Early Events of Innate Immune Response to Influenza Virus in a Complex Cellular Environment Revisited with Multi-Parametric Flow Cytometry

Abstract 4931

The development of an innate immune response against viruses involves multiple cellular interactions implying infected cells and the cells endowed with innate immune functions. These cells have to act in unison to develop a potent response. Although multiple interactions sustained the development of the innate response, much of the knowledge on the function of human innate immunity comes from investigations of isolated cells or dual partner coculture. Our aim was to investigate the early interactions of influenza virus with innate immune cells in a complex cellular environment in order to deepen our knowledge on the dynamics of the innate immune response. Polychromatic flow cytometry has been the appropriate method of exploration. We designed panels of 12 markers to explore PBMC stimulated with live-influenza virus or a mix of TLR agonists mimicking the interactions of influenza virus with TLR7/8 and TLR4. The DC populations were identified as lineage⁻HLA-DR⁺ cells and we took advantage of the polychromatic cytometry to split the lineage markers into separate channels to analyze NK, T cells and monocytes simultaneously to DC. Intra-cellular expression of IFN-a, TNF-a, IL-12, IL-6, IFN-g, and CD107 were analyzed in a time-dependent fashion. Our results demonstrate the benefits of using complex cell culture for the understanding of innate immune response to virus. We show a new field of application for multi-parametric flow cytometry as a powerful tool of investigation of innate immune responses in complex cell systems, with a sensibility to detect subtle modifications comparable to isolated cell systems. The multiple time-dependent results allowed to approach innate immune activation as correlated dynamic events. Dynamic studies lead to re-visit the relative roles of infection and TLR induced-DC response in the early specific features of Flu induced innate activation.