The frequency and function of nucleoprotein-specific CD8 + T cells are critical for heterosubtypic immunity against influenza virus infection

ABSTRACT Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8 + T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8 + T cells. Our results indicate that both CD8 + T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8 + T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8 + T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8 + T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8 + T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.