Infection with the pathogenic fungus, Histoplasma capsulatum (Hc) is initiated by coincidental inhalation of conidia and hyphal fragments. In lungs, these elements convert into yeast cells which are the form that is responsible for the clinicopathological manifestations of disease. Successful resolution of infection requires a collaborative interaction between T cells and macrophages. The principal mechanism whereby T cells contribute to effective clearance of the fungus is by release of cytokines that arm macrophages to inhibit the intracellular growth of yeast cells. Although several lines of evidence support the integral role of T cells in host resistance, much of the work has analyzed polyclonal T cells from lymphoid organs. In this proposal, Dr. Deepe will examine the T cell receptor (TCR) repertoire in lungs of naive and immune mice exposed to Hc. The hypothesis is that there is a bias in the TCR repertoire in the lungs of mice and that the overrepresented cells are key to host defenses. Preliminary data indicate that in naive mice there is a bias in the TCR repertoire during the first 21 days of infection.
In Specific Aim 1) the investigator will analyze the VB repertoire in immune mice reexposed to Hc using reverse transcription-polymerase chain reaction combined with Southern hybridization and the results confirmed with flow cytometry; 2) determine if the V beta repertoire varies among Hc isolates; 3) determine if the anatomic location of prominent V beta cells in inflamed lungs differs from the other V beta bearing T cells, and 4) determine if the overexpressed V beta cells are of clonal origin.
In Specific Aim 2, the investigator will analyze the functional properties of overexpressed V beta cells. Dr. Deepe will 1) determine if the course of pulmonary Hc or the inflammatory response to Hc is altered by elimination of one or more T cell populations bearing the most prominent V beta families; 2) determine if passive transfer of prominent V beta bearing T cell clones alters the course of pulmonary Hc and 3) determine if the prominent V beta cells are the principal generators of lymphokines involved in protection in Specific Aim 3, the investigator will determine the immunobiological importance of antigens that stimulate overexpressed V beta . Dr. Deepe will 1) identify antigens that are recognized by the prominent V beta using an epitope-tagged cDNA library, and 2) determine if deletion of the gene encoding the antigen(s) causes deviation of the immune response to Hc. Hence, there will be an understanding at the clonal level the critical function(s) of these T cells.
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