May 1998

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Sarah Goodwin, 404/727-3366 -

Using novel assays described as the new "gold standard" for measuring specific immune responses to viruses, Emory University School of Medicine researchers have found that the response of CD8+ T cells, which provide the front line of protection against invading viruses, is considerably larger and much more targeted to specific viral antigens than scientists previously believed.

Although it is well known that T cells multiply in the body in response to viral infection, it has been difficult to measure the number of cells and exactly what they are responding to. The most common assay used to measure the response, the limiting dilution analysis (LDA) has led scientists to believe that only a small fragment (1 percent to 5 percent) of activated CD8+ T cells are responding to specific viral antigens, and that most of the T-cell response is a general, non-specific one.

Rafi Ahmed, Ph.D., Georgia Research Alliance Eminent Scholar in the Emory Department of Microbiology and Immunology and director of the Emory Vaccine Center; Kaja Murali-Krishna, Ph.D., a postdoctoral dellow in Dr. Ahmed's laboratory; and John Altman, Ph.D., Emory assistant professor of microbiology and immunology, used three novel assays to test the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) in mice. They found that more than 70 percent of the activated CD8+ T cells were responding to specific antigens of LCMV - a number they termed "remarkably high," and one that was 20 to 100 times greater than numbers based on LDA testing.

Based on these results, the Emory investigators calculated that virus-specific CD8+ T cells can rapidly expand >30,000 fold (about 15 divisions) in one week with an estimated doubling time of six to eight hours during the peak phase of the response.

The research was reported in the February issue of Immunity. Nobel laureate Peter C. Doherty, in a commentary in the April 10 issue of Science, says that the new findings have "triggered a revolution" in our understanding of the virus-specific T cell responses by "finally developing accurate methods" to measure these responses.

"The size of the antiviral response is much greater than our previous estimates, and radically changes our perception of the T cell response to viruses," says Dr. Ahmed. "This research should prove very important for vaccine development, because you want a vaccine to induce a good response, and this research allows us to see directly what a good response is."

Dr. Ahmed believes the findings about virus-specific responses will most likely apply to viral infections in general, including infection with EBV (infectious mononucleosis), HIV and human T cell leukemia virus type 1 (HTLV-1), all of which are characterized by massive increases in the number of CD8+ T cells. "It is likely that most of the expanded CD8+ T cells in these infections are specific to those particular viruses also," he says.

One of the assays used in the study - tetramer staining - was developed by Dr. Altman while a researcher in the laboratory of Dr. Mark Davis at Stanford University. In the tetramer technique, small peptides (fragments of viral protein) of the virus are placed inside an MHC Class I (major histocompatibility complex) molecule. MHC Class I molecules are proteins that "present" pathogens to T cell receptors, which in turn recognize the pathogens and attack them. A tetramer consists of four MHC molecules containing particular viral peptides. Labeled tetramers are then applied to a mixture of white blood cells and bind only to those CD8+ T cells that are specific for the MHC plus viral peptide combination.

"The beauty of the tetramer technology is that it can be applied to study T cell responses to any pathogen, from influenza to HIV to malaria. And tetramers give you both a more accurate and a much more rapid picture of the immune response," Dr. Altman explains. In his Science article, Dr. Doherty says that tetramer staining is "set to be the gold standard for quantifying virus-specific CD8+ T cells."

The Emory researchers also tested the CD8+ T cell response using sensitive assays that measure the production of interferon-gamma, a cytokine (a small protein secreted from a cell) produced by virus-specific T cells that is important in controlling the infection. The interferon-gamma assay also demonstrated a strong, virus-specific response by CD8+ T cells.

Studies using the LDA technique have underestimated the virus-specific T cell response, explains Dr. Ahmed, partly because it requires culturing the cells for one to two weeks, and only T cells that are capable of dividing and surviving for that long are counted. Since activated T cells are prone to die when restimulated with viral antigens, many of the antigen-specific T cells either die or do not divide enough to be counted.

After the initial CD8+ T cell viral response, about 95 percent of the T cells die and the remainder persist to become memory cells poised to respond to future invasions by the same virus. The assays used in the Emory study were also able to detect memory CD8+ T cells specific to the LCMV virus more than one year after infection.

Dr. Altman's initial findings have confirmed this prediction for HIV infection and he continues to actively pursue investigations of the CD8 T cell response to HIV in chronically infected patients on antiretroviral therapy.

"One of the hardest things to do is to identify T cells responding to a particular pathogen," explains Dr. Ahmed. "This work greatly revises our thinking on the dynamics of T cell activation and will lead us to re-examine our models of viral-induced T cell proliferation."

This research was supported by grants from the National Institutes of Health and the American Cancer Society.


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