ABSTRACT Immunosurveillance for intracellular pathogens is primarily mediated by conventional TCR CD8 T lymphocytes that recognize pathogen-derived oligopeptides presented by the highly polymorphic classical or MHC class Ia molecules. Yet, there is growing appreciation that these oligomorphic nonclassical or MHC class Ib molecules may also present pathogen-derived antigens. Evidence to date, however, has suggested that MHC-Ib-restricted CD8 T cell-mediated immunity is relegated to host defense against intracellular bacterial infections. Using the mouse polyoma virus (PyV) model, we recently discovered that mice lacking MHC class Ia molecules but retaining MHC class Ib molecules (i.e., Kb-/-Db-/- mice) are as resistant as their wild type counterparts to the oncogeneic potential of this virus, efficiently control acute and persistent phases of infection, and do so in a CD8 T cell-dependent manner. We identified the viral peptide and its MHC class Ib-restricting molecule, constructed MHC-I tetrameric reagents, and tracked the evolution, functional integrity, and maintenance of these novel unconventional CD8 T cells throughout the course of PyV infection. Importantly, we determined that these antiviral CD8 T cells confer protection against PyV infection, and constitute a previously unappreciated component of the antiviral CD8 T cell repertoire. Our new data thus provides the first evidence for a defined MHC class Ib-restricted antiviral CD8 T cell response that contributes to host defense. The overall goal of this application is to use the mouse PyV infection system to comprehensively define the requirements for eliciting these unconventional CD8 T cells, and to apply insights from these studies to evaluate candidate interventions to promote recruitment of antiviral CD8 T cells across MHC class Ia allogeneic barriers. Three Specific Aims are proposed: (1) to define the requirements for selection and maintenance of PyV-specific, MHC class Ib-restricted CD8 T cells; (2) to investigate mechanism(s) to explain the finding that the majority of these T cells are functionally compromised; and (3) to define the contributions of CD4 T cells and select costimulatory pathways in generating and maintaining functional anti-PyV MHC class Ib-restricted CD8 T cells in order to promote control of PyV infection in mice of MHC-Ia allogeneic strains. PUBLIC HEALTH RELEVANCE: These studies should motivate efforts to uncover MHC class Ib-restricted CD8 T cell responses in other viral infections, and, given the limited polymorphism of MHC class Ib molecules, provide a platform for developing peptide-based viral vaccines having broad coverage across MHC haplotypes.