Figure 3.

Airway exosomes contain influenza virus antigen and are capable of initiating a cellular immune response. (A) Proteomic analysis measuring levels of CD9, MHC II, CD74, and MHC I expressed by exosomes isolated from BALf over the course of an influenza virus infection. (B,C) Exosomes purified from the BALf of mice infected 1–4 days prior with 10⁴ PFU of X31-OVA (1) were cultured with 5 × 10⁴ CFSE-labeled OT-I CD8⁺ T cells with or without 1 × 10⁴ DCs. T cell proliferation was measured 60 h later. (B) Representative OT-I.CD8+ T-cell proliferation profiles with exosomes recovered from the BALf on days 2 and 3 p.i., cultured with or without DCs. (C) The absolute number of divided OT-I.CD8+ T cells. Data pooled from 3 to 9 independent experiments. Symbols represent the mean ± sem (two-way ANOVA, Sidak's multiple comparison). Dotted line represents the background level of T-cell division. (D) PKH26-labeled exosomes were intranasally administered into mice and 1 h later the lungs were recovered and the proportion of DCs and macrophages that had engulfed PKH67+ exosomes was profiled. (E) Mice injected with 2 × 10⁶ CFSE-labeled OT-I.CD8⁺ T cells were intranasally administered either saline (naïve) or x31-OVA (1) or 0.75 mg of exosomes recovered from the BAL of naïve mice or mice infected with x31-OVA (1) 2 days earlier and the absolute number of divided OT-I cells in the mLN was measured 4 days later. (E) Representative flow cytometry profiles gated on CD8+ T cells. (F) Graph depicts the absolute number of divided cells. Data pooled from 4 independent experiments (one-way ANOVA, Dunnett's multiple comparison). *p < 0.05, ***p < 0.001, ****p < 0.0001.