Fig. 4. In vitro screening for immunogenicity of IAV and IBV epitopes in human PBMC.

a, b Frequencies of IFN-γ⁺CD8⁺ T-cells after PBMCs were expanded with IAV (a) or IBV (b) peptide pools for 13 days and restimulated with C1R.A24 cells pulsed with the corresponding peptide pools (left panel; IAV n = 5 biologically independent samples, IBV n = 4 biologically independent samples). Right panels show dissection of each peptide from IAV pools 1 and 2 (LIFT n = 5, non-LIFT n = 3–5) (a) or IBV pool 10 (LIFT n = 9 biologically independent samples, non-LIFT n = 5 biologically independent samples) (b) using single peptide-pulsed C1R.A24 cells after 15 days of peptide-pool T-cell expansions in LIFT and non-LIFT. a, b Pie charts depict protein origin of immunogenic peptides and the median contribution to the total IFN-γ response for Indigenous (LIFT) and non-Indigenous (non-LIFT) donors. Colored bars below the bar chart indicate the protein origin of characterized peptides. c, d Individual IBV peptide IFN-γ⁺CD8⁺ T-cell responses in non-LIFT donors following expansion with B/Malaysia/2506/04-infected C1R.A24 cells for 15 days and ICS. c For the ICS, peptides derived from B/Malaysia/2506/04 or d comparison between autologous (v1) and the alternative circulating variant (v2) was used. Symbols indicate individual donors screened (n = 5 biologically independent samples). d IBV PB2_550–558 v1 and v2 responses after virus expansion versus IBV PB2_550–558 v1 peptide expansion are shown on the right. a–d Bars indicate median. e Cross-reactive PB2 responses of IAV PB2, IBV PB2v1, and IBV PB2v2 peptides following PBMC expansion with IAV (A/HKx31) (left panels; n = 4 biologically independent samples) or IBV (B/Malaysia/2506/04) infected (right panels; n = 5 biologically independent samples) C1R.A24 cells from 5 non-LIFT donors with representative concatenated FACS plots. f Crystal structures of HLA-A*24:02 presenting the PB2 peptide variants IAV PB2 (red, left) and IBV PB2v1 (pink, middle), and an overlay of both peptides (right).