Fig. 5: IN/IT immunization with MPV/S-2P induces S-specific CD4⁺ and CD8⁺ T-cell responses in the blood and lower airways that are restimulated after boosting.

S-specific CD4⁺ and CD8⁺ T-cells in blood (A–C) or bronchoalveolar lavage (BAL) (D–F). Peripheral blood monocytic cells (PBMC) and bronchoalveolar lavage (BAL) cells from indicated days post-immunization (pi) or post-challenge (pc) were left unstimulated or stimulated with overlapping SARS-CoV-2 spike (S) or (BAL only) nucleoprotein (N) peptides, and processed for flow cytometry (see Fig. S3B for gating). Dot plots showing interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression by CD4⁺ or CD8⁺ T-cells from PBMC (A) or BAL (D) of representative MPV (top) or MPV/S-2P-immunized (bottom) macaques. Background-corrected frequencies of S-specific IFNγ⁺/TNFα⁺ CD4⁺ (B, E) or CD8⁺ T-cells (C, F) from PBMC (B, C) or BAL (E, F) on indicated days. Expression of proliferation marker Ki-67 by IFNγ⁺/TNFα⁺ S-specific CD4⁺ (red) or CD8⁺ (purple) T-cells from blood (G, H) or BAL (I, J) of MPV/S-2P-immunized macaques. Gating and histograms showing evolution of Ki-67 expression by IFNγ⁺/TNFα⁺ CD4⁺ or CD8⁺ S-specific T-cells in blood (G) or BAL (I) from a representative MPV/S-2P-immunized macaque. IFNγ⁻/TNFα⁻ T-cells (gray) are shown for comparison. Frequencies of Ki-67⁺ T-cells in IFNγ⁺/TNFα⁺ T-cells from blood (H) or BAL (J). B, C, E, F, H, J Medians (lines), min and max values (whiskers), 25th to 75th quartiles (boxes), and individual values are shown for MPV primed (n = 4), MPV/S-2P primed (n = 8) and boosted (n = 4) macaques. MPV/S-2P-primed and primed/boosted macaques are represented by circles or triangles. Source data are provided in the Source Data file.