Fungal pathogens and most infectious agents enter the body at mucosal surfaces. Mucosal immune priming induces both systemic and mucosal resistance, and is believed to be the ideal strategy to prevent or treat infections that arise from a mucosal surface. Systemic priming, in turn, usually induces poor mucosal immunity and is judged to be less desirable against mucosal infections. Contrary to this paradigm, we find that a live-attenuated, vaccine strain of Blastomyces dermatitidis can protect mice against a lethal pulmonary infection (via Th1 CD4+ T cells) when the attenuated strain is delivered only by the subcutaneous route, and not by the respiratory route. Here, we will investigate the fundamental mechanisms by which these 2 routes of antigen (Ag) delivery trigger CD4+ T cells during the priming and the recall phases. We will endeavor to uncover the cellular and molecular mechanisms that lead to effective or failed priming of T cells in the skin versus the lung. T cell receptor (TCR) transgenic (Tg) systems are arguably the best immunological tools to decipher the activation of clonal T cells in vivo, but there are no such reagents available for studying T-cell immunity to the dimorphic fungi. Therefore, we propose to create a Blastomyces-specific CD4+ TCR Tg mouse to monitor and analyze the activation of Ag-specific CD4+ T cells in these two immunologically disparate settings. We hypothesize that the subcutaneous delivery of Ag will prime TCR Tg, Blastomcyes-specific CD4+ T-cells in the skin-draining lymph nodes to become activated Th1 effector cells, and protect against lung challenge upon recall. Mucosal priming, on the other hand, will lead to the generation of functionally impaired Th1 effectors unable to provide resistance to challenge. Our specific aims are to: 1) Develop an adoptive transfer system to monitor Blastomyces-specific CD4+ T cells in vivo. Fungal-specific TCR Tg mice will be created from a T cell clone that protects mice against experimental infection. TCR Tg cells will be used to establish an autologous adoptive transfer system to track the phenotypic and functional properties of protective Ag-specific T cells during the priming phase in the skin, and on recall in the lung. 2) Identify whether and how mucosal priming fails to engender Ag-specific Th1 CD4+ cells. TCR Tg cells will let us track the critical stages of the CD4+ T cell response after Ag exposure at the respiratory mucosa: recruitment, activation, proliferation and differentiation in the lung and draining lymph nodes during the priming and recall phases. These events will be compared/contrasted to those after Ag exposure in the skin. Our studies will yield new insight into the evolution of T cell responses and immunity in mucosal tissues. This knowledge will contribute to the development of effective strategies to induce acquired, protective immunity for improved control of fungal and other microbial infections at these sites. PROJECT NARRATIVE: Fungal and other pathogens enter the body through the lung and cause disease at this site or after dissemination. The studies proposed here will create a uniquely powerful transgenic mouse to investigate the induction of protective immune responses in the lung. Our findings will provide the mechanistic basis to design strategies that prevent and/or treat infections with fungi and other microbial infections.