White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. In the unactivated state, these cells circulate in the blood and accumulate in lymphoid tissues such as lymph nodes and spleen. Upon encounter with foreign materials (antigens) on the membranes of specialized antigen presenting cells (dendritic cells), these resting T-cells become activated, undergo numerous cell divisions, and differentiate into effector cells. The effector cells leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also invade normal tissues where their activity can cause autoimmune pathology. After elimination of an infecting organism, most of the activated T-cells die, but some remain as memory cells, to provide a more rapid and vigorous response if the same pathogen is encountered in the future. Recent work has suggested that this general scheme applies to both CD4 and CD8 T-cells, but that there are also important differences in the signals that control the extent of proliferation and the survival of memory cells for these two T-cells lineages. Furthermore, there are also data suggesting that memory cells may be of more than one type, with some recirculating in lymphoid compartments and others patrolling peripheral tissues. The former may provide the major source of new cells upon re-infection, whereas the latter may mediate the earliest effector response to the infection. The proper balance of both types may be critical for effective T-cell mediated host defense. Other lymphocytes such as NK cells and regulatory T cells contribute to both the enhancement and suppression of these T cell responses through direct and indirect means. This project attempts to gain both a qualitative (especially tissue-specific 4 dimensional space and time) and a quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells. Issues such as the route, amount, and frequency of antigen exposure, as well as the presence or absence of adjuvants that stimulate the innate immune system, are being studied for their effects on the generation and tissue distribution of effector and memory CD4 and CD8 T-cells. The movement of activated T-cells into non-lymphoid tissues is being analyzed using both conventional cellular immunological methods and newer imaging techniques that allow high resolution dynamic observation of how cells migrate, interact, and carry out their effector functions. Through this research, a better understanding of lymphocyte dynamics during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines and to strategies for the amelioration of autoimmune processes. During the past year we have established a robust system for vaccination using the non-replicating pox vector MVA, to permit in situ analysis of immune cell behavior in response to a clinically used vaccine vector. Variants of the virus have been developed that encode both fluorescent proteins and model antigens that are recognized by TCR transgenic T cells expressing distinct fluorescent proteins. This allows for tracking of the sites of viral infection and of the location and dynamic behavior of antigen-specific T cells during immune responses in situ, using our advanced 2-photon intravital imaging methods. Preliminary studies have suggested unexpected locations for some of the cells initially infected by MVA within draining lymph nodes, the rapid loss of directly infected cells, a role for cross-presentation as well as direct presentation in the activation of CD8 T cells, and a paradox in terms of where and when CD8 T cells and CD4 T cells respond to the MVA-encoded antigens. In the latter case, we are now engaged in trying to understand how what appears to be dispersed initial antigen activation of these two cell types on different antigen-presenting cells can be reconciled with data from other laboratories and from the LBS that indicate a requirement for antigen co-presentation to the CD8 and CD4 T cells on the same dendritic cells for induction of robustimmune memory. We have also established a model for examine the effect of regulatory T cells (Tregs) on the MVA-induced response and have obtained preliminary data showing a striking differential effect of these cells on CD8 T cells responding to different determinants of the MVA-encoded antigen and also on the balance of highly polarized effectors versus multifunctional/ memory T cells emerging after priming.
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