Fig. 7: Detection of SARS-CoV-2-N-specific CD8⁺ T lymphocytes in K18-hACE2 mice three months after i.m. injection with either Nef^mut- or Nef^mut/N- expressing DNA vectors (4 per group).

a Detection of N-specific CD8⁺ T cells in spleens of injected mice. A total of 2.5 × 10⁵ splenocytes were incubated overnight with or without 5 μg/ml of either unrelated or SARS-CoV-2-N-specific peptides in IFN-γ EliSpot microwells. Shown are the numbers of SFUs/well calculated as mean values of triplicates after subtraction of mean spot numbers calculated in wells of splenocytes treated with an unspecific peptide. Reported are intragroup mean value. One-tailed Mann–Whitney U Test, p = 0.0143, error bars, s.e.m. b Raw data from two ICS/flow cytometry analysis for the detection of IFN-γ expressing CD8⁺ Trm lymphocytes within immune cells isolated from lungs of K18-hACE2 mice injected with either Nef^mut- or Nef^mut/N-expressing DNA vectors. The lungs of immunized mice were explanted after intravenous injection of fluorescently labeled anti-CD45 mAb. Then, pools each formed by the cells extracted from the lungs of two mice injected with either Nef^mut- (pools # 1 and 2) or Nef^mut/N- (pools # 3 and 4) DNA vectors were cultivated in the presence of either N-specific or unspecific peptides. Reported are the percentages of IFN-γ expressing cells over the total of the CD8⁺ Trm sub-populations. c Percentages of CD8⁺ Trm cells expressing IFN-γ over the total of CD8⁺/CD44⁺ T lymphocytes isolated from lungs of mice injected with the indicated DNA vectors. Shown are mean values of percentages of positive cells from cultures treated with specific peptides after subtraction of values detected in cells treated with an unrelated peptide. The results are from two cell cultures each formed by the pool of cells from two immunized mice.