The normally quiescent immune system has evolved to respond rapidly and specifically to the wide variety of infectious agents. During the initiation of a response, antigens (unique proteins, lipids or carbohydrates) of pathogens are recognized by specific antigen receptors on T and B lymphocytes (TCR and BCR, respectively). Such recognition events are converted into biochemical signals that initiate cellular activation that contribute to effective immune responses. When T cells are activated, they produce lymphokines (soluble hormones of the immune system) or develop the capacity to kill infected cells. Similarly, the activation of B cells leads to the production of specific antibodies that protect us from extracellular pathogens.
We are interested in understanding the biochemical signals that are initiated by TCR and BCR that induce rare antigen-specific cells to proliferate and to exert their potent effector functions. We would like to know how these biochemical signals are controlled, in order to avoid or control autoimmunity and tissue damage. Understanding the signaling mechanisms responsible and their functions may lead to the development of new therapies for pathological immune responses (e.g., asthma, lupus, or transplant rejection).
Although a number of different molecules are required for an effective T cell response, the TCR plays the central role in antigen recognition. This is an extraordinarily complex structure. It consists of an α/β-chain disulfide-linked heterodimer that recognizes antigen. The α/β-chains are associated with invariant chains of the CD3 complex (γ, δ, and ε) and a ζ-containing dimer that are responsible for initiating intracellular signals.
The CD3 and ζ chains communicate with intracellular signal transduction machinery via a 17–amino acid sequence motif (termed ITAM, for immunoreceptor tyrosine-based activation motif) that couples the TCR to cytoplasmic enzymes involved in signal transduction. ITAMs are also contained within other receptors involved in antigen recognition on natural killer cells, B cells, mast cells, and basophils, thereby representing a common signaling mechanism within the hematopoietic lineage. Interestingly, membrane proteins of some viruses, including the Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, contain ITAMs, allowing them to usurp the host lymphocyte-signaling machinery to their benefit.
How do ITAMs initiate signals? Protein-tyrosine phosphorylation, which can modify the function or location of proteins in the cell, is the earliest event associated with TCR stimulation. We showed that ITAMs interact sequentially with intracellular protein-tyrosine kinases (PTKs), enzymes that can catalyze the transfer of phosphate groups to tyrosine residues of cellular proteins. Lck associates with the cytoplasmic domains of the CD4 and CD8 coreceptors which colocalize with the TCR during antigen recognition. Lck initiates TCR signaling by phosphorylating the CD3 and ζ-chain ITAMs. Phosphorylation of the ITAMs creates binding sites for members of a second family of PTKs that includes Zap70. Zap70, which is recruited to the TCR, is then phosphorylated and activated by Lck. In collaboration with John Kuriyan's lab (HHMI, University of California, Berkeley), we have solved the structure of Zap70 in the autoinhibited conformation. Our structural data and more recent functional studies suggest that binding to a doubly phosphorylated ITAM induces a conformational change in Zap70 that facilitates its activation via phosphorylation by Lck.
Zap70, identified and cloned by our lab, is normally only expressed in T cells and natural killer cells. Its importance is highlighted by the human severe combined immunodeficiency syndrome that results from inactivating Zap70 mutations. Spontaneous or induced mutations of Zap70 that decrease its function in mice alters T cell development and leads to autoimmune diseases resembling rheumatoid arthritis or systemic lupus erythematosus. The expression of Zap70 in approximately 50 percent of cases of human chronic lymphocytic leukemia, a B cell malignancy, is associated with a poor prognosis.
These studies of Zap70 in various human diseases or mouse models of disease suggest that Zap70 may be a valid therapeutic target. However, no specific inhibitor of Zap70 exists. In collaboration with Kevan Shokat's lab (HHMI, University of California, San Francisco), we have developed a model small molecule inhibitor for a mutant form of Zap70 that we have substituted for wild-type Zap70 in cell lines and in mice. Inhibition of Zap70 catalytic function blocks most, but perhaps not all, T cell responses to antigen. We are beginning to define the kinase-dependent and -independent functions of Zap70 in various experimental models. Our studies have provided new insights into how T cells kill other cells (see figure) and the developmental transitions that occur during T cell development. We are poised to determine whether inhibition of Zap70 kinase function might be useful therapeutically in autoimmunity, asthma, or transplantation.
The TCR-regulated signaling pathway is not inert in the unstimulated T cell. It is tonically active, balanced by the action of positive and negative regulators of signaling. As must be evident, the activity of PTKs is tightly regulated. CD45, a very abundant transmembrane protein-tyrosine phosphatase (PTPase), plays a critical role in regulating Lck and is required for TCR signaling; CD45 functions to dephosphorylate the carboxyl-terminal negative regulatory site of Lck. This negative regulatory site in Lck and that of all Src-family kinases is phosphorylated by the cytoplasmic kinase Csk. To study this dynamic regulation of the CD45 phosphatase and the Csk kinase, we have developed a chemical genetic approach to block Csk function, as we did for Zap70. These studies suggest that the opposing actions of CD45 and Csk help to establish a dynamic basal state poised to resist inappropriate cell activation but to respond rapidly and robustly to appropriate stimulation of antigen receptors.
We are also studying the function of other receptor-like PTPases in T and B cells, including CD148 and PTPα. Recently, we have inactivated CD148 and find that it is functionally important in regulating ITAM-dependent receptors. Our studies suggest that the structurally distinct CD45 and CD148 PTPases have overlapping functions in regulating Src-family kinases in B cells and in macrophages. In collaboration with Yotis Senis and Steve Watson (University of Birmingham, U.K.), we showed that platelet responses to collagen via GPVI, an ITAM-coupled receptor, are impaired in CD148-deficient mice. This results in defective thrombosis and increased bleeding in the deficient mice. Our ongoing studies are aimed at understanding the unique functions of the phosphatases and their regulation in various hematopoietic cell lineages.
The complex activation of lymphocytes is regulated in extraordinarily diverse ways. We are beginning to understand superficially the biochemical events that control lymphocyte responses. With such understanding, insights into the pathogenesis of human disease will emerge and effective therapies will follow.
Portions of this work are supported by grants from the National Institutes of Health, the American College of Rheumatology, and the Arthritis Foundation.
As of February 28, 2014