On a global scale, infectious agents as a group are the leading cause of mortality in infants, children, and adults. With its focus on disease prevention, vaccination is one of the most efficacious and cost effective means for infectious disease control. However in order to design safe and effective vaccines, a better understanding of how adaptive immune responses are generated and the specific immunological conditions during antigen exposure that will confer long-lived protection is needed. In addition to their role in triggering innate immune responses, the Toll-like receptor (TLR) family of molecules is believed to play essential roles in controlling the adaptive immune response by facilitating the generation of antigen-specific Thl-(IFN-yproducing) CD4 T cells. In the absence of TLRs or the downstream signaling molecule, MyD88, most infections or immunizations trigger an exclusive Th2-(no IFN-y produced, and IL-4 and IL-13 produced instead) immune response. Listeria monocytogenes (Lm) is a major pathogen in the neonate and in other immune-compromised hosts; and in the absence of TLR/MyD88, Lm infection retains the ability to generate IFN-y producing CD4 and CDS T cells. An analysis of the mechanistic basis for TLR/MyD88-independent generation of Thl immunity will be important for designing vaccines that aim to trigger this type of immune response, and will require me to become proficient with cellular immunology techniques that I yet do not have. We hypothesize (1) that entry into the cell cytoplasm and induction of type I-IFNs distinguish Lm from other infection and immunization models and allow Lm to induce Thl-immunity in the absence of TLR/MyD88 and (2) that regulatory T cells (normally inhibited by TLR-dependent pathways) can also be inhibited during Lm infection despite the lack of TLR-mediated cell activation. To test the role of Lm cytoplasmic entry and induction of type I-IFNs, we will examine the immune response following infection with Lm mutants that cannot gain access to the cell cytoplasm, and in mice with targeted disruption of type IIFN receptor. To test the role of regulatory T cells, we will isolate these cells during Lm infection and examine their function both in vitro and following adoptive transfer in vivo. The studies proposed will begin to characterize the mechanistic details of a TLR-independent pathway by which Thl-adaptive immune responses can be generated, and serve as the preliminary experiments for my transition into an independent investigator in the fields of immunology and infectious disease. ? ? ?
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