Frontline: The p85alpha isoform of phosphoinositide 3-kinase is essential for a subset of B cell receptor-initiated signaling responses

Phosphoinositide 3-kinase (PI3K) is a ubiquitously expressed signaling enzyme that plays an integral role in development and activation of B cells. B cell receptor (BCR)-driven proliferation is completely blocked either in cells lacking the p85alpha regulatory isoform of PI3K or in wild-type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85alpha to early signaling events has not been fully investigated. Here we show that B cells lacking p85alpha have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85alpha results in partial reductions in Ca2+ mobilization and IkappaB phosphorylation, whereas ERK phosphorylation is not diminished. Moreover, although Akt phosphorylation is partially reduced, phosphorylation of several proteins downstream of Akt is preserved. These partial impairments suggest that there are other routes to PI3K activation in B cells apart from p85alpha-associated catalytic subunits. Notably, addition of phorbol ester restores BCR-mediated proliferation in p85alpha-deficient cells but not wild-type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85alpha is a failure to activate diacylglycerol-regulated signaling enzymes, most likely protein kinase C.     

Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cells

B‐cell receptor (BCR)‐mediated signals provide the basis for B‐cell differentiation in the BM and subsequently into follicular, marginal zone, or B‐1 B‐cell subsets. We have previously shown that B‐cell‐specific expression of the constitutive active E41K mutant of the BCR‐associated molecule Bruton's tyro s ine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low‐level expression of the E41K or E41K‐Y223F Btk mutants was associated with reduced follicular B‐cell numbers and significantly increased proportions of B‐1 cells in the spleen. Crosses with 3‐83μδ and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B‐1 B‐cell fate choice, but resulted in selective expansion or survival of B‐1 cells. Residual B cells were hyperresponsive and manifested sustained Ca²⁺ mobilization. They were spontaneously driven into germinal center‐independent plasma cell differentiation, as evidenced by increased numbers of IgM⁺ plasma cells in spleen and BM and significantly elevated serum IgM. Because anti‐nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B‐cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B‐cell activation.     

PI3K and Btk differentially regulate B cell antigen receptor-mediated signal transduction

Phosphoinositide-3 kinase (PI3K) is thought to activate the tyrosine kinase Btk. However, through analysis of PI3K-/- and Btk-/- mice, B cell antigen receptor (BCR)-induced activation of Btk in mouse B cells was found to be unaffected by PI3K inhibitors or by a lack of PI3K. Consistent with this observation, PI3K-/- Btk-/- double-deficient mice had more severe defects than either single-mutant mouse. NF-kappaB activation along with Bcl-xL and cyclin D2 induction were severely blocked in both PI3K-/- and Btk-/- single-deficient B cells. Transgenic expression of Bcl-xL restored the development and BCR-induced proliferation of B cells in PI3K-/- mice. Our results indicate that PI3K and Btk have unique roles in proximal BCR signaling and that they have a common target further downstream in the activation of NF-kappaB.     

Distinct signaling mechanisms activate the target of rapamycin in response to different B-cell stimuli

Phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), a downstream kinase, are both required for proliferation of splenic B cells. However, the functions of PI3K and mTOR in response to different stimuli and among B cell subsets have not been fully elucidated. We used flow cytometry and magnetic cell sorting to examine the requirement for PI3K and mTOR in responses of splenic B cell subsets to BCR and LPS stimulation. BCR-mediated phosphorylation of Akt and Erk is sensitive to the PI3K catalytic inhibitor wortmannin in both marginal zone (MZ) and follicular (FO) cells. BCR-mediated mTOR activation in both subsets is inhibited by wortmannin, though less strongly in MZ cells. In contrast, LPS-induced mTOR signaling is strikingly resistant to wortmannin in both subsets. Similarly, functional responses to LPS are partially wortmannin resistant yet sensitive to mTOR inhibition by rapamycin. We also observed mitogen-independent mTOR activity that is regulated by nutrient availability, and is significantly elevated in MZ cells relative to FO cells. These data define both similarities and differences in PI3K/mTOR signaling mechanisms in MZ and FO cells, and suggest that mTOR signaling can occur in the absence of PI3K activation to promote B cell responses to LPS.     

p53 regulates Btk-dependent B cell proliferation but not differentiation

Btk is critical for B cell development and proliferation. Mice lacking Btk have a defect in B cell development, resulting in a loss of mature B cells and decreased proliferative responses following B cell receptor cross-linking. In contrast, mice deficient in the tumor suppressor p53 display increases in developing B cell populations in the bone marrow. To investigate the potential role of p53 in Btk-dependent B cell development and function, we generated mice doubly-deficient in p53 and Btk. Btk/p53-deficient mice showed an increase in splenic B220+ cell numbers compared with Btk-deficient mice, although there was no recovery in B cell subset differentiation. In contrast to the lack of recovery of B cell development, there was a recovery in lipopolysaccharide and anti-immunoglobulin M (IgM) plus interleukin-4-induced proliferation of Btk/p53-deficient B cells, although there was no recovery to anti-IgM stimulation alone. Thus, p53 promotes B cell expansion and proliferation, but p53 deficiency cannot compensate for Btk deficiency in the development of B cell subsets.     

Deletion of PI3K-p85alpha gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells

Class IA phosphotidylinositol-3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that are important in regulating signaling events in B and T cells. However, their role in natural killer (NK) cells is not understood. Here, using mice that lack the regulatory p85alpha subunit and its alternatively spliced variants p55alpha/p50alpha (collectively termed as p85alpha(-/-)), we defined the role of PI3K in NK cell development and function. p85alpha(-/-) mice had impaired lineage commitment leading to reduced NK cellularity in the bone marrow and liver. p85alpha(-/-) NK cells showed a defective Ly49 subset specification and a decreased expression of CD43. Lack of p85alpha severely reduced the NK-mediated cytotoxicity against tumor cells representing 'induced-self' and 'missing-self'. More importantly, NKG2D and NK1.1 receptor-mediated cytokine and chemokine generation was significantly compromised in p85alpha(-/-) NK cells. These results reveal a previously unrecognized role of p85alpha in the development, terminal maturation, cytokine/chemokine generation and tumor clearance of NK cells.     

Bruton's tyrosine kinase inhibition induces rewiring of proximal and distal B-cell receptor signaling in mice

Bruton′s tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B- cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B-cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF-κB and Akt/S6 signaling was decreased in Btk-deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk-effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho-CD79a and phospho-PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells.     

Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function

Phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) phosphatase serve essential functions in the regulation of cell growth, differentiation and survival by modulating intracellular phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) concentrations. Here we show that the conditional deletion of Pten in B cells led to the preferential generation of marginal zone (MZ) B cells and B1 cells. PTEN-deficient B cells were hyperproliferative in response to mitogenic stimuli, and exhibited a lower threshold for activation through the B cell antigen receptor. Inactivation of PTEN rescued germinal center, MZ B and B1 cell formation in CD19-/- mice, arguing that recruitment and activation of PI3K are the dominant roles for CD19 in these B cell subpopulations. These findings establish the central role of PI-3,4,5-P3 regulation in the differentiation of peripheral B cell subsets.     

BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation

Tyrosine phosphorylation of adaptor proteins permits the B cell antigen receptor (BCR)-associated protein tyrosine kinases to regulate downstream effector molecules. Here, we report the identification of a novel B cell adaptor for phosphoinositide 3-kinase (PI3K), termed BCAP. Tyrosine phosphorylation of BCAP is mediated by Syk and Btk, thereby providing binding site(s) for the p85 subunit of PI3K. Disruption of the BCAP gene in the DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to impaired Akt response. Moreover, recruitment of PI3K to glycolipid-enriched microdomains (GEMs) is significantly attenuated in the absence of BCAP. Hence, these data suggest that BCAP bridges BCR-associated kinases to the PI3K pathway by regulating PI3K localization.     

Attenuation of phosphoinositide 3-kinase δ signaling restrains autoimmune disease

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn^?/?p110δ^+/KD mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE.     

Functional folding of a cytoplasmic single-chain variable fragment and its use as elutable protein purification tag

The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85alpha) of phosphatidylinositol-3-kinase (PI3K) (isc-p85alpha), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85alpha fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85alpha. This indicates that the iscFv part of isc-p85alpha does not negatively influence p85alpha folding and interaction with p110. Moreover, successful incorporation of the p85alpha-moiety of isc-p85alpha into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.     

Expression and selection of productively rearranged TCR beta VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1(+) alpha beta T cells

The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta VDJ rearrangements and pre-T alpha mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3 zeta/Lck double-deficient mice fail to express TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCR beta VDJ genes but before expression of TCR beta proteins. Most NKT cells of TCR alpha-deficient mice exhibit cell surface gamma delta TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.     

Defective development and function of Bcl10-deficient follicular,marginal zone and B1 B cells

Bcl10 is an intracellular protein essential for nuclear factor (NF)-kappaB activation after lymphocyte antigen receptor stimulation. Using knockout mice, we show that absence of Bcl10 impeded conversion from transitional type 2 to mature follicular B cells and caused substantial decreases in marginal zone and B1 B cells. Bcl10-deficient B cells showed no excessive apoptosis. However, both Bcl10-deficient follicular and marginal zone B cells failed to proliferate normally, although Bcl10-deficient marginal zone B cells uniquely failed to activate NF-kappaB efficiently after stimulation with lipopolysaccharide. Bcl10-deficient marginal zone B cells did not capture antigens, and Bcl10-deficient (Bcl10-/-) mice failed to initiate humoral responses, leading to an inability to clear blood-borne bacteria. Thus, Bcl10 is essential for the development of all mature B cell subsets.     

Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells

RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria.     

Phosphatidylinositol 3-kinase improves the efficiency of positive selection

We have generated transgenic mice expressing the amino-terminal fragment of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110ABD) in thymocytes. Expression of p110ABD results in constitutive activation of PI3K and in significant increases in the numbers of mature, single-positive thymocytes. We previously reported that the increase in mature cells was in part due to a defect in thymic emigration. In this study we identify another component to this phenotype. Expression of p110ABD results in an enhancement of positive selection, without alterations in thymocyte lifespan or negative selection. Since PI3K can affect activation of Btk, which in turn potentiates calcium fluxes, during B cell development, our results suggest that PI3K could play a role in the regulation of Itk kinases in T cells, and that both cell types share a common signaling network to modulate calcium responses downstream of their antigen receptor.