotype of PKC ß-deficient mice, which have BCRdefects identical to the deficiencies of the other components(17). The other possibility is that PI3K activity is requiredfor the activation of Akt, with the major isoform being Akt1.However, mice deficient in neither Akt1 nor Akt2 have been reportedto have B-cell defects comparable to those seen in PKC ß-deficientmice (4, 5).

The other essential components of the BCR complex, based onthe phenotypes of deficient mice, are the adapter protein Blnk(18, 21), the PI3K binding protein BCAP (37), and members ofthe Vav family of GTP exchange factors. The adapter proteinBlnk is critical for the recruitment and activation of PLC ${gamma}$ 2through direct interaction of the SH2 domains of PLC ${gamma}$ 2 with tyrosine-phosphorylatedBlnk, and Blnk deficiency results in the elimination of theCa²⁺ signal in BCR signaling. A deficiency in BCAP also createsa very similar phenotype and, biochemically, is also associatedwith loss of PLC ${gamma}$ 2 function but retention of the recruitmentand tyrosine phosphorylation of PLC ${gamma}$ 2 in the BCR complex. Basedon these observations it can be postulated that BCAP is requiredto recruit, activate, and/or stabilize the p85/p110 ${delta}$ complexin the context of the BCR signalsome. In the case of Vav, adeficiency of Vav-1 has a severe consequence for T-cell developmentand a lesser consequence for B-cell development (8, 39). However,the absence of both Vav-1 and Vav-2 dramatically affects BCRsignaling and results in a phenotype comparable to that seenin p110 ${delta}$ -deficient mice (27). Importantly, the absence of Vav-1and Vav-2 eliminates the Ca²⁺ flux induced by BCR engagement,and the activation of Vav-1 and Vav-2 has been proposed to requirebinding of PIP₃ by their pleckstrin homology domains. The precisenature by which Vav-1 and Vav-2 contribute to the BCR complexsignaling is not known.

In contrast to the case for the BCR complex, the deficiencyin p110 ${delta}$ did not affect signaling through aggregation of theTCR complex with anti-CD3 either in terms of a mitogenic responseor in the induction of a Ca²⁺ flux. However, there was a reductionin the much weaker responses of T cells to PHA and ConA, thesignificance of which is not known. Although the absolute numbersof splenic and lymph node T cells are slightly reduced, it islikely that this reduction is related to the overall reducedsize of the spleen and lymph nodes. In addition to there beingno differences in peripheral blood lymphocyte numbers betweenwild-type and p110 ${delta}$ -deficient mice, no significant differencesin thymic size or cellularity was observed. Importantly, manyof the components of the BCR complex are distinct from thoseof the TCR complex, although often family members are. For example,PLC ${gamma}$ 2 is not required for a Ca²⁺ flux following TCR aggregation,although PLC ${gamma}$ 1 likely is essential. Similarly, the TCR complexrequires Rlk and Itk (24) and not Btk. As noted above, it hasbeen suggested that Vav-1 is critical to TCR signaling, whileVav-1 and Vav-2 redundantly are required for BCR signaling.In the TCR complex the adapter protein Blnk is replaced by thefamily member SLP-76. Further downstream, the critical roleof PKC ß in B-cell responses is replaced by PKC ${theta}$ inT cells (25). Thus, although the ultimate function of the receptorcomplex might be comparable, the functional components of thecomplex are different and are likely to have evolved to functionsynergistically.

Hypogammaglobulenima in humans is associated with mutationsin the BCR complex components Btk and BLNK. It might be anticipatedthat other components, identified through gene disruptions inmice, would contribute to those cases in which an etiology isnot known. Recent studies (34), however, failed to identifycausative mutations in PLC ${gamma}$ 2 in 32 such cases. It should be noted,however, that the deficiency of PLC ${gamma}$ 2 in mice affects many receptorsof the immunoglobulin superfamily, including the collagen receptorcomplex on platelets, and therefore a spectrum of phenotypicchanges would be expected in individuals with a deficiency inPLC ${gamma}$ 2. In contrast, as described here, p110 ${delta}$ deficiency resultsin a very selective defect in B cells and consequently is apotential candidate for hypogammaglobulenima in humans.

Following submission of this paper, an article appeared whichdescribed the properties of a mouse strain in which homologousrecombination was used to create a dominant negative form ofp110 ${gamma}$ (20). Similar to the properties of mice in which a nullmutation exists, signaling through the BCR complex was affected,although the degree of loss of the calcium response was less.This may due to a role for p110 ${delta}$ in forming a stable BCR complexin addition to its enzymatic role, possibly through its interactionwith BCAP. A significant difference existed, however, in theconclusions regarding T-cell responses. As noted above, we havenot detected any consistent differences in the response of Tcells to TCR engagement in numerous experiments. In mice expressingthe dominant negative form of p110 ${delta}$ , the responses to anti-CD3were reduced 30 to 50% while the responses to anti-CD3 plusanti-CD28 were either not decreased or increased. It was concludedthat the reduction in the T-cell responses was responsible forthe dramatic reduction in T-dependent antibody responses, aphenotype also seen in our null mutants. As noted above, however,the absence of CD19 also results in a loss of T-dependent responses,and its role in the BCR complex might equally explain the lossof T-dependent antibody responses, independent of a functionalrole for p110 ${delta}$ in T-cell responses. Lastly, we have not observedan inflammatory bowel disease in mice containing a null mutationsimilar to that described for mutant mice containing the dominantnegative mutation. This difference could potentially be dueto differences in animal housing conditions coupled with theB-cell defects. Alternatively, the dominant negative proteinmay create a phenotype in other lineages of cells.

ACKNOWLEDGMENTS

We acknowledge Ken Sato, Jean-Christophe Marine, Evan Parganas,Catriona McKay, Richard Moriggl, Mei-Hwei Chang, and Kai-HsinLin for valuable discussions. We also thank John Raucci forthe injection of ES cells into blastocysts and the staff ofthe Animal Resources facility for help in animal maintenance.The FACS sorting and Ca²⁺ flux studies were performed by RichardAshmun and Mahnaz Paktinat, and general technical support wasprovided by Linda Synder and Kristen Rothammer. The ES cellswere kindly provided by Jan van Deursen.

S.-T.J was supported by the National Taiwan University Hospitaland Taiwan National Science Council (grants NSC89-2314-B-002-399and NSC90-2314-B-002-181). This work is supported by CancerCenter CORE grant CA21765, by grant RO1 DK42932 to J.N.I., bygrant PO1 HL53749, and by the American Lebanese Syrian AssociatedCharities (ALSAC).

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN 38105. Phone: (901) 495-3422. Fax: (901) 525-8025. E-mail: james.ihle{at}stjude.org.

Present address: Department of Pediatrics, National Taiwan UniversityHospital, Taipei 100, Taiwan.

Present address: Blood Research Institute and Department ofMicrobiology and Molecular Genetics, Medical College of Wisconsin,Milwaukee, WI 53226.

REFERENCES

Top