Figure 5.

Ag-Specific T Cell Responses Are Not Impaired following i.m. or i.n. Delivery of Ad Vectors Expressing Exosome Display EGFP in Mice

(A and B) Mice were vaccinated i.m. with 1 × 10⁸ IFU Ad5-EGFPΔ (gray box), Ad5-EGFP_C1C2 (green box), ChAd-EGFPΔ (gray circle), ChAd-EGFP_C1C2 (green circle), or PBS (naive) in a final volume of 50 μL of PBS. Two weeks later, anti-EGFP IFN-γ⁺ T cell responses in the spleen were measured by ELISpot. Splenocytes were stimulated overnight with an EGFP peptide pool spanning the entire EGFP Ag (EGFP PepMix) (A) or with the H2-Kd-restricted peptide EGFP_200–208 (B). Solid line indicates median (n = 5 mice/group). (C and D) Figures show anti-EGFP IFN-γ⁺ T cell responses in the spleen detected by ELISpot stimulating with EGFP PepMix (C) or EGFP_200-208 (D) following i.n. vaccination, using identical vaccines as described above, administered in a 50-μL volume. Line indicates median (n = 5 mice/group). (E–J) Ag-specific T cell responses in the lung (E–G) and BAL (H–J) were quantified using cell surface staining with ICS by flow cytometry. Cells were stimulated with a peptide pool spanning the entire EGFP Ag (EGFP PepMix), DMSO (negative control), or PMA/ionomycin (positive control). Legend indicates cytokine-secreting cells as a percentage of total, live CD8⁺ T cells. CD4⁺ responses were below the limit of detection for all cytokines analyzed (data not shown). Solid lines indicates geometric mean and represent n = 3–5 mice/group for lung and BAL, with the exception of n = 1 for BAL PBS due the low numbers of cells present in the BAL of PBS-treated mice. The lower limit of detection frequency was determined by the equation: [(1/minimum CD8⁺ count across DMSO and GFP stimulations) × 100]. Statistical significance was determined using a non-parametric Kruskal-Wallis test with a post hoc Dunn’s multiple comparison test. *p < 0.05, NS = p > 0.05. IFN-γ, interferon-γ; ELISpot, enzyme-linked immunospot assay; SFU, spot forming unit; DMSO, dimethyl sulfoxide, PMA, phorbol 12-myristate 13-acetate; LLD, lower limit of detection.