We previously reported a new method for the direct confocal microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue. These studies showed that individual T cells bind stably to antigen-bearing dendritic cells for several hours before activation-associated events lead to dissociation of the cell pairs and rapid migration of the T cells in the lymphoid tissue. This experimental system has now been improved by moving to multiphoton rather than confocal imaging and true intravital observation methods have been developed. We are now able to routinely image peripheral organs and tissues such as the liver, kidney, bone marrow, and skin so that effector functions during infectious processes can be observed. Using these new methods, we are in the midst of a long-term study of lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver, with our first set of results showing for the first time the changes in cell positioning involved in mycobacterial granuloma formation, the differences in mobility between myeloid and lymphoid components of these granulomas, and the specific changes in granuloma structure that accompany anti-TNF treatment. We have also analyzed the early events following natural bite infection of animals with Leishmania, revealing the extensive infiltration of neutrophils, their uptake of these organisms but failure to destroy the parasites, and the role of this neutrophil invasion in the capacity of the parasite to successfully infect its preferred host cell, the macrophage. Ongoing work is also aimed at developing tools for in situ imaging of the pulmonary tract during influenza, BCG, and TB infection and the liver in a transgenic model of hepatitis infection. We are also evaluating the effect of viral and other infections on the state and function of non-hematopoietic cells (fibroblastic reticular cells) in secondary lymphoid tissues. New transgenic mice with cells ubiquitously expressing fluorescent proteins are being generated to provide new tools for long-term tracking of cells and genetically engineered strains of mice that have fluorescent reporters under the control of cytokine gene regulatory elements are being employed to relate the physical behavior of immune cells to their function.
