Influenza viruses lead to substantial morbidity and mortality including ~3-5 million cases of severe illness and ~290,000-650,000 deaths annually. One of the major hurdles regarding influenza vaccine efficacy is generating a durable, robust cellular immune response. Appropriate stimulation of the innate immune system is key to generating cellular immunity. Cross-talk between innate dendritic cells (DC) and natural killer (NK) cells plays a key role in activating virus-specific T cells, yet the mechanisms used by influenza A viruses (IAV) to govern this process remain incompletely understood. Here, we used an ex vivo autologous human primary immune cell culture system to evaluate the impact of DC-NK cell cross-talk and subsequent naïve T cell activation at steady-state and after exposure to genetically distinct IAV strains–A/California/07/2009 (H1N1) and A/Victoria/361/2011 (H3N2). Using flow cytometry, we found that exposure of DCs to IAV in co-culture with NK cells led to a decreased frequency of CD83+ and CD86+ cells on DCs and an increased frequency of HLA-DR+ on both DCs and NK cells. We then assessed the outcome of DC-NK cell cross-talk on T cell activation. At steady-state, DC-NK cell cross-talk increased pan T cell CD69 and CD25 expression while exposure to either IAV strain reduced pan T cell CD25 expression and suppressed CD4+ and CD8+ T cell IFN-γ and TNF production, following chemical stimulation with PMA/Ionomycin. Moreover, exposure to A/Victoria/361/2011 elicited lower IFN-γ production by CD4+ and CD8+ T cells compared with A/California/07/2009. Overall, our results indicate a role for DC-NK cell cross-talk in T cell priming in the context of influenza infection, informing the immunological mechanisms that could be manipulated for the next generation of influenza vaccines or immunotherapeutics.

Influenza viruses lead to substantial morbidity and mortality including ~3-5 million cases of severe illness and ~290,000-650,000 deaths annually. One of the major hurdles regarding influenza vaccine efficacy is generating a durable, robust cellular immune response. Appropriate stimulation of the innate immune system is key to generating cellular immunity. Crosstalk between innate dendritic cells (DC) and natural killer (NK) cells plays a key role in activating virus-specific T cells, yet the mechanisms used by influenza A viruses (IAV) to govern this process remain incompletely understood. Here, we used an ex vivo autologous human primary immune cell culture system to evaluate the impact of genetically distinct IAV strains on DC-NK cell crosstalk and subsequent T cell activation. We report that the addition of NK cells to cultures containing both DCs and naïve T cells led to an increase in the frequency of CD69+ and CD25+ T cells and elevated levels of IFN-γ, TNF, and IL-10. However, upon IAV infection of DCs, the addition of NK cells to cultures no longer increased the frequency of CD25+ T cells nor elevated IFN-γ, TNF, and IL-10 cytokine levels. Investigation of the impact of IAV infection on DC-NK crosstalk revealed that exposure of DCs to influenza virus in co-culture led to an increased frequency of HLA-DR+ and a decreased frequency of CD83+ and CD86+ cells -molecules involved in stimulating T cell activation. An expansion of an HLA-DR+ NK cell subset was observed following culture with influenza-infected DCs in a contact-dependent and cytokine independent-manner. Overall, our results indicate a role for DC-NK cell crosstalk in T cell priming in the context of influenza infection, informing the immunological mechanisms that could be manipulated for the next generation influenza vaccine or immunotherapeutic.