AbstractCD8+ and CD4+ T cells provide cell-mediated cross-protection against multiple influenza strains by recognising epitopes bound as peptides to human leukocyte antigen (HLA) class I and -II molecules respectively. Two challenges in identifying the immunodominant epitopes needed to generate a universal T cell influenza vaccine are: A lack of cell models susceptible to influenza infection which present population-prevalent HLA allotypes, and an absence of a reliable in-vitro method of identifying class II HLA peptides. Here we present a mass spectrometry-based proteomics strategy for identifying viral peptides derived from the A/H3N2/X31 and A/H3N2/Wisconsin/67/2005 strains of influenza. We compared the HLA-I and -II immunopeptidomes presented by ex-vivo influenza challenged human lung tissues. We then compared these with directly infected immortalised macrophage-like cell line (THP1) and primary dendritic cells fed apoptotic influenza-infected respiratory epithelial cells. In each of the three experimental conditions we identified novel influenza class I and II HLA peptides with motifs specific for the host allotype. Ex-vivo infected lung tissues yielded few class-II HLA peptides despite significant numbers of alveolar macrophages, including directly infected ones, present within the tissues. THP1 cells presented HLA-I viral peptides derived predominantly from internal proteins. Primary dendritic cells presented predominantly viral envelope-derived HLA class II peptides following phagocytosis of apoptotic infected cells. The most frequent viral source protein for HLA-I and -II was matrix 1 protein (M1). This work confirms that internal influenza proteins, particularly M1, are a rich source of CD4+ and CD8+ T cell epitopes. Moreover, we demonstrate the utility of two ex-vivo fully human infection models which enable direct HLA-I and -II immunopeptide identification without significant viral tropism limitations. Application of this epitope discovery strategy in a clinical setting will provide more certainty in rational vaccine design against influenza and other emergent viruses.Author SummaryInfluenza infections present a significant global health challenge. High rates of mutation require reformulation of vaccines annually. Vaccines are designed to induce antibody responses to the surface proteins of the influenza virus, but the contribution of T cells to overall immunity is unclear. Here, we used several totally human laboratory models to show how the viral proteins are presented to the T cells to induce immunity. We found that CD8 T cells, which kill infected cells, and CD4 T cells which support the CD8 T cells as well as the antibody-producing B cells, mainly see proteins from inside the viral particle, not the surface ones which are targeted by antibodies. These internal viral proteins are more similar between different viral strains than the surface proteins, and therefore suggest that vaccines designed to induce T cell responses could be better protective if they target internal viral proteins.