Volume 16 Number 3, SPRING 1999

t cells’ actions covert no longer

Researchers rush to adopt Stanford immunologist Mark Davis' strategy for keeping tabs on T cells as they fight disease.

BY MITCH LESLIE

A DYING CANCER PATIENT RECEIVES AN EXPERIMENTAL VACCINE INTENDED TO MOBILIZE THE IMMUNE SYSTEM TO BATTLE THE TUMOR. Within a few weeks, the cancer is melting away. * This outcome is no dream. Some cancer patients have entered remission after treatment with experimental cancer vaccines. But scientists have reacted cautiously to these apparent successes because they lacked the tools to monitor the activity of the T cells that combat cancer and infections. With only a blurry picture of the immune system's workings, it wasn't possible to distinguish cases of successful treatment from inexplicable remissions.

That should change now that a Stanford team led by immunologist Mark Davis, PhD, has devised a quicker and more accurate way to tally the number of T cells in a blood sample by labeling the cells with sticky fluorescent tags. Though announced nearly three years ago, the technique has entered wide use only within the last year, says Davis, a professor of microbiology and immunology and a Howard Hughes Medical Institute investigator. He predicts that the method will not only help guide the developers of cancer vaccines but it will also provide a bonanza of information on any disease that involves a T-cell response, including autoimmune diseases and viral diseases like AIDS.

T cells are among the most specialized cells in the body. Each one bristles with protein receptors that are specialized to recognize a particular antigen, or snippet of foreign protein. Antigens, which the immune system treats as signs of invasion, occur on pathogens like bacteria and viruses and on some kinds of cancers.

The traditional way to count T cells primed to apprehend a particular antigen is very crude. The technique, known as limited dilution analysis, entails steeping a blood sample in a solution containing the antigen, thus stimulating some T cells targeted against the antigen to multiply. After an incubation time of up to two weeks, the cells are tagged with fluorescent antibodies and counted with the fluorescence-activated cell sorter (FACS). This benchtop machine, fondly known as the "fax" machine, was invented in the 1970s by husband-and-wife team Leonard and Leonore Herzenberg, both professors of genetics at Stanford. The sorter funnels T cells through a tiny channel and counts the flashes as labeled cells are illuminated by a laser.

However, Davis notes, the results encompass all T cells in the sample and therefore provide an imprecise count. "Somewhere in that haystack of T cells there were some against your particular antigen," he says. The standard method has another serious shortcoming -- it's slow. Coaxing sufficient numbers of T cells to grow can take from three days to two weeks. And it does not reveal anything about whether the T cells are active or inactive.

Recognizing these failings, Davis and John Altman, PhD, then a postdoctoral researcher in Davis's lab and now at Emory University Medical Center in Atlanta, began searching for a more specific and faster assay. You could achieve the required specificity, they reasoned, by exploiting the affinity for antigens exhibited by T-cell receptors.

Receptors on a T cell will bind to their target antigen only if they "see" it attached to a molecule called the major histocompatibility complex (or MHC) that is found on the surface of cells. In effect, MHC is a billboard on which cells announce their health. If a cell is infected with a virus, it will hang viral antigens on its MHC molecules, signalling "Come and destroy me" to the body's T cells. Likewise, cancer cells often drape their MHC molecules with antigens that attract T cells.

Davis and Altman wanted to construct a chemical marker that would fasten firmly to T cells so they could be counted in the FACS machine. The researchers first tried simple tags made from an antigen stitched to an MHC molecule and capped with a fluorescent compound. But these tags bound weakly to the T cells and, as a result, would fall off easily. Next, the researchers constructed more elaborate markers consisting of two antigen-MHC complexes, but these too were prone to slip off. Their eventual solution to the problem was a tetramer made from four antigen-MHC complexes and the fluorescent molecule, a combination that clasped the T cells tightly.

It's possible to make tetramers that will lock onto almost any T cell, as long as the antigen recognized by that T cell has been isolated. But the technique offers other advantages, too. Instead of days or weeks, it requires only a few hours. What's more, tagged cells can be isolated for further study, after which they can even be injected back into the patient. Moreover, the speed of the analysis makes it possible to track the waxing or waning of T-cell numbers over the course of a single day. That information will allow scientists to better understand how the immune system mobilizes to fend off an infection. Scientists will also be able to check whether cancer vaccines actually stimulate the production of functional, anti-cancer T cells.

Using the new assay, Davis and colleagues have already made a surprising discovery about the body's response to cancer. When they analyzed blood samples from patients with metastatic melanoma, they found that half of the patients were producing numerous T cells targeted against the cancer. Why didn't the immune system in these patients purge the cancer? Once the T cells were isolated, the answer became apparent. For some reason, the cells were inactive and wouldn't attack the cancer, Davis and colleagues reported in the June issue of the journal Nature Medicine. Davis thinks this inhibition might reflect an inherent restraint against attacking one's own cells, something that cancer vaccine designers will have to overcome.

Though Davis and colleagues debuted their work in Science in 1996, the idea stirred little interest until last summer, when 12 papers applying the technique appeared within just two months, Davis says. Why the delay? Davis says there are two reasons. First is the natural skepticism toward new techniques. The other reason is that his lab has a reputation for doing technically challenging work, and other labs may have been hesitant to give the new technique a try. However, he points out that any lab already doing T-cell counts should be able to handle this procedure. SM