MS spectra were acquired at resolution of 120,000 at 300C1,500 m/z, RF lens 60% and an automatic gain control (AGC) ion target value of 4.0e5 for a maximum of 100 ms and an exclusion duration of 40s. proteomics, and the relative quantities of each protein was decided as described in the methods. Quantities are expressed as the percentage of the intensity of the top 3 peptides from each protein from 3 technical replicates.(PDF) ppat.1009894.s006.pdf (260K) GUID:?AC4AE35B-E687-47C1-9630-48A90C6D2003 S3 Fig: Infection rates in A549 cells prior to DC engulfment. A549 cells were infected at an MOI of 1 1.0 for 12 h, achieving 80% contamination (Data are mean contamination rates from replicate samples stained independently, n = 3 +/- SD).(PDF) ppat.1009894.s007.pdf (127K) GUID:?CCB5C720-1CED-4ABE-8D94-CE7B4CC17E5B S4 Fig: Flow cytometric gating strategy for identification of influenza infection in lung tissue sample cell subsets. Lung tissue explants Actarit were infected with IFV-A ex-vivo and incubated post-infection for 24 h. Tissue samples were then enzymatically dispersed TNF-alpha and the cells stained with monoclonal antibodies conjugated to cell-specific markers. Cell markers were used to identify (A) leukocytes (CD45-Horizon). (B) CD45+ cells were then gated to identify Actarit and exclude T cells (CD3-PECy7). (C) CD45+/CD3-/HLA-DR+ cells were macrophages. (D) CD45-CD326+ cells were identified as epithelial cells. (E) NP1/FITC staining was then used to identify infected macrophages (HLA-DR-APC/Cy7+/FITC+) and epithelial cells (F) (CD45-/CD326-PerCP/Cy5.5+/NP-FITC+).(PDF) ppat.1009894.s008.pdf (906K) GUID:?BB60B6D0-7CCC-4BDA-8125-9289111CC0AA Attachment: Submitted filename: em class=”submitted-filename” plos-response-to-reviewers-letter-2020-12-02.pdf /em ppat.1009894.s009.pdf (238K) GUID:?1E2BE879-361D-4C0A-A83D-511ED8C52AD9 Data Availability StatementThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022884. All other relevant data is available in the manuscript and supporting information files. Abstract CD8+ 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 contamination 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 Actarit 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 power of two ex-vivo fully human contamination 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 Actarit influenza and other emergent viruses. Author summary Influenza 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 computer virus, but the contribution of T cells to overall immunity is usually 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 comparable between different viral strains.