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 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 multiple human disorders including primary immunodeficiencies, autoimmunity and cancer. 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. We are particularly interested in how these pathways affect responses to infectious diseases and immunization.

 Research:
I. Tec Kinases: Over the last 12 years, we have studied members of the Tec family of tyrosine kinases. Mutations affecting the prototypical member, Btk, are responsible for the human genetic disorder X-linked agammmaglobulimemia, characterized by abnormal B cell development and function. Confirming the importance of these kinases, a recent report described two siblings with a profound lethal immunodeficiency who had mutations affecting Itk, a Tec kinase expressed in T lymphocytes; polymorphisms in the Itk promoter have also been associated with increased risk for asthma.
 We have shown that mutations affecting Tec kinases expressed in T cells, Itk and Rlk, impair T lymphocyte development and function in mice and alter responses to infections and allergic challenges in vivo. In addition to their recognized roles in T cell receptor induced activation of phospholipase-c gamma and Ca++ mobilization, we have shown that the Tec kinases play important roles in regulating the actin cytoskeleton and cell adhesion, which are critical for T cells to exert 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. 
Over the last two years, we have further examined the role of Tec kinases in CD4+ T helper cell differentiation and regulation of patterns of cytokine production important for distinct types of immune responses, including their roles in regulating the function and differentiation of TH17 cells, an effector cell subclass important for responses to extracellular bacteria and which contribute to the pathology of autoimmune and inflammatory disorders, including asthma. In work carried out in this period, we have further examined the role of Tec kinases in regulating the balance between Th17 and regulatory T cells. We are also evaluating the potential role of Itk in responses to Epstein Barr Virus (EBV), given the description of Itk deficiency in patients with fatal infectious mononucleosis. 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), characterized by fatal infectious mononucleosis, B cell lymphomas, and antibody defects. SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We previously generated mice deficient in SAP and found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. 
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 are T cell intrinsic, ie SAP deficient T cells fail to provide essential signals to B cells for generating germinal centers and long-term antibody responses, which are critical for successful vaccination. In collaboration with Dr. R. Germain of NIAID, we used intravital microscopy to show that SAP-deficient CD4+ T cells have a selective defect in adhesion to B cells in vivo and in vitro, preventing them from delivering the contact-dependent signals required for B cells to form germinal centers and generate long-term humoral responses. This work, (Qi et al, Nature, 2008), 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. 
Although our studies are primarily performed in mice, our results have provided the basis for several confirmatory studies showing similar defects in germinal centers, long term antibody and B cell memory responses in XLP patients. Moreover, it provided potential insight into the B-cell centric phenotypes in XLP, including defective T cell help for B cell antibody responses and an inability of T cells and NK cells to kill EBV-infected B cells (Schwartzberg, Nat Rev. Immunol 2009). In unpublished work, we have confirmed these adhesion defects in cells from XLP patients.
 We are continuing this work, focusing on two major areas. The first is to evaluate the role of SAP in regulating interactions between T and B cells and whether such defects account for the abnormal responses to infection with EBV (which infects B cells). The second area focuses on the role of SAP in the development and function of Tfh cells, a helper T cell population required for providing signals to B cells for germinal center formation.
We have used these mice to help show requirements for the differentiation of Tfh cells and their cross-regulation with other T cell lineages (Lu, K et al, In Press, Immunity). Understanding the cellular interactions and signals defective in these mice is therefore of high importance for understanding the pathophysiology of this disease as well as the requirements for successful vaccine development.