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Spotlight
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Laboratory of Cell-Mediated Immunity: Immunological Monitoring and Assay Development
It is well known that our immune system not only protects us against infectious diseases, but also plays a central role in protecting us against cancer and helping to fight cancer after it has developed.
NIH investigators are conducting numerous clinical trials in which a patient’s immune system has been “boosted” via a cancer vaccine. Unlike traditional vaccines, which are used to prevent disease, cancer vaccines are given after someone gets cancer. Proteins, peptides, or whole tumor cells are used as antigens to create a vaccine aimed at: 1) stopping or slowing the growth of cancer cells, 2) destroying cancer cells, or 3) keeping cancer from spreading to other parts of the body. This approach, known as immunotherapy (or biological therapy), has shown promise in treating some forms of cancer, including melanoma and lymphoma. Studies are also underway to help treat patients with kidney, breast, ovarian, prostate, colon, and rectal cancers. Selection and application of appropriate immunological assays (tests) to measure immune reactivity due to vaccination are essential. Correlating information gained from these assays to clinical outcome helps identify the potential protective ability of the vaccine.
Monitoring Patient Immune Responses
The Laboratory of Cell Mediated Immunity (LCMI), headed by Dr. Anatoli Malyguine helps integrate NIH’s clinical and basic research by testing, developing, and enhancing immunological assays that are used to evaluate a patient’s response to a particular vaccine. Part of the Clinical Services Program, SAIC-Frederick, Inc., the LCMI works closely with the NCI Vaccine Working Group Steering Committee (Dr. Jay Berzofsky, Chair), which is responsible for prioritizing and overseeing the development of immunological assays used in many vaccine clinical trials. Dr. Malyguine and members of his staff meet with the Steering Committee on a regular basis to present the lab’s latest testing and validation results. This feedback, in turn, assists the Steering Committee in evaluating and integrating new methodologies that are critical to this relatively new area of clinical research. Moreover, LCMI can serve as an expert, independent laboratory for monitoring clinical trials. A significant part of LCMI’s work is immunological monitoring of several cancer vaccine trials conducted by NCI researchers. LCMI analyzes samples from vaccinated patients with melanoma, lung cancer, lymphoma, HIV-associated malignancies, and others. LCMI also performs immunological assays for basic NCI/NIH research. The laboratory comprises highly trained personnel with expertise in the development, execution, and analysis of complex immunological assays.
Since its inception, the LCMI has focused much of its efforts on developing, validating, optimizing, and standardizing a variety of immunoassays, including standard tests and state-of-the-art techniques such as the ELISPOT (Enzyme-linked ImmunoSpot) assay. ELISPOT is an extremely sensitive test that provides clinical investigators with information on both the type of immune protein that is being released and the number of responding cells. Antibody-coated membranes are used to detect locally secreted immune proteins that have been released by individual cells during the patient’s immune response. This release of proteins from activated cells results in spot formation. At appropriate cellular concentrations, each spot formed represents a single reactive cell. The data obtained from this assay help determine whether a new vaccine has been successful in strengthening the patient’s immune response.
Assays Available to Clinical Investigators
Currently, the following assays approved by the Steering Committee are available to NIH investigators for clinical monitoring of human samples: peptide IFN (interferon)-γ ELISPOT, whole protein IFN-γ ELISPOT, autologous tumor IFN-γ ELISPOT, granzyme B (GrB) ELISPOT, cell proliferation, cytokine induction, cytotoxic T lymphocyte (CTL) induction, and 51Cr (chromium)-release. The LCMI has also adapted ELISPOT assays for mouse studies. Combining these assays helps the clinical investigator perform more comprehensive immunological monitoring of patients participating in cancer vaccine trials.
The peptide IFN-γ ELISPOT is used to measure cytotoxic T lymphocyte (CTL) reactivity to HLA.A2-binding, 9- or 10-mer peptides. The whole- protein IFN-γ ELISPOT, a unique assay developed by the LCMI, monitors immune responses to  vaccinations with whole proteins or peptides greater than 10-mer.
The desired outcome of cancer vaccination is to induce a potent T-cell response that can specifically recognize and eliminate the patient’s tumor cells in vivo. Accordingly, immunological assays that recognize native tumor cells (i.e., are tumor specific) may be more clinically relevant than assays that recognize just a particular tumor protein or peptide (i.e., are antigen specific). To address this issue, the LCMI developed an alternative approach, the autologous tumor IFN-γ ELISPOT assay, which monitors the patient’s immune response against his/her own whole tumor cells. This assay can be directly applied when patient tumor cells are available, when tumor-specific antigens have not been fully identified, or when whole or lysed tumor cells are used as the immunogen.
Another ELISPOT assay modification, the Granzyme B (GrB) ELISPOT, can be used for direct ex vivo monitoring of antigen-specific CTL. One main function of CTL is to recognize and kill tumor cells, thereby eliminating them from the body. A key mediator of target cell death is GrB, found in granules of CTL and natural killer (NK) cells. The release of this protein can be used to measure NK and CTL ability to kill tumor cells (i.e., cytotoxicity). LCMI testing has shown that the GrB ELISPOT is a superior alternative to the 51Cr-release assay (which measures the lysis of target cells) because it is more sensitive and uses fewer effector cells. In addition, unlike the IFN-γ ELISPOT, the GrB ELISPOT directly measures cytotoxic cell activity. The LCMI team has adapted this assay specifically for monitoring cancer vaccine trials.
Future Plans
The LCMI plans to further characterize the particular immune functions elicited in response to various vaccines. That is, utilize several assays in conjunction to better delineate the particular functional ability of tumor-specific T cells.
One novel way to better evaluate T-cell function is to study the release of multiple immune proteins in an ELISPOT assay within the same test well. Currently LCMI is developing dual-color ELISPOT assays that measure T-cell ability to produce IFN-γ and/or GrB. Cells that produce only IFN-γ will be represented by red spots, cells that produce only GrB by blue spots, and cells that produce both IFN-γ and GrB by purple spots. Alternatively, the proteins of interest can also be measured using fluorescence (red, green, and yellow).
In addition, assays that measure other molecules important for killing tumor cells, such as perforin, are now being optimized for single- or dual-color (GrB and perforin) analysis. LCMI staff members also have been working to develop a flow cytometric cytotoxicity assay to assess the cytolytic activity of human or mouse killer lymphocytes in vitro. This assay will allow measurement of cytotoxic lymphocyte activation and target cell death simultaneously in one sample. The method is non-radioactive, fast, sensitive, reliable, and allows evaluation of cell-mediated cytotoxicity using limited amounts of patient or animal cells.
From Emergency Room to Laboratory
Dr. Malyguine graduated from the First Leningrad Medical Institute (U.S.S.R.) in 1972. As a first year medical student, he became interested in cancer research and started research work in cancer immunology as a volunteer in the Institute of Cytology of the U.S.S.R. Academy of the Sciences, Leningrad (now St. Petersburg). As a result of his research, he published several scientific papers during his student years.
Dr. Malyguine began his medical career as an emergency doctor, where he continued his research; he says, “It was different from what I was planning to do, but it was in some respects better. With this kind of job you are not saving the world, but helping one person at a time. And during your shift you are the one responsible for the health and lives of thousands and thousands people in your service area, whatever happens. Combining this with research work was a very challenging and rewarding experience.”
After receiving his PhD in immunology in 1976, Dr. Malyguine served first as a staff scientist and then as a senior scientist at the Institute of Cytology. In 1989, he helped to organize the Laboratory of Clinical Immunology in City Hospital and directed this lab part-time.
In 1990, he accepted a visiting scientist position at Helsinki University, Finland, where he worked for the Department of Pathology in Dr. Timonen’s lab, studying human NK cell biology and monitoring clinical trials. “Taking a position in Helsinki, I came back from management to bench work. It was a very exciting time. Everything was different and new—people, language, resources, the general atmosphere,” says Dr. Malyguine.
In 1993, Dr. Malyguine moved to the U.S.A., working in the Department of Immunology, Duke University Medical Center, NC. While at Duke, Dr. Malyguine performed research in NK and T-cell biology and studied the role of these cells in xenotransplantation. “The problem of transplantation immunology seems to be the opposite of cancer immunology. In the first case, our goal is to suppress immunity, in the second to activate it. However, many aspects of these two disciplines are interwoven and represent, in fact, different sides of the same problem—to find efficient ways of modulating immune responses,” Dr. Malyguine explains. He joined the Clinical Services Program in 1997 as a senior scientist and in 2000 became a head of a new lab—the LCMI. “All of my previous career was directly or closely related to medicine and my current position seems to be a logical continuation of all I’ve done before,” he says.
Requesting LCMI Services
Although NCI clinical investigators are the primary requesters of LCMI’s services and expertise, this core laboratory is open to all NIH intramural investigators. To learn more about the LCMI, please contact Dr. Malyguine (amalyguine@ncifcrf.gov, 301-846-1890) or Susan Strobl, Lab Supervisor (sstrobl@ncifcrf.gov, 301-846-6922). To formally request services, go to the NCI Yellow Task Web site at http://web.ncifcrf.gov/campus/yellowtask/
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