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IMMUNOLOGY Live T Cell Action in Lymph Nodes: Dating, Mating, Procreating Most people know about the macho side of T cells, which bravely seek out and destroy pathogens in our bodies or just as brazenly make an allergic reaction even worse. From left, Thorsten Mempel, Ulrich von Andrian, and Sarah Henrickson peered into the lymph nodes of living mice and found three phases of T cell activation. (Photo by Leah Gourley) In the ultimate reality show, new microscopic movies peek deep into lymph nodes located behind the knees of living mice and show how naive T cells prepare for their careers as warriors of the immune system. The T cells in training learn how to choose their battles in three distinct stages, according to a report in the Jan. 8 Nature from researchers in the lab of Ulrich von Andrian, HMS professor of pathology at the CBR Institute for Biomedical Research. In the lab, the three-step process of T cell activation has been dubbed "dating, mating, and procreating." "For the first time at a single cell level, you can actually see the immune response in a live animal." --Ulrich von Andrian For a T cell, one of the tricky parts of the job is to find the rare foreign antigen it was born to fight. This information comes from dendritic cells. Immature dendritic cells patrol tissues and engulf microorganisms, dead cells, and cellular debris, von Andrian said. Infections or inflammation send them scurrying to the nearest lymph node, maturing on the way as they ready their antigen alerts to present to those few T cells that can understand. Restless, naive T cells constantly prowl through the body, hanging around each lymph node for a day or two before moving on. An estimated 25 to 100 million different types of T cell circulate in the blood of each adult, but there are probably only 100 to 1,000 copies of any one type specialized to attack a particular invading pathogen, said postdoctoral fellow and first author Thorsten Mempel, unless a person's immune system has been boosted by a vaccine or previous identical infection. With those daunting odds, it is no wonder that fidgety T cells may have to keep pushing on to the next node in their search. "Now we've shown that once a T cell initially enters a node and meets a dendritic cell with an antigen it recognizes, it doesn't immediately establish a long-lasting relationship," Mempel said. "For the first few hours, T cells attach briefly, moving from one dendritic cell to the next and scanning for antigen. Only after this dating period do they start to form long-lasting relationships, the mating stage." Birds and the Bees The first eight hours of brief, serial encounters surprised the researchers. "They buzz around like bumble bees sampling flowers," von Andrian said. Meanwhile, dendritic cells shoot out their namesake tendrils and retract them, allowing more interactions with T cells. After this sampling phase, most of the mingling T cells settled down monogamously with each one's favorite dendritic cell. As seen in one-hour sample recordings, two thirds of the conjugates lasted longer than 30 minutes, and more than half lasted for an hour or more, according to analysis by a cell motility software program written by co-author Sarah Henrickson, a second-year medical student in the MD-PhD program. The researchers measured cytokines and other biochemical changes that eventually help the T cells find the site of inflammation and destroy infected cells. This mating stage, or phase two, lasted about 12 hours. One day after homing to a node, T cells wiggled free from their partner dendritic cells and rapidly divided into thousands of copies of themselves. These offspring eventually acquire full effector potential, von Andrian said, which allows them to charge off together to slay their dragons. Videos of the node show that T cells in this procreation phase returned to rapid motion and short phase-one style encounters with dendritic cells. "For the first time at a single cell level, you can actually see the immune response in a live animal," said von Andrian. "There seem to be certain preprogrammed steps. Each step has its own dynamics. At each phase, if we look on the surface of T cells, we see distinct markers likely to reflect distinct biochemical events within each cell." T Cell Tango The four-dimensional results from living animals (see http://cbr.med.harvard.edu/investigators/vonandrian/lab and select Videos) may reconcile two opposing beliefs about T cell activation studied in vitro, the researchers said. The traditional view held that once the T cell and dendritic cell recognized each other, they immediately connected for hours until the T cell reached an activation threshold that triggered it to propagate and turn into an infection-fighting effector T cell. Other studies also have proposed that the interface is formed by an immunological synapse that matures during contact. Four years ago, on the other hand, another research group reconstructed the lymph collagen matrix and instead found dynamic, transient interactions between T cells and dendritic cells. The new paper says both ideas may be right, but at different times. The researchers used CD8 T cells, and their preliminary work shows CD4 T cells behave similarly. They injected antigen-loaded dendritic cells from genetically identical donor mice, followed by an artificially high dose of matching T cells from other genetically identical donor mice. In an innovative twist, the researchers devised a way to synchronize T cell activity by letting the cells migrate into lymph nodes for only a short period before injecting an antibody that blocks subsequent T cell entry into lymph nodes. Eventually, the findings might have implications for developing more effective vaccines. "Most vaccines work at the level of the lymph node," von Andrian said. "Dendritic cells gobble up the antigen after it is deposited in a peripheral tissue by a doctor or nurse and carry it to the draining lymph node, where the immune response occurs." In the meantime, the new intravital microscopy techniques will allow the researchers to explore more details about the nature and timing of the finely choreographed activities inside living lymph nodes. --Carol Cruzan Morton