Fig. 4.

Improved re-activation of memory T cells derived from I.N. vaccinated donors in recipient lungs upon challenge. (a) Diagram illustrating the experimental design of adoptive transfer of purified Thy1.2⁺ T cells from M7 + S-RBD-vaccinated donors (via S.C. or I.N. routes) into Thy1.1⁺ naïve recipients, followed by I.N. challenge with S protein. (b) Flow cytometry plots indicating the presence of donor Thy1.2⁺CD8⁺ T cells in the lungs of vaccinated recipient mice, but not control mice, 5 days after challenge. Full gating strategy provided in Supplementary Figure S4A. (c) Donor Thy1.2⁺CD8⁺ T cells constituted a minor portion of haemopoietic cells in the lung following challenge and did not differ in frequency between I.N. or S.C. vaccinated groups, but were not detected in control mice. (d) Histogram of TNF expression on donor (Thy1.2⁺) and recipient (Thy1.1⁺) CD44⁺CD8⁺ T cells representative of each group indicates an increase in TNF expression by donor T cells in lungs from vaccinated mice. Downsampling of 600 T cells was used to facilitate comparisons of equal numbers of cells for each sample. The percentage of TNF⁺ cells of total CD44⁺CD8⁺ T cells is indicated in the legend. (e and f) The MFI for (e) TNF expression and (f) CD69 expression were compared for Thy1.2⁺ donor CD8 T_MEM cells in the lungs following S challenge. For (e and f), vaccinated groups were not compared to control groups since there were insufficient donor memory T cells in the lungs of unvaccinated control groups for TNF expression quantification. N = 4–5 mice per group derived from two independent experiments. ∗p < 0.05 by Student’s unpaired t-test.