The central role of T-cells in acquired resistance to granulomatous fungal disease is supported by a large body of experimental data in animals, but relatively little work has addressed these defense mechanisms and their clinical significance in humans. The goal of this proposal is to examine regulatory and effector functions and genetic control of major histocompatibility complex (MHC)-restricted, antigen-specific T-cells in humans infected with the dimorphic fungus Blastomyces dermatitidis. The hypothesis to be tested is that patterns of cytokine production, cytolysis, and T-cell receptor (TCR) gene usage of cells that recognize an immunodominant fungal antigen will be influenced by the epitopes displayed by MHC class I and II molecules on antigen presenting cells. Selected patterns will determine the disease severity or profile in individual patients. A well-defined 120 kD surface protein on B.dermatitidis yeasts (designated WI-1), or peptides of this molecule, will be used for cloning and in vitro assays of T-cells or binding of MHC molecules. In years 1 and 2, Blastomyces-specific T-cell clones derived in response to WI-1 using peripheral blood lymphocytes from a patient will be characterized further, and T-cells from peripheral blood and infected skin lesions of additional patients cloned and studied. Cytokine production, cytolytic effects directed against antigen labeled and Blastomyces-infected targets, and TCR gene usage by the clones will be examine. In years 2 and 3, T-cell clones responsive to WI-1 will be used to probe peptide fragments of the antigen to identify immunodominant epitodes. Selected fragments will be sequenced and the WI-1 DNA amplified using polymerase chain reaction. Amplified gene products will be sequenced to extend peptide sequence data and allow preparation of synthetic, immunogenic peptides. In years 3 and 4, MHC molecules that display WI-1 peptide antigen(s) to T-cell clones will be defined. HLA-loss variants of an immunoselected lymphoblastoid B-cell line (LCL 721) with deletions of selected MHC class I and II molecules will be used as antigen presenting cells to define peptide antigen restricting elements. HLA-loss variants transfected with individual restricting elements will be used to confirm the results. In years 4 and 5, WI-1 peptides will be radiolabeled and studied for in vitro binding to MHC class I or II molecules on LCL cells from blastomycosis patients. MHC binding of peptide will be analyzed with respect to the corresponding function and TCR gene usage of cells that respond to peptide and interpreted in light of patient HLA background and disease outcome. Findings from this work will advance basic knowledge about the cell- mediated defense mechanisms that control blastomycosis and other systemic mycoses, and may yield new information about the immunologic basis of unique susceptibilities to granulomatous diseases described in humans.
