Signal Transduction Events Involved in Lymphocyte Activation and Differentiation
Summary: Arthur Weiss studies the biochemical signal transduction events that control lymphocyte responses. He is particularly interested in the mechanisms involved in signal transduction by antigen receptors, and how abnormalities in these mechanisms can lead to autoimmune diseases.
To protect us from infectious organisms, the normally quiescent immune system has evolved to respond rapidly and specifically to the wide variety of pathogens. 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 must be converted into biochemical signals that initiate cellular responses. The events that lead to the activation of T lymphocytes are critical to protect us from intracellular pathogens. 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 controlled by antigen receptors 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 extraordinarily complex structure consists of an α/β-chain disulfide-linked heterodimer, which is noncovalently associated with the invariant chains of the CD3 complex (γ, δ, and ε), and a ζ-containing dimer. The α/β chains recognize antigen. The CD3 and ζ chains initiate intracellular signals.
We previously showed that 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. Multiple ITAM copies are present in the ζ chain and single copies are present in each of the CD3 chains, perhaps representing a strategy for signal amplification. 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 herpesvirus, contain ITAMs, allowing them to communicate with and take advantage of the host lymphocyte-signaling machinery.
How do ITAMs initiate signals? Protein-tyrosine phosphorylation, which can modify the function or location of proteins in the cell, is one of the earliest events 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, a Src-family member that associates with the cytoplasmic domains of the CD4 and CD8 coreceptors, 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 consists of Syk and ZAP-70. ZAP-70, which is recruited to the TCR, is then subjected to activation via its phosphorylation by Lck. In recent studies, done in collaboration with John Kuriyan's lab (HHMI, University of California, Berkeley), we have solved the structure of ZAP-70 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 ZAP-70 that facilitates its activation via phosphorylation by Lck.
ZAP-70, 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 ZAP-70 mutations. Spontaneous or induced mutations of ZAP-70 that decrease its function in mice can alter T cell development and lead to autoimmune diseases resembling rheumatoid arthritis or systemic lupus erythematosus. ZAP-70 also seems to play an important role in the behavior of the most common form of adult leukemia, chronic lymphocytic leukemia (CLL). In collaboration with Thomas Kipps (University of California, San Diego), we have shown that ZAP-70 protein is expressed in about 50 percent of CLL cases and that its expression, at a certain threshold level, is associated with a poor prognosis. We have developed a simple blood test, based on ZAP-70 expression levels, that is now frequently used clinically for prognostic purposes. Our studies suggest that ZAP-70 heightens signal transduction by the ITAM-containing BCR in CLL cells and may, thereby, alter the biologic behavior of these cells.
All of these studies of ZAP-70 in various human diseases or mouse models of disease suggest that ZAP-70 may be a valid therapeutic target. However, no specific inhibitor of ZAP-70 exists. Recently, in collaboration with Kevan Shokat's lab (HHMI, University of California, San Francisco), we have used a model chemical genetic inhibitor approach in which we can specifically inhibit the kinase function of a mutant allele but not the wild-type allele of ZAP-70 that we have expressed in cell lines and in mice. Inhibition of ZAP-70 catalytic function blocks most, but perhaps not all, T cell responses to antigen. We are now well positioned to define the kinase-dependent and -independent functions of ZAP-70 in experimental models and to determine whether inhibition of ZAP-70 kinase function might be useful therapeutically in autoimmunity, asthma, or transplantation. At the same time, based on this information and in collaboration with the Kuriyan lab, we are undertaking an effort to block the activation of wild-type ZAP-70 in T cells with small molecules by inhibiting the conformational change of ZAP-70.
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 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 are using various alleles of CD45 and a chemical genetic approach to block Csk function, as we did for ZAP-70.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.
As of May 30, 2012