Dissecting the Role of IL-12 in Naive CD8+ T cell Activation by Dendritic Cells

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DESCRIPTION (provided by applicant): Advancements in the development of vaccine vectors and adjuvants has vastly improved the quality of public health in recent decades. Interleukin 12 (IL-12) is a cytokine that has been shown to possess potent adjuvant activity, thus this cytokine could improve treatments designed against a variety of intracellular pathogens as well as tumors. A complete understanding of how IL-12, and its antagonist interleukin 10, dictates cytotoxic lymphocyte activation (CTLs) remains to be determined. The goal of this research project is to determine the mechanisms by which interleukin 12 (IL-12) and interleukin 10 (IL-10) produced upon dendritic cell infection with the model pathogen, Listeria monocytogenes regulate naive CD8+ T cell (CTL) activation and the development of a protective population of memory cells. Our lab has demonstrated that the neutralization of IL-10 in culture augments naive CTL activation during priming, while the neutralization of IL-12 has the opposite effect. Thus, we hypothesize that the cytokines IL-12 and IL-10 regulate the interactions between naive CD8+ T cells and Listeria-infected dendritic cells during priming and this regulation dictates the number of effectors that acquire the ability to confer protective immunity. In order to test this hypothesis we propose the following Specific Aims: Specific Aim 1: To determine how the cytokines IL-12 and IL-10 generated upon listerial cytoplasmic entry in dendritic cells regulate effector function of naive T cells as measured by interferon gamma in vitro and the protective capacity of these cells in vivo. Specific Aim 2: To determine if IL-12 or IL-10 regulate the frequency and duration of interactions between naive CD8+ T cells and Listeria- infected dendritic cells in vitro. In order to address these questions naive CTLs will be exposed to Listeria infected wild-type, IL-12 knockout, or IL-10 knockout DCs in vitro. The affects of the removal of these cytokines will be analyzed by monitoring interferon gamma production, IL-12 receptor signaling and polarization, and in vivo protection assays against Listeria monocytogenes after the adoptive transfer of naive CTLs. In addition, the duration and frequency of interactions between CTLs and DCs when IL-12 or IL-10 is neutralized will be monitored via time lapse video microscopy and flow cytometric based conjugate assays. By completion of my thesis project, I will have elucidated how IL-12 and IL-10 dictate the functional outcome of CD8+ T cells on the molecular level. The findings generated through this work could be translated into the improvement of currently used vaccines and therapeutics against a variety of intracellular pathogens and tumors.