Most viral peptides displayed by class I MHC on infected cells are immunogenic

Abstract

CD8⁺ T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells. Next, we screened each peptide for immunogenicity in multiple virus-infected mice, revealing a wide range of immunogenicities. A surprisingly high fraction (>80%) of pMHCI were immunogenic in at least one infected mouse, and nearly 40% were immunogenic across more than half of the mice screened. The high number of peptides found to be immunogenic and the distribution of responses across mice give us insight into the specificity of antiviral CD8⁺ T cell responses.

Keywords: CD8+ T cells; MHC class I; antigen presentation; vaccinia virus; virus.

Fig. 1.

Identification of a viral immunopeptidome. Cells were infected with VACV for 6 h, and MHCI-bound peptides were identified by LC-MS/MS. (A) ProteinPilot confidence score for VACV peptide sequences. Known immunogenic peptides are in black. Number of peptides above 50 and 95% confidence levels are shown at the top. (B) Example of a comparison between synthetic (red, Lower) and eluted (black, Upper) peptide spectra. Dominant y and b ions are indicated along with Pearson r correlation between spectra. (C) Example of MRM detection of a peptide, showing the native (Upper) sequence only in infected cells and spiked heavy peptide (Lower) in mock and infected samples. Dot-product (dotp) of eluted detection is indicated. (D) Pearson r correlation values (left axis) between synthetic and eluted spectra across 129 peptides. Filled circles are peptides with a correlation P < 0.05; circles in green are eluted peptides with ProteinPilot confidence >95%. The right axis shows MRM dotp values for each peptide validated by MRM. (E) Distribution of binding affinities of VACV peptides for MHCI.

Fig. 2.

The protein sources of a viral immunopeptidome. (A) The number of H-2^b–presented peptides from each VACV protein. The viral proteome is ordered by kinetic class and then position in genome, from left to right. Gaps are proteins from which no peptide was detected. (B) Peptide distribution according to the kinetic class of source proteins, normalized to protein length. (C and D) Abundance of pMHC from MS precursor signal intensity (C) and number of times each peptide was sequenced by MS (D), according to the kinetic class of source proteins, normalized to protein length. (E) Comparison of number of peptides identified here versus those identified previously. Epitopes identified in Moutaftsi et al. (4) and all others from the IEDB are shown separately. Kruskal–Wallis multiple comparison tests were used to determine significance between all possible pairs in B–D; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

The majority of members in a viral immunopeptidome are immunogenic. Peptides were tested for reactivity against CD8⁺ T cells in spleens from mice infected with VACV. Each peptide was tested in eight mice. (A) The distribution of data from individual mice for a selection of 18 pMHC, two to represent each possible number of times positive (as noted on top). The dashed line represents the average of three times the SD of negative controls across the eight mice, and closed circles are values that were above the threshold for immunogenicity. (B) The distribution of pMHCI according to the number of times that each was immunogenic out of eight tests. (C) Comparison of the cumulative size of the response for VACV peptides identified by LC-MS/MS here versus those identified previously. (D) Summed size of the CD8⁺ T cell response of the major and minor peptides. (E) Comparison of the cumulative response to the top 90 most immunogenic VACV epitopes against the total anti-VACV CD8⁺ T cell response (GzmB⁺, CD62L⁻) 7 d after infection. ***P = 0.0003 by paired t test.

Fig. 4.

Factors associated with immunogenicity for viral pMHC. (A) The cumulative size of CD8+ T cell responses to each viral protein when ordered as in Fig. 2A. Gaps are nonimmunogenic proteins. (B) Distribution of major epitopes and (C) immunogenicity of viral proteins across the kinetic classes, top 10% of immunogenic proteins are significantly reduced for late proteins (Fisher’s exact test, P = 0.04033). (D and E) Abundance of pMHC estimated by precursor ion intensity (D) and number of times sequenced by MS (E) across major, minor, and nonimmunogenic (Non) peptides. (F) Comparison of IC₅₀ for major, minor, and nonimmunogenic peptides. Kruskal–Wallis multiple comparison tests were used to determine significance between all possible pairs in D–F; *P < 0.05, ***P < 0.001, ****P < 0.0001.