Fig. 4. Detection of N-specific CD8⁺ T cells in both blood and lungs of mice injected with N-expressing DNA (n = 10).

a Western blot analysis of 30 μg of lysates from HEK293T cells transfected with DNA vectors expressing either Nef^mut/N or N. The same assay was carried out on equal volumes of buffer where purified EVs were resuspended after differential centrifugations of respective supernatants. As control, conditions from mock-transfected cells were included. Filters were revealed by anti-flag tag, anti-β-actin, and anti-Alix (i.e., an EV marker) antibodies. Molecular markers are given in kDa. The results are representative of two independent experiments. Blots derived from the same experiment and were processed in parallel. b Detection of N-specific CD8⁺ T lymphocytes in PBMCs of K18-hACE2 mice i.m. injected with either Nef^mut/N- or N-expressing DNA vectors (5 mice per group). Shown are results from IFN-γ EliSpot analysis carried out on 1 × 10⁵ PBMCs incubated overnight with 5 μg/ml of either unrelated or N-specific peptides. Intragroup mean values are indicated, error bars, s.e.m. c Percentages of cells simultaneously expressing IFN-γ, IL-2, and TNF-α over the total of CD8⁺/CD44⁺ T cells isolated from lungs of mice injected with the indicated DNA vectors. d Percentages of lung CD8⁺ Trm cells (i.e., CD49a⁺, CD69⁺, CD103⁺) expressing IFN-γ over the total of lung CD8⁺/CD44⁺ T lymphocytes from mice injected with the indicated DNA vectors. For both c and d panels, shown are mean values of percentages of positive cells from pooled cell cultures treated with specific peptides after subtraction of values detected in cultures treated with an unrelated peptide.