Anne B. Satterthwaite

Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication.

Antibodies against nuclear self-antigens are characteristic of systemic autoimmunity, although mechanisms promoting their generation and selection are unclear. Here, we report that B cells containing the Y-linked autoimmune accelerator (Yaa) locus are intrinsically biased toward nucleolar antigens because of increased expression of TLR7, a single-stranded RNA-binding innate immune receptor. The TLR7 gene is duplicated in Yaa mice because of a 4-Megabase expansion of the pseudoautosomal region. These results reveal high divergence in mouse Y chromosomes and represent a good example of gene copy number qualitatively altering a polygenic disease manifestation.

Xid-like Phenotypes: A B Cell Signalosome Takes Shape

The molecular events linking BCR stimulation to Ca2+ flux are coming into focus. Considering together the results of biochemical studies and mouse genetic analyses, strong support now exists for a “signalosome” model of B cell activation. In this view, the central response regulator (PLCγ2) is activated by multiple independent inputs that are organized by interactions with membrane phosphoinositides and a docking protein (BLNK). A further level of organization may be provided by localization in lipid rafts. It should be emphasized that the picture of the B cell signalosome is probably incomplete. It is likely that other proteins that interact with Btk may be involved in signal integration and modification. It will be important to apply genetic tests of function to these components. Another challenge for the future is to determine whether distinct signalosomes are organized by other receptors on B cells. The application of gene targeting technology, including conditional inactivation and RAG chimeric approaches, should continue to clarify the complexities of lymphocyte activation.§To whom correspondence should be addressed (e-mail: owenw@microbio.ucla.edu).

Activation of bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain

Brutons tyrosine kinase (BTK) is a nonreceptor tyrosine kinase critical for B cell development and function. Mutations in BTK result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Using a random mutagenesis scheme, we isolated a gain-of-function mutant called BTK* whose expression drives growth of NIH 3T3 cells in soft agar. BTK* results from a single point mutation in the pleckstrin homology (PH) domain, where a Glu is replaced by Lys at residue 41. BTK* shows an increase in phosphorylation on tyrosine residues and an increase in membrane targeting. Transforming activity requires kinase activity, a putative autophosphorylation site, and a functional PH domain. Mutation of the SH2 or SH3 domains did not affect the activity of BTK*. Expression of BTK* could also relieve IL-5 dependence of a B lineage cell line. These results show that transformation activation and regulation of BTK are critically dependent on the PH domain.

Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome

Mice with a targeted disruption of the gene encoding a lymphoid-expressed orphan G protein-coupled receptor, G2A, demonstrate a normal pattern of T and B lineage differentiation through young adulthood. As G2A-deficient animals age, they develop secondary lymphoid organ enlargement associated with abnormal expansion of both T and B lymphocytes. Older G2A-deficient mice (>1 year) develop a slowly progressive wasting syndrome, characterized by lymphocytic infiltration into various tissues, glomerular immune complex deposition, and anti-nuclear autoantibodies. G2A-deficient T cells are hyperresponsive to TCR stimulation, exhibiting enhanced proliferation and a lower threshold for activation. Our findings demonstrate that G2A plays a critical role in controlling peripheral lymphocyte homeostasis and that its ablation results in the development of a novel, late-onset autoimmune syndrome.

Redundant and Opposing Functions of Two Tyrosine Kinases, Btk and Lyn, in Mast Cell Activation

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcεRI). In this study, we have made the following observations on growth properties and FcεRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcεRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-γ1 and C-γ2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcεRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-α and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Cα and protein kinase CβII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

The role of Bruton’s tyrosine kinase in B‐cell development and function: a genetic perspective

Summary: Mutations in Bruton’s tyrosine kinase (Btk) result in the B‐cell immunodeficiencies X‐linked agammaglobulinemia in humans and X‐linked immunodeficiency in mice. These diseases are characterized by blocks in B‐cell development at multiple stages and impaired function of residual mature B cells. This review focuses on a series of in vivo genetic studies that have begun to define the mechanism by which Btk regulates B‐cell development and function. The functional interactions between Btk and other signaling molecules defined by this approach are more complex than initially appreciated from in vitro biochemical and cell culture studies.

Btk plays a crucial role in the amplification of Fc RI-mediated mast cell activation by Kit

Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cγ1 activation and calcium mobilization, and to phosphorylate NFκB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments FcϵRI-mediated mast cell activation.

Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

Brutons tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle.

Constitutive membrane association potentiates activation of Bruton tyrosine kinase

Mutations in the nonreceptor tyrosine kinase Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Genetic and biochemical evidence implicates Btk as a key component of several B cell signaling pathways. Activation of Btk by a point mutation (E41K) within the PH domain (Btk*) results in fibroblast transformation and is correlated with increased membrane localization of Btk. When wild type Btk is activated by coexpression with Lyn, the tyrosine phosphorylated pool of Btk is highly enriched in the membrane fraction. To determine whether membrane association is sufficient to activate Btk, we targeted Btk to the plasma membrane using a series of fusion proteins including GagBtk, CD16Btk and CD4Btk. Constitutive membrane association greatly enhanced the ability of Btk to transform Rat2 fibroblasts in the presence of high levels of Src activity. All membrane targeted forms of Btk were highly tyrosine phosphorylated. Transformation required membrane localization, Btk kinase activity, transphosphorylation by Src family kinases, and an intact SH2 domain but not the PH or SH3 domains. These data suggest that membrane localization is a critical early step in Btk activation.

SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.