Genetic and Biochemical Approaches to Tyrosine Kinase Function

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Summary: Our laboratory studies signal transduction in the immune system with a focus on tyrosine phosphorylation, non-receptor tyrosine kinases and downstream effector molecules. These proteins are required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth, differentiation and development involved in autoimmunity, immunedysregulation and cancer. Mutations affecting such signaling pathways have been found to give rise to multiple human disorders including primary immunodeficiencies. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how signaling molecules and pathways contribute to normal function of cells in the immune system and their dysfunction in disease and how manipulation of signaling pathways can aid in the development of therapeutics. Research: I. Tec Kinases: Over the last 10 years, we have studied members of the Tec family of tyrosine kinases. Mutations affecting the prototypical member of this family, Btk, are responsible for the human genetic disorder X-linked agammmaglobulimemia and Btk is required for normal B cell development and function. We have shown that mutation of Tec family kinases expressed in T cells, Itk and Rlk, can also severely impair T lymphocyte development and function in mice and influence responses to infections in vivo. In addition to their previously recognized roles in antigen receptor induced activation of phospholipase-c gamma, a key enzyme required for Ca++ mobilization, we have shown that the Tec kinases play important roles in regulation of the actin cytoskeleton and cell adhesion in T lymphocytes, which are critical for T cells to exert proper effector cell functions. We have also shown that mutations of these kinases affect patterns of cytokine production by CD4+ T helper lymphocytes and the ability of mice to respond to distinct types of infectious diseases. As an extension of these studies, we have noted that many signaling pathways regulated by the Tec kinases are important for infection of T cells by HIV. Hypothesizing that Itk may affect HIV infection, we examined this issue by inhibiting Itk expression or function using siRNA, dominant-negative mutants of Itk or a published inhibitor of Itk. Indeed, we found that inhibition of Itk impairs HIV infection of CD4+ T cell lymphocytes. Inhibition of Itk affected multiple stage of HIV replication, including viral entry, viral gene expression and virion production. This study, which was published this year in PNAS (PMCID: PMC2365562), provides a model for how cellular proteins can be therapeutic targets for inhibiting HIV replication, an important issue since viral targets are subject to the rapid development of resistance. In continuing work, we are also examining the effects of mutations affecting Tec kinases on T cell development and differentiation. We have recently found that mutation of the Tec kinases affects development of conventional T lymphocytes and that most of the CD8 cells that develop in these mice resemble a normally rare population of innate-like T cells that are important for immediate early responses to bacterial infections. Since these mice develop a large number of innate-type T cells, we have used them to help define the requirements for the development of conventional and innate type lymphocytes. As an extension of this work, recently published in Immunity PMCID: PMC2377064, we have provided evidence that these innate-type phenotypes result from a distinct selection pathway on hematopoietic cells in the thymus. We have further uncovered a critical role for signaling from SLAM family receptors of costimulatory receptors and the adaptor molecule SAP (see below), which are expressed on hematopoietic cells, in this process. We have further examined the role of Tec kinases in CD4+ T helper cell differentiation. We and others have previously demonstrated that Itk, the Tec kinase most highly expressed in T lymphocytes, is required for the production of TH2 cytokines and in vivo TH2 responses, a subclass of T cell responses important for responses to certain parasitic infections as well as allergens. In contrast, expression of Rlk/Txk, a related kinase, has been linked to TH1 responses (which are important for inflammation and responses to intracellular pathogens). In recent work using transgenic mice that over-express Rlk/Txk on the Itk-deficient background, we have provided evidence that these effects are secondary to the patterns of expression of these kinases, where Itk is normally the predominant Tec kinase expressed in TH2 cells (J. Immunol. in press). Itk is an active target for potential therapeutic development for asthma, a TH2 mediated disease; these findings help further delineate its role in TH2 cytokine regulation. II. SAP: As an extension of these studies, we are examining other signaling molecules involved in T helper cell differentiation including SAP, a small SH2 containing adaptor protein, mutations of which are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We had previously generated mice deficient in SAP and have found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. SAP is required for TH2 cytokine expression in response to TCR stimulation. We have shown this function requires its ability to recruit Fyn. In the last several years, we have demonstrated that SAP also plays a critical role in responses to immunization. We further showed that the impaired antibody responses in these mice is secondary to a defect in CD4+ T cells, ie SAP deficient T cells fail to provide an essential signal to B cells for generating germinal centers and long-term antibody responses, a critical step for the development of successful immunization and vaccine development. Understanding the cellular interactions and signals that are defective in these mice is therefore of high importance or understanding the requirements for successful vaccine development. To understand the defect in T cells in the SAP-deficient mice, we have continued to examine T cell function and biochemistry of T cell activation in cells from SAP-deficient mice. In collaboration with Dr. R. Germain of NIAID, we have used intravital microscopy to show that SAP-deficient CD4+ T cells have a selective defect in adhesion to B cells, preventing them from delivering the contact-dependent signals required for B cells to form germinal centers and generate long-term humoral responses. We have further confirmed this result using adhesion assays in vitro and shown that the ability of SAP to mediate cell adhesion is independent of its ability to signal via the Fyn kinase. This work, which is in press at Nature, has provided new insight into the requirements for T:B cell interactions and cellular collaborations required for long-term antibody responses, a critical feature of successful immunization. Moreover, it provides potential insight into several of the B-cell dependent phenotypes in XLP, including humoral immune defects and an inability of T cells and NK cells to kill EBV-infected B cells. III. Mouse genetics: Most of our studies rely on the generation of gene-targeted mice. In an effort to increase efficiency of generating and screening for gene-targeted mice, we have shown that quantitative PCR is an efficient screening tool for homologous recombination and have used this technique to highlight potential problems using full-length Bacterial artificial chromosomes for gene-targeting (Nucl. Acids Res, 2008, Pubmed citation in progress by journal).