Though we think of them as blood cells, lymphocytes spend much of their time in distinct locations in the body. Now teams at three University of California schools have for the first time captured, in vivo, immune cells in motion as they differentiate and respond to antigen within these compartments.

The key technology underlying the two papers: two-photon microscopy. In two-photon microscopy fluorophores are excited simultaneously by two photons of twice the wavelength of the single, smaller-wavelength photons used in traditional confocal microscopy. Longer wavelength photons have lower energies and penetrate further into tissue than short wavelength photons, making the technique ideal for imaging deep into live tissue samples.

"Unlike any other imaging modality, we can see directly into the lymph nodes at some depth. So we can track the cells as they move about, say within 400 microns of the surface of the tissue," says Michael Cahalan of UC Irvine, an author on one of the papers.

In one study, Ellen Robey and colleagues at UC Berkeley used two-photon microscopy to study the migration of developing T cells (thymocytes) in intact mouse thymuses.[1] During development, thymocytes receive signaling cues that determine if they will mature to become killer- or helper-T cells or undergo apoptosis. "We know that when cells get signaled to mature, they move from the [cortex] to the [medulla], but we haven't had any information about the details of that process and how it occurs in real time," says Robey.

Robey's team found that most thymocytes in the region of the cortex containing immature cells move slowly and nondirectionally, whereas a small fraction moves quickly and directionally. "When we analyze [the directional cells'] direction we find that most of them are moving towards the center of the tissue towards where the medulla would be," Robey explains. The researchers posited that the fast-moving cells were those selected to mature, and confirmed this in transgenic mouse models.

Sebastian Amigorena of the National Institute for Health and Medical Research (INSERM) in Paris, points out that although two-photon microscopy has been used for several years to study the movement of developing thymocytes, Robey's paper was the first to show directional movements of lymphocytes in vivo. "The technology ... is more precise and developed, but it's not revolutionary. What they observed, however, is very new and important," Amigorena says.

Previous experiments either exposed cells in vitro to a chemokine that caused them to migrate, or looked at cells in tissue slices, inferring migration patterns from the positions of cells at fixed time points. These new studies, however, used tissue explants maintained at body temperature and perfused with an oxygen-rich medium that keeps the tissue healthy and alive. Robey says results from experiments using such explants have been shown in the past to correlate well with data from experiments in which the tissue to be studied is surgically exposed in a live, immobilized animal.

In the second article, Cahalan collaborated with Jason Cyster of UC San Francisco, to study the movement of T cells and B cells in lymph nodes after B-cell activation.[2] Previous studies showed that before activation, lymphocytes move in a random walk, Cahalan explains. By tracking the behavior of cells in real time following immunization, the authors showed that activated B cells in fact migrate directionally toward the follicle edge along a CCL21 chemokine gradient. "We believe that our work is one of the clearest examples of a chemotactic event occurring in multicellular organisms," says Cyster.

The researchers also tracked interactions between B cells and helper T cells during the initial stages of antigen-induced migration. B cells and T cells are normally found in specific zones in the lymph nodes, but after each cell encounters antigen, they migrate to the edges of their respective zones and interact, initiating an antibody response. During this phase, B cells and T cells formed one-to-one conjugate pairs that appeared to "dance" and exchange partners, with the B cell "leading" the T cell, Cahalan says.

This observation conflicts with a prior study showing that T cells act as leaders.[3] Ron Germain of the National Institute of Allergy and Infectious Diseases notes that further experiments will be needed to determine "who's leading the march."