Using intravital 2-photon imaging methods developed in the LBS over the past several years, we are now able to routinely image peripheral organs and tissues such as the liver, kidney, bone marrow, and skin so that immune effector cell behavior and to some extent effector functions during infectious processes can be observed. Using these new methods, we previously described lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver, the differences in mobility between myeloid and lymphoid components of these granulomas, and the specific changes in granuloma structure that accompany anti-TNF treatment. During the past year, we have extended these studies to compare the local effector responses of antigen-specific and unspecific CD4+ T cells within granulomas and to relate the migration properties of the T cells to their effector activity. These data show that only a small fraction of the antigen-specific T cells within a granuloma show migration arrest at any time and that the small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma, as assessed by isolation and intracellular staining of the cells isolated from the infected liver. Confocal imaging data show that these effectors cells polarize their secretion of the effector cytokine towards sites of antigen load (bacteria). The limited migration arrest of antigen specific T cells and the low number of cytokinesecreting cells making just detectable levels of IFNgamma is not an artifact of the analytic method, because injection of high levels of the cognate antigenic peptide into the infected animals results in both migration arrest of nearly all the T cells and production of 1-2 logs more IFNgamma per cell by 80-90% of the T cells under these conditions. These data suggest that during normal immune responses to mycobacteria in liver granulomas, there is very limited antigen presentation just sufficient at any moment to activate small fraction of all available effector cells into a cytokine-secretory state, and to do so just at the margin of quantitative response potential. These findings provide entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. Many of these observations have been repeated in M tuberculosis-infected animals, arguing that it is not the low pathogenicity of the BCG that leads to such limited effector responses, or the differential dynamic behavior of macrophages or T cells within granulomas. These data are currently being prepared for publication. We have also analyzed the early events following natural bite infection of animals with the parasite 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. These data showed that a prevailing model of Leishmania infection the Trojan Horse model is incorrect and that infection of tissue macrophages involves uptake of the parasite after release from infected neutrophils. It also highlighted the paradoxical infection-promoting role of neutrophil infiltration in this infectious disease. Ongoing work is also aimed at developing tools for in situ imaging of the pulmonary tract during influenza, BCG, and TB infection. We are also evaluating the effect of viral and other infections on the state and function of non-hematopoietic cells (fibroblastic reticular cells and other stromal elements) in secondary lymphoid tissues. During the past year we have developed new staining protocols that permit as many as 8 colors to be used to detail the distribution of stoma cells subsets and immune (hematopoietic) cells within lymphoid tissues, and begun to assess using 2 photon imaging the effects of LCMV infection on the stromal components of lymph nodes and spleen, along with concomitant changes in chemokine expression and T cell migration.
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