PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization.

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

Autonomous maturation of alpha/beta T lineage cells in the absence of COOH-terminal Src kinase (Csk).

The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of alpha/beta T lineage cells in T cell receptor (TCR) beta-deficient or recombination activating gene (rag)-1-deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as alpha/beta T cell development is blocked in lck(-/)-fyn(-/)- csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples alpha/beta T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the alpha/beta TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I-restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR-MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.

The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells.

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases.

The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells. The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs. Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated. This raises a question about the mechanism of interaction between these enzymes. Here we present Cbp--a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk. Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src. In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling. These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs.

B-1 cells: orthodox or conformist?

B-1 cells differ from conventional peripheral B cells (B-2) by anatomical location, surface marker expression, antibody repertoire and growth properties. The lineage hypothesis of B-1 cell development attributes the properties of B-1 cells to their unique origin. The induced differentiation hypothesis suggests the surface-immunoglobulin-driven development of B-1 cells from common B-1/B-2 cell progenitors. In both models self-antigen-induced signalling plays the central role in positive selection of B-1 cells. The ability of B-1 cells to be positively selected by self-antigens raises questions about the mechanism of this phenomenon.

The B-cell-specific Src-family kinase Blk is dispensable for B-cell development and activation.

The B-cell lymphocyte kinase (Blk) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells of mice. The early onset of Blk expression during B-cell development in the bone marrow and the high expression levels of Blk in mature B cells suggest a possible important role of Blk in B-cell physiology. To study the in vivo function of Blk, mice homozygous for the targeted disruption of the blk gene were generated. In homozygous mutant mice, neither blk mRNA nor Blk protein is expressed. Despite the absence of Blk, the development, in vitro activation, and humoral immune responses of B cells to T-cell-dependent and -independent antigens are unaltered. These data are consistent with functional redundancy of Blk in B-cell development and immune responses.

Negative regulation of CD4 lineage development and responses by CD5.

CD5 deficiency results in a hyper-responsive phenotype to Ag receptor stimulation. Here we show that the development and responses of CD4 lineage T cells are regulated by the function of CD5. Thymocytes expressing the I-Ad-restricted DO11.10 TCR undergo abnormal selection without CD5. In H-2d mice, the absence of CD5 causes deletion of double-positive thymocytes, but allows for efficient selection of cells expressing high levels of the DO11.10 clonotype. By contrast, there is enhanced negative selection against the DO11.10 clonotype in the presence of I-Ab. T cell hybridomas and DO11.10 T cells are more responsive to TCR stimulation in the absence of CD5. Such hypersensitivity can be eliminated by expression of wild-type CD5, but not by a form of CD5 that lacks the cytoplasmic tail. Finally, CD5 deficiency partially suppresses the block of CD4 lineage development in CD4-deficient mice. Taken together, the data support a general role for CD5 as a negative regulator of Ag receptor signaling in the development and immune responses of CD4 lineage T cells.

An influence of CD5 on the selection of CD4-lineage T cells.

Combining CD5-null, MHC-deficient and lineage-specific reporter animals, we have investigated the influence of CD5 on positive selection and the choice of CD4- versus CD8-lineage commitment on broad populations of thymocytes. CD5 has no obvious quantitative effect in wild-type mice. In mice lacking MHC class II molecules, however, increased numbers of transitional, class I-selected CD4+ CD8(int) CD3(hi) cells were positively selected in the absence of CD5. Importantly, they were committed to the CD4 lineage. Our results indicate that CD5 negatively regulates the differentiation of CD4-committed cells in suboptimal conditions, thus perhaps serving to tighten the correlation between restriction of the TCR and lineage choice.

Csk controls antigen receptor-mediated development and selection of T-lineage cells.

The development and function of alphabetaT lymphocytes depend on signals derived from pre-T and alphabetaT cell receptors (preTCR and alphabetaTCR) (reviewed in refs 1, 2). The engagement of these receptors leads to the activation of Lck and Fyn, which are protein tyrosine kinases (PTKs) of the Src family. It remains unclear to what extent the activation of Src-family PTKs can direct the differentiation steps triggered by preTCR and alphabetaTCR. Here we show that the inactivation of the negative regulator of Src-family PTKs, carboxy-terminal Src kinase (Csk), in immature thymocytes abrogates the requirement for preTCR, alphabetaTCR and major histocompatibility complex (MHC) class II for the development of CD4+ 8+ double-positive and CD4+ single-positive thymocytes as well as peripheral CD4 alphabetaT-lineage cells. These data show that Csk and its substrates are required to establish preTCR/alphabetaTCR-mediated control over the development of alphabetaT cells.

The molecular mechanism of B cell activation by toll-like receptor protein RP-105.

The B cell-specific transmembrane protein RP-105 belongs to the family of Drosophila toll-like proteins which are likely to trigger innate immune responses in mice and man. Here we demonstrate that the Src-family protein tyrosine kinase Lyn, protein kinase C beta I/II (PKCbetaI/II), and Erk2-specific mitogen-activated protein (MAP) kinase kinase (MEK) are essential and probably functionally connected elements of the RP-105-mediated signaling cascade in B cells. We also find that negative regulation of RP-105-mediated activation of MAP kinases by membrane immunoglobulin may account for the phenomenon of antigen receptor-mediated arrest of RP-105-mediated B cell proliferation.

Abnormal development of secondary lymphoid tissues in lymphotoxin beta-deficient mice.

The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) alpha and LTbeta form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTalpha homotrimers can be secreted as well. Mice with a disrupted LTalpha gene lack lymph nodes (LN), Peyer's patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTalpha homotrimers or LTalphabeta heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTbeta was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTbeta reveal severe defects in organogenesis of the lymphoid system similar to those of LTalpha-/- mice, except that mesenteric and cervical LN are present in most LTbeta-deficient mice. Both LTbeta- and LTalpha-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTbeta-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTalpha can signal independently from LTbeta in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.

Xid and Xid-like immunodeficiencies from a signaling point of view.

The analysis of Btk-associated molecules and ligand-induced Btk phosphorylation has suggested the existence of a complexed Btk-associated signaling network involved in the activation of B lymphocytes and mast cells. Recent gene targeting experiments have revealed protein kinase C betaI/II (PKCbetaI/II) as a critical component of the Btk-dependent signaling chain and have highlighted a potential role for the Btk-PKCbetaI/II interaction in the amplification of B cell receptor mediated signaling.

CD2 regulates the positive selection and function of antigen-specific CD4- CD8+ T cells.

The CD2 glycoprotein has been implicated in both positive and negative regulation of T-cell mitogenesis. To study the involvement of CD2 in T-lymphocyte development and immune responses, we have analyzed two lines of CD2-null mice, each expressing a distinct class I major histocompatibility complex (MHC)-restricted T-cell receptor (TCR). In both situations, the absence of CD2 appeared to promote the positive selection of cells in a manner that is similar to that which occurs in the absence of CD5. Consistent with this, compound homozygotes that lacked both CD2 and CD5 showed evidence of enhanced positive selection even in the absence of a transgenic TCR. Despite the observed enhancement of positive selection, the lack of CD2 was associated with defects in proliferative responses and interferon-gamma production when transgenic thymocytes and mature T lymphocytes were stimulated with the appropriate antigens. These findings raise the possibility that impaired sensitivity to selecting ligands in the thymus may provide a selective advantage that improves the efficiency of positive selection for certain TCRs. Furthermore, the results highlight the potential for a differential role for CD2 in thymocyte selection and T-cell immune responses.

CD5-mediated negative regulation of antigen receptor-induced growth signals in B-1 B cells.

A subset of B lymphocytes present primarily in the peritoneal and pleural cavities is defined by the expression of CD5 and is elevated in autoimmune diseases. Upon signaling through membrane immunoglobulin M (mIgM), splenic B lymphocytes (B-2) proliferate, whereas peritoneal B cells (B-1) undergo apoptosis. However, in CD5-deficient mice, B-1 cells responded to mIgM crosslinking by developing a resistance to apoptosis and entering the cell cycle. In wild-type B-1 cells, prevention of association between CD5 and mIgM rescued their growth response to mIgM crosslinking. Thus the B cell receptor-mediated signaling is negatively regulated by CD5 in normal B-1 cells.

Immunodeficiency in protein kinase cbeta-deficient mice.

Cross-linking of the antigen receptor on lymphocytes by antigens or antibodies to the receptor results in activation of enzymes of the protein kinase C (PKC) family. Mice homozygous for a targeted disruption of the gene encoding the PKC-betaI and PKC-betaII isoforms develop an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B cells, which is similar to X-linked immunodeficiency in mice. Thus PKC-betaI and PKC-betaII play an important role in B cell activation and may be functionally linked to Bruton's tyrosine kinase in antigen receptor-mediated signal transduction.

Critical role for the common cytokine receptor gamma chain in intrathymic and peripheral T cell selection.

The common cytokine receptor gamma chain (gammac), which is a functional subunit of the receptors for interleukins (IL)-2, -4, -7, -9, and -15, plays an important role in lymphoid development. Inactivation of this molecule in mice leads to abnormal T cell lymphopoiesis characterized by thymic hypoplasia and reduced numbers of peripheral T cells. To determine whether T cell development in the absence of gammac is associated with alterations of intrathymic and peripheral T cell selection, we have analyzed gammac-deficient mice made transgenic for the male-specific T cell receptor (TCR) HY (HY/gammac- mice). In HY/gammac- male mice, negative selection of autoreactive thymocytes was not diminished; however, peripheral T cells expressing transgenic TCR-alpha and -beta chains (TCR-alphaT/betaT) were absent, and extrathymic T cell development was completely abrogated. In HY/gammac- female mice, the expression of the transgenic TCR partially reversed the profound thymic hypoplasia observed in nontransgenic gammac- mice, generating increased numbers of thymocytes in all subsets, particularly the TCR-alphaT/betaT CD8+ single-positive thymocytes. Despite efficient positive selection, however, naive CD8+ TCR-alphaT/betaT T cells were severely reduced in the peripheral lymphoid organs of HY/gammac- female mice. These results not only underscore the indispensible role of gammac in thymocyte development, but also demonstrate the critical role of gammac in the maintenance and/or expansion of peripheral T cell pools.

A role for CD5 in TCR-mediated signal transduction and thymocyte selection.

CD5 is a transmembrane protein that is expressed on the surface of T cells and a subset of B cells. The absence of CD5 rendered thymocytes hyperresponsive to stimulation through the T cell antigen receptor (TCR) in vitro. Selection of T cells expressing three distinct transgenic TCRs was also abnormal in CD5-deficient mice. These observations indicate that CD5 can influence the fate of developing thymocytes by acting as a negative regulator of TCR-mediated signal transduction.