Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5⁺ ILC2 responses, and induced a dominant IL-13⁺ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ–deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR⁺ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Innate lymphoid cells (ILCs) and T cells represent critical populations of cells that have diverse roles in inflammation and protection (1, 2). Both cell populations consist of subsets that differ in cytokine expression and function. While T cells are important for viral clearance, they can also exacerbate lung immunopathology (1, 3–5) Among ILC subsets, ILC2s play a critical role in pulmonary immunity, particularly in maintaining the lung barrier surface (6–9). During infection, ILC2s respond to the epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin, and produce the type 2 cytokine IL-5 (10–12). This, in turn, can lead to increased eosinophil recruitment and airway hyperreactivity (AHR) (8, 13–15). Like T cells, ILC2s can play both beneficial and detrimental roles during viral lung infection (6–8).It is known that host cytokines can regulate the activity of ILC and T cell subsets. For example, we previously found that interferon (IFN)-γ deficiency results in enhanced ILC2 activity and increased survival from challenge with the 2009 pandemic strain A/California/04/2009 (CA04) influenza A virus (8). However, our current studies have shown no effect of IFN-γ following challenge with the Puerto Rico/8/1934 (PR8) influenza A virus, a strain that is a commonly used model for the highly virulent 1918 pandemic influenza virus. Although both strains are H1N1 influenza A viruses, they have striking differences in expression of functional PB1-F2, a viral proapoptotic protein that is associated with immunopathology and mortality (16). While the PR8 viral strain expresses full-length PB1-F2, the PB1-F2 gene in the CA04 strain is truncated and nonfunctional (16–20). As a result, the PR8 virus exhibits significantly increased virulence compared to the CA04 viral strain. However, the impact of PB1-F2 on the lymphocyte function that is critical for protection during influenza is not known. A better understanding of the role of pathogen virulence factors in regulating immune cell activity during influenza may aid in designing future therapies for human use.We hypothesized that the PB1-F2 virulence protein can differentially regulate ILC2 and T cell activity in conjunction with host IFN-γ signaling. To test this hypothesis, we have investigated pulmonary immunity in wild-type (WT) and IFN-γ–deficient BALB/c mice infected with PB1-F2 gene reassortant PR8 and CA04 viruses. Our findings demonstrate that viral virulence genes, together with host factors, play critical roles in regulating both ILC2 and T cell responses during influenza, and this, in turn, determines host survival.