Trypanosoma cruzi infects up to 18 million individuals and is the major cause of heart disease in most of Latin America. Most of the studies of immunity to T. cruzi have focused on the role of the antibody response in controlling the parasite load in a host. The role of cellular immunity in the anti-parasite response is less well understood. In particular, little is known about the ability of the immune system to recognize and destroy parasite infected host cells. T. cruzi enters and divides in the cytoplasm of a variety of types of host cells. The location of the parasite in the cytoplasm makes it likely that molecules released by the parasite are processed and presented in association with class I MHC molecules and are available for recognition by CD8+ T cells. An intact CD8 T cell compartment is necessary for mice to survive T. cruzi infection, however the mechanism(s) by which these CD8+ T cells influence the infection is not known. In this study, we will focus on the activity of CD8+ as cytotoxic cells for T. cruzi-infected cells. This effort will include further refinement of techniques already in use in the lab to generate and test the activity of T. cruzi-specific CTL. Infected, antigen-pulsed. osmotically loaded, and peptide-sensitized host cells will be used to elicit and measure anti-T. cruzi CTL activity. We will construct transfected cell lines expression parasite antigens and use these lines to both elicit and measure CTL activity. Protocols which stimulate strong anti-T. cruzi CTL activity will be evaluated for their ability to generate protective immunity to challenge with T. cruzi and for their effect on pathogenesis during the infection. CTL lines recognizing parasite antigens will be passively transferred to normal and SCID mice to analyze their protective capacity. Once the system for generation and measurement of T. cruzi-specific CTL has been firmly established, the antigens recognized by T. cruzi-specific CTL will be analyzed. This analysis will take two approaches: the analysis of candidate antigens which are already in hand or are soon to be available (SSP-4, the major amastigote surface protein, and heat shock proteins) and a more extensive screening of parasite proteins or peptides which have not been previously described. The screening for prospective CTL target antigens will make use of a panel of amastigote-specific monoclonal antibodies, metabolic labeling of infected cells and analysis of peptides expressed in association with class I MHC on the surface of infected host cells. Completion of this project will provide information on an aspect of the immunology of T. cruzi infection which has been largely ignored. In addition, this work will extend beyond the study of a phenomenon and simple identification target antigens to provide information on the cell biology of this intracellular parasite.
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