Adam Zweifach - US grants

DESCRIPTION: The long term goal of this proposal is to understand the role of intracellular calcium ([Ca2+]i) signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virally infected cells and cancer cells, and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. CTL function is therefore of critical relevance to health and disease, and detailed knowledge of how CTLs kill is of key importance to understanding the etiology of viral diseases such as AIDS, cancer autoimmune disorders, and to preventing rejectio of transplanted organs. Therapeutic strategies may be suggested by detailed knowledge of CTL function. One of the two mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzyme contained in specialized CTL lytic granules into an area of close apposition formed with the target. Granule exocytosis is a two step process, involving relocation of lytic granules followed by their fusion, and is known to require increased [Ca2+]i caused by influx across the plasma membrane. However, the specific role(s) of Ca2+ in driving granule exocytosis is unknown. Critically, granule exocytosis has not been studied using the powerful physiological approaches that have advanced our understanding of exocytosis in other cell types. The specific aims of this proposal are to use patch clamp recording, capacitance measurements and digital video imaging techniques to: 1) determine the mechanism of Ca2+ influx in CTLs; 2) investigate the idea that Ca2+ channels are localized to create functional Ca2+ gradients; and 3) determine the role(s) played by Ca2+ in granule exocytosis. These specific aims will establish a firm physiological foundation for understanding the mechanism of this critical exocytotic event.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the role of intracellular calcium signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virus-infected cells and cancer cells and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. Understanding CTL function is therefore important for preventing and treating naturally occurring viral diseases such as AIDS and influenza, and viral diseases such as smallpox used as biological weapons. It is also important for understanding and treating cancers and autoimmune diseases. Finally, the ability to suppress CTL function is vital for successful organ transplantation. One of the main mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzymes contained in specialized lytic granules into an area of close apposition formed with the target. Granule exocytosis is known absolutely to require increased intracellular calcium caused by influx across the plasma membrane. However, the specific role(s) of calcium in granule exocytosis are unknown, the number of calcium-dependent steps is unclear, and molecules that confer calcium-dependence have not been identified. The specific aims of this proposal will use a battery of techniques, including novel fluorescence imaging methodologies we have developed, to: 1) determine whether bulk cytosolic calcium increases are sufficient to support granule exocytosis, or whether higher-than-cytosolic calcium increases in microdomains are required. 2) Investigate the calcium dependence of granule reorientation and of reorientation-independent exocytosis. 3) Determine whether immunological synapse formation is calcium dependent, and acts as a slow step in granule reorientation. 4) Investigate the role of the calcium-dependent phosphatase calcineurin in granule exocytosis. These studies will significantly further our understanding of the role of calcium influx in lytic granule exocytosis.

An award has been made to the University of Connecticut under the direction of Dr. David A. Knecht to acquire a spinning disc confocal microscope to be used for observing living cells in real time. The instrument will allow real-time, fast imaging of cells to study a variety of cellular processes, including motility, auxin transport, vesicular transport, and auxin localization in plants, slime molds, and fish. The microscope will increase viability of cells and allow observations to be made over long periods of time. The institution has strong record in several programs for recruiting underrepresented groups to science, and the instrument will enhance the experiences of students in these programs. Most of the users of the confocal microscope will be graduate students, and undergraduate and high school students will be given demonstrations with the instrument.

DESCRIPTION (provided by applicant): Target cell killing by cytotoxic T lymphocytes (CTLs) is critical for the immune response to viruses and tumors, is involved in transplant rejection and contributes to autoimmune disease pathogenesis. A key mechanism CTLs use to kill target cells is secretion of cell-killing agents from specialized lysosomal lytic granules. Screening a small molecule library for compounds that inhibit lytic granule exocytosis would 1) serve as an assay for blockers of the known signals that control the process; 2) reveal the presence of and define chemical probes for as-yet-unknown signals and 3) lead to novel immunomodulatory drugs that could enhance our ability to transplant organs and stem cells and to treat autoimmune disorders. CTL granule exocytosis can be monitored using fluorescently-labeled antibodies to detect the externalization of lysosome-associated membrane protein 1 (LAMP-1) from lytic granules to the plasma membrane in a simple no-wash assay protocol suitable for flow cytometry. We plan to capitalize on this simple assay together with the unique HTS flow cytometry capabilities at the University of New Mexico Center for Molecular Discovery (UNMCMD) to establish a phenotypic screen and to discover chemical probes that inhibit granulocyte exocytosis. We propose three specific aims. In the first, we will optimize assay conditions, determine DMSO tolerance, and apply repeated measures tests to assess assay robustness. We will measure in a low-throughput format dose-response curves for known inhibitors of different signaling pathways involved in granule exocytosis, then determine whether and at what concentrations these inhibitors can reliably be detected in a mock small-scale trial of the assay. In our second aim, also to be conducted in Year 1, we will transfer the assay to the UNMCMD, perform the validation steps needed to confirm assay performance in a high-throughput format, then screen the Prestwick Library of pharmaceuticals, a 7-plate validation set from the Molecular Libraries Small Molecule Repository (MLSMR) and the ICCB library library of 480 known bioactive compounds. We believe that completion of this aim by itself could substantially advance our knowledge of the signals controlling granule exocytosis. Finally, in year 2 (and of course contingent on success in aims 1 and 2) we will conduct the full screen of compounds in the MLSMR at the UNMCMD. PUBLIC HEALTH RELEVANCE: Cytotoxic T cell lytic granule exocytosis is important for killing virus infected cells, tumor cells and transplants, and is also involved in the etiology of some autoimmune disorders. Identifying small molecule inhibitors of granule exocytosis via high-throughput screening could lead to new clinically-important drugs that could be used as immunosuppressive agents, and could also provide important insights into the signaling pathways that control the process. This application is designed to determine whether a simple and robust high-throughput assay of lytic granule exocytosis based on the externalization of lysosome-associated membrane protein can be developed. If it can, we will use it to screen a large scale compound library for inhibitory compounds.

Therapies designed to enhance immunity are becoming important components of cancer therapies, and could be used to increase anti-viral immunity and augment the efficacy of vaccines. Many strategies focus on biologic agents such as recombinant cytokines or monoclonal antibodies. However, the success of imiquimod and lenalidomide, the two available small molecule immune-enhancers, suggests that additional agents of this type could be useful. One reason that there are so few small-molecule immune enhancers is that there has historically been no way to conduct high-throughput screening (HTS) to find them. Imiquimod and thalidomide analogs were found to enhance immune responses by accident. We have developed an assay that should finally make it possible to screen large compound libraries for immune-enhancing small molecules. The assay is a powerful revision of an HTS that we applied successfully to the NIH's Molecular Libraries Small Molecule Repository. In the new enhanced assay, we stimulate TALL- 104 human leukemic cytotoxic T lymphocytes (CTLs) with beads coated with anti-CD3 antibodies, generating submaximal exocytosis specifically in the bead-bound population. We monitor exocytosis by measuring binding of a fluorescently-labeled antibody against LAMP-1 (CD107a) to cells using flow cytometry. This allows us to conduct a no-wash assay that can detect compounds that enhance of exocytosis and discriminate them from compounds that cause exocytosis on their own. TALL-104 CTLs serve in our strategy as both a model of an immunologically-relevant cell type as well as a surrogate for other immune cell types for which it would be difficult to devise HTS-ready assays. Conducting a screen with TALL-104 cells followed by assessment of the effects of hits on other important immune functions will likely lead to the identification of therapeutic leads or to probes that could be used to identify novel cellular targets that can be exploited to produce immune enhancement. Our three aims are designed are to 1) develop and optimize a highly sophisticated assay that will assess the effects of treating cells with compounds for different lengths of time in a single read step, then validate the optimized assay by screening the Prestwick Compound Library; 2) develop a set of secondary tests to examine the effect of hits on target cell killing by CTLs and representative functions of helper T cells, B cells and dendritic cells and 3) validate the HTS/ follow-up strategy by screening the Broad Institute's DOS informer collection of ~10K compounds. Completing our aims will provide an assay and follow-up workflow suitable for screening larger compound collections, as well as generating a number of candidate molecules obtained from screening the Prestwick and Broad collections that can be pursued in future experiments.

Immunosuppressants are absolutely essential for successful organ transplantation and are useful in the treatment of autoimmune disorders. Analyzing their mechanism of action can, as in the case of cyclosporine and calcineurin, yield important insights into basic features of T cell biology. We recently embarked upon an ambitious three-step project intended to use chemical biology approaches to identify unknown pathways involved in lymphocyte function. The over-arching rationale underlying the project was that compounds that inhibit T cell activation and work via unknown molecular mechanism (MMOA) could be developed into chemical probes that could be used to identify novel cellular targets, which would reveal currently-unknown aspects of basic T cell biology and might become the basis for new classes of immunosuppressant agents. The first step of the project- screening the NIH's Molecular Libraries Small Molecule Repository of ~375,000 compounds and identifying compounds with unknown MMOA- succeeded. We monitored lytic granule exocytosis using TALL-104 human cytotoxic T lymphocytes as a model, measuring externalization of LAMP-1/ CD107a using flow cytometry. Among hits with unknown MMOA was 2-N-[(2-methoxyphenyl)methyl]-4-N-[(4- propan-2-ylphenyl)methyl]thieno[3,2-d]pyrimidine-2,4-diamine, CID 49792547, which is the subject of this application. This compound inhibits lytic granule exocytosis with potency in the micromolar range, but does not work via any of the mechanisms we tested. It inhibits IL-2 production by Jurkat human leukemic T cells, confirming that has broad immunosuppressive activity. It is a drug-like molecule that is amenable to synthesis and the generation of diverse analogs. The project's intended second step was to generate analogs for structure-activity analysis, then use that information to design chemical probes to use in the third and final step, applying affinity-based and/or photo- crosslinking approaches to identify the unknown target that underlies the compound's activity. However, Chemistry Center support was withdrawn when the NIH MLPCN program ended. This application for an R03 is intended to allow us to continue to pursue the overall goals of the project by creating probes for target identification. We will create analogs of CID 49792547, then test their effects on lytic granule exocytosis, IL-2 secretion and toxicity. This information will allow us to identify the highest possible affinity analogs and will reveal sites that can be used to attach linkers which will allow the compound to be coupled to biotin for creation of an affinity matrix and to bifunctional photo-crosslinking/ click-chemistry groups that can be used to label target proteins. Future efforts will be directed towards target identification using probes developed in this application and in a companion application submitted previously focused on another compound.