The IAV peptides isolated from H-2Db and Kb molecules were quantitated using an LC-MRM (liquid chromatography-multiple reaction monitoring) approach as previously reported for other viral epitopes4 (Supplementary Data?2)

The IAV peptides isolated from H-2Db and Kb molecules were quantitated using an LC-MRM (liquid chromatography-multiple reaction monitoring) approach as previously reported for other viral epitopes4 (Supplementary Data?2). influenza A virus (IAV)-peptides following either direct or cross-presentation. All these peptides, including seven novel epitopes, elicit T cell responses in infected C57BL/6 mice. Directly presented IAV epitopes maintain their relative abundance across distinct cell types and reveal a broad range of epitope abundances. In contrast, cross-presented epitopes are more uniform in abundance. We observe a clear disparity in the abundance of the two key immunodominant IAV antigens, wherein direct infection drives optimal nucleoprotein (NP)366C374 presentation, while cross-presentation is optimal for acid polymerase (PA)224C233 presentation. The study demonstrates how assessment of epitope abundance in both modes of antigen presentation is necessary to fully understand the immunogenicity and response magnitude to T cell epitopes. influenza A virus Quantitation of directly presented IAV peptides Having established the detectable repertoire of IAV peptides directly presented by DC2.4 cells at 8 hpi, we next quantitated the abundance of each peptide. The IAV peptides isolated from H-2Db and Kb molecules were quantitated using an LC-MRM (liquid chromatography-multiple reaction monitoring) approach as previously reported for other viral epitopes4 (Supplementary Data?2). Stable isotope-labeled viral peptides LY2409881 were synthesized and LC-MRM parameters individually optimized for each peptide to provide a complete suite of internal quantitative standards (see Table?1, Supplementary Tables?1C3, and Supplementary Figs.?1 and 2). The abundance of the 21 IAV-derived peptides spanned three orders of magnitude, ranging from 1C2 copies/cell of PB1653C660 to an average of 3871 copies/cell of NP366C374 (Fig.?1a). Of the well-characterized CD8+ T-cell epitopes, the immunodominant NP366C374 and subdominant NS2114C121 peptides were the most KLRK1 abundant, being present at an average of 3871 LY2409881 and 2464 copies/cell, respectively, while the subdominant epitopes PB1-F262C70 and PB1703C711 were substantially lower at 684 and 294 copies/cell, respectively. One of the least abundantly presented species was the immunodominant epitope PA224C233, at only 7 copies/cell. Thus, the abundance of LY2409881 peptides presented following direct infection of the DC2.4 cells did not predict the CTL immunodominance hierarchy. Open in a separate window Fig. 1 Detection and quantitation of MHCI-bound IAV peptides following direct infection. DC2.4 cells (1??108) were mock treated or infected for 8?h with the PR8 strain of IAV at an MOI of 5, epitopes were eluted from immunoaffinity-purified Kb and Db MHCI molecules and analyzed by LC-MRM. a Absolute quantitation of peptide abundance shown as peptide LY2409881 copies/cell. values. Dashed lines represent the limit of detection Given that productive IAV infection is restricted to respiratory epithelial cells, which are also the targets of the IAV-specific CTL response, we next investigated the relative abundance of LY2409881 IAV-derived peptides presented on H-2Db and Kb molecules expressed on the surface of an infected lung epithelial cell line (LET1 cells)11 (Supplementary Data?2). Although the infection efficiency of LET1 cells was similar to DC2.4 cells (~80C85%) (Supplementary Fig.?3a), the expression of surface H-2Kb and Db complexes was lower in LET1 cells (Supplementary Fig.?3b), resulting in an overall reduction in the yield of peptides/cell (Fig.?1b). However, there was a significant correlation (RCC?=?0.6327, values Characterization of T-cell responses to the identified IAV peptides The above data represent the first in-depth analysis of the differential abundance of virus peptides presented by MHC class I complexes via the routes of direct versus cross-presentation. In order to relate this information to the immunogenicity of each peptide in vivo, we systematically characterized the immune response elicited to each peptide identified in this study after IAV infection. The.