Figure 4.

Airway exosomes generated following infection with a replication incompetent virus evoke inflammation and influenza virus-specific cellular immune responses. (A,B) MDCK cells were infected in the presence of trypsin at a multiplicity of infection (moi) of 10 with influenza virus or a S-FLU, or were cultured with 0.75 μg of exosomes purified from the BALf of mice infected with 2 days earlier with S-FLU. The proportion of influenza virus NP+ cells was determined 14 hrs later by flow cytometry. Data representative of 2 independent experiments. (B) Titres of infectious influenza virus released from virus-infected cells were quantitated using a virospot assay. Bars represent the mean + sd. (C) Absolute numbers of neutrophils (Ly6g⁺CD11b⁺) in the lung of mice 4 days following intranasal delivery of 0.75 μg of exosomes recovered from BALf of mice 2 days after intranasal delivery of either influenza virus (x31) or S-FLU. Data pooled from 2 independent experiments. Symbols represent individual mice (n = 2–5 mice per group, one-way ANOVA, Tukey's multiple comparison). (D,E) Mice received 0.75 μg of exosomes recovered from the BALf of mice infected with either influenza virus (x31) or S-FLU 2 days earlier and the absolute number of influenza virus nucleoprotein-specific cells (NP-tetramer+) in the mLN was measured 7 days later. (D) Representative flow cytometry profiles gated on CD8⁺ T cells. (E) Graph depicts the absolute number of divided CD8+ NP-tetramer+ cells. Data pooled from 2 independent experiments (one-way ANOVA, Dunnett's multiple comparison). *p < 0.05, **p < 0.01.