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Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response

Nature Immunology volume 24, pages 1933–1946 (2023)Cite this article

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Abstract

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of ‘cryptic’ viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

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Fig. 1: Discovery of co-immunodominant CTL response directed against M-SL9, an epitope encoded by a noncanonical IAV matrix ORF.
Fig. 2: M-SL9-specific CD8 T cells are restricted to the nonclassical MHC class Ib, Qa-1.
Fig. 3: Immunophenotype and clonal diversity of M-SL9/Qa-1-specific CD8 T cells.
Fig. 4: Distinct anatomical distribution and kinetics of M-SL9/Qa-1-specific CD8 T cells.
Fig. 5: Efficient lung-resident memory cell differentiation of M-SL9/Qa-1-specific CD8 T cells.
Fig. 6: Recall of M-SL9/Qa-1-specific CD8 T cells during a secondary IAV infection.
Fig. 7: M-SL9 presentation depends on the second AUG codon of the matrix gene segment.
Fig. 8: M-SL9/Qa-1-specific CD8 T cells are raised by mRNA vaccination and mediate cytolysis in vivo.

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Data availability

Raw mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifiers PXD045025 and https://doi.org/10.6019/PXD045025. Data supporting TCR sequence analysis are deposited in GitHub at the URL https://github.com/mhogan240/NatImmuno2023. Data supporting M-SL9 sequence variation analysis are publicly available from BV-BRC. Other data and reagents that support the findings of this study are available from the corresponding authors Michael J. Hogan and Laurence C. Eisenlohr upon request. Source data are provided with this paper.

Code availability

Code supporting analysis of TCR sequences and M-SL9 variant sequences is deposited in GitHub at the URL https://github.com/mhogan240/NatImmuno2023.

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Acknowledgements

We thank F. Tuluc, J. Murray and J. Lora of the Children’s Hospital of Philadelphia Flow Cytometry Core Facility for technical advice and services; L. Spruce, H. Fazelinia and S. Seeholzer (formerly) of the Children’s Hospital of Philadelphia Proteomics Core Facility for technical guidance and services; the NIH Tetramer Core Facility for providing tetramers for this study; J. R. Melamed and D. Weissman for technical advice on LNP generation; R. Serafin for providing related data; J. J. Rim for assistance with manuscript preparation; and D. F. Jenkins for data management support. We gratefully acknowledge the contributors to the Influenza Research Database, BV-BRC and the GISAID database, including the laboratories and authors responsible for obtaining specimens, generating genetic sequences and sharing data via the GISAID Initiative. M.J.H. was supported by the Cancer Research Institute as a Cancer Research Institute Irvington Fellow and by the Roberts Family–Katalin Karikó Fellowship in Vaccine Development from the Aileen K. and Brian L. Roberts Family Foundation via the University of Pennsylvania Institute for Immunology & Immune Health (I3H). N.M. was supported by the Roy and Diana Vagelos Molecular Life Sciences Program and by a College Alumni Society Research Grant from the University of Pennsylvania. N.P. was supported by NIH R01AI146101 and R01AI153064. S.P.R. is supported by research supplement 3R01AI046709-18S1 to promote diversity and L.B. is supported by NIH R01AI046709. K.W.L. and B.E.B. are supported by R01AI125524. L.C.E. and N.T. were supported by NIH R21AI153978. This work was funded in part by contract #75N93021C00015 from NIH NIAID. BioRender.com was used to create panels in Figs. 1, 2 and 8.

Author information

Author notes

  1. Nikita Maheshwari

    Present address: Department of Pathology, University of Chicago, Chicago, IL, USA

  2. Michael A. Miller

    Present address: Century Therapeutics, Philadelphia, PA, USA

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Michael J. Hogan, Nikita Maheshwari, Emma J. Hedgepeth & Laurence C. Eisenlohr

  2. School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA

    Nikita Maheshwari

  3. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Bridget E. Begg & Kristen W. Lynch

  4. The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Annalisa Nicastri & Nicola Ternette

  5. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Hiromi Muramatsu & Norbert Pardi

  6. Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA

    Michael A. Miller

  7. Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA

    Shanelle P. Reilly & Laurent Brossay

  8. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Laurence C. Eisenlohr

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Contributions

M.J.H. conceived the project, designed the studies, performed experiments, analyzed and interpreted the data and wrote the paper. N.M. co-conceived the project, co-designed studies, co-performed the immunoprecipitation, ELISpot and antigen presentation experiments and analyzed and interpreted data. B.E.B. performed primer extension assays and provided interpretation together with K.W.L. A.N. and N.T. performed LC–MS2, analyzed data and provided interpretation. E.J.H. provided essential support for animal studies. H.M. and N.P. contributed mRNA reagents and related expertise. M.A.M. isolated the B6.23 hybridoma. S.P.R. and L.B. provided reagents and expertise regarding MHC-Ia and Qa-1b−/− bone marrow. L.C.E. co-conceived and advised the project and interpreted the data. All authors provided critical scientific feedback, aided in the preparation of the manuscript and agree with the conclusions.

Corresponding authors

Correspondence to Michael J. Hogan or Laurence C. Eisenlohr.

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Competing interests

N.T. is or has been a paid consultant to Roche Pharma, Enara Bio, Grey Wolf Therapeutics, T-Cypher Bio and Infinitopes on the topic of cancer antigen discovery. All other authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks Katherine Kedzierska and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: S. Houston, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Peptide analysis by mass spectrometry (MS).

a, b, Histograms of peptide lengths of unique peptide species identified from (a) any origin and (b) IAV origin. c–f, NetMHCIIpan 4.0 was used to predict peptide:MHC-II affinity (KD) values for core epitope sequences of 9 amino acids from MS-identified peptides of all lengths. Sequence logo diagrams were prepared using unique core epitopes predicted to bind to (c,d) I-Ab or (e,f) I-Ed with a KD < 2,000 nM for peptides of (c,e) any origin or KD < 10,000 nM for peptides of (d,f) IAV origin. These sequence logos clearly exhibit the expected MHC-II ligand sequence motifs (based on ref. 34). g, The sequence logo diagram for all unique 9-mer peptide identifications (appearing as a small local peak in panel ‘a’) shows strong similarity to the H2-Kb and H2-Db sequence motifs, but not to the Qa-1 sequence motif (motifs available from NetMHCpan 4.0 at ref. 82). In sequence logo diagrams, ‘bit’ is a unit of relative amino acid frequency that is inversely related to the Shannon entropy of each position.

Extended Data Fig. 2 Identification of M-SL9 by mass spectrometry and validation of immunogenicity in C57Bl/6 mice.

a, MS2 spectrum resulting in M-SL9 identification, with b and y ion fragments indicated, along with mass/charge (m/z), retention time (RT), and P-value. b, c, Spleens were recovered from C57Bl/6 (N = 3) or BALB/c (N = 4) mice 9 days after infection with IAV PR8, and either bulk spleen cells or isolated spleen CD4 T cells were stimulated overnight with synthetic M-SL9 peptide, positive control peptides, or DMSO vehicle, and secreted IFN-γ was detected by ELISpot. b, For C57Bl/6 mice, the MHC-Ia control peptide was NS2109-121; the MHC-II control peptides were NP264-280 and NP306-322; and DC2.4 cells were used as the APC to stimulate CD4 T cells. c, For BALB/c mice, the MHC-II control peptides were NP55-71 and HA121-137, and A20 cells were used as the APC to stimulate CD4 T cells. Data points represent individual mice in one independent experiment; bars are the mean +/− s.e.m.

Extended Data Fig. 3 Annotation of M-SL9 coding sequence within the full IAV PR8 matrix gene segment.

The positive-sense RNA sequence is shown on top. Primary and secondary AUG codons are underlined and labeled, M-SL9 amino acids are highlighted in yellow, the M1 protein sequence is in light blue, M-MG16 nucleotides including stop codon are colored red, and relevant splice sites are labeled. The first nucleotide of each codon is aligned with the single-letter amino acid code and the first digit of the nucleotide number. This sequence was used to generate PR8 for this study and matches the sequence in GenBank accession AF389121.

Extended Data Fig. 4 Intracellular cytokines, cytolytic markers, and FoxP3 expression in individual mice.

a–d, Representative gating strategy for intracellular cytokine and cytolytic marker staining (lower boxes from two different experiments/stains). b–e, Lymphocytes were isolated from naïve (N = 21) or PR8 flu-infected (N = 34) C57Bl/6 mouse lungs, stimulated with indicated peptides, and stained for the indicated markers. b–d, Data for individual mice are shown in the same order for each epitope. c, Comparison of intracellular cytokine responses following infection with 40 FFU PR8 (N = 7), 160 FFU PR8 (N = 7), or no virus (naïve; N = 5), showing more consistent M-SL9 responses to 160 FFU. Female and male mice are indicated by purple and orange bars underneath the graphs. e, CD8 T cells (both total unstimulated as well as peptide-restimulated IFN-γ+ cells) from PR8-infected mice (N = 11) stain do not upregulate FoxP3 expression relative to the naïve (N = 4) mouse baseline. Gray events are all CD3+ cells; blue events and blue percentages represent CD3+ CD8+ cells. Bars show the mean +/- s.d. and P-values of interest are shown from a two-way ANOVA with Sidak’s multiple comparisons test comparing naïve and PR8-infected conditions. GzmB: granzyme B.

Extended Data Fig. 5 Hybridoma clone B6.23 recognizes two forms of M-SL9 present in isolates of PR8.

Amino acid sequences are shown for the originally identified M-SL9, present in pDZ PR8, and M-SL9-P, present in other PR8 isolates (for example GenBank V01099). B6-CIITA fibroblasts served as APCs and were co-cultured overnight with B6.23 cells in the presence of the indicated peptide concentrations. A sigmoidal curve was fit to the data points above (mean +/− s.d.), representative of three independent experiments. The geometric mean half-maximal effective concentration (EC50) values across all three experiments were computed as 940 ng/ml for M-SL9 and 51 ng/ml for M-SL9-P.

Extended Data Fig. 6 Evidence supporting Qa-1 restriction of M-SL9.

a, The MHC-Ia molecules H2-Db and H2-Kb are not stabilized on RMA-S cells by M-SL9 peptide. RMA-S cells bearing unstable empty MHC-I molecules (due to TAP deficiency) were incubated in the presence of the indicated synthetic peptides, and surface expression of H2-Db and H2-Kb was measured by flow cytometry. Mean fluorescence intensities of each stain were normalized to the negative control condition using HA91-107, an I-Ab-binding epitope with no known binding to H2-Db or H2-Kb, and shown as averages +/- s.d from 3 independent experiments. H2-Db-binding NP366-374 and H2-Kb-binding SIINFEKL were used as positive controls. b-e, Validation of HeLa cell lines and BMDCs showing Qa-1 restriction of M-SL9. b, The sufficiency of Qa-1b expression for M-SL9 presentation to its cognate T hybridoma was confirmed using a HeLa cell line transduced with full-length, wild-type Qa-1b and an untransduced parental HeLa cell line as a control. Bars are mean +/- s.e.m. from triplicate technical replicates, representative of 3 independent experiments, and P-values were calculated by Welch’s t-test (two-tailed). c, Qa-1 expression on cell lines used in b was validated by flow cytometry. d, The expected staining pattern was confirmed for HeLa cell lines used in Fig. 2; these lines were transduced with retroviruses encoding chimeric MHC-Ib molecules containing the α3 domain (D3) from H2-Db to allow efficient staining with the H2-Db D3-specific mAb 28-14-8. e, The expected staining pattern was also confirmed for BMDCs used in Fig. 2.

Extended Data Fig. 7 Qdm/Qa-1b tetramer co-stains with NKG2A/C/E.

a, Lung lymphocytes from naïve C57Bl/6 mice were stained with an anti-NKG2A/C/E mAb and Qdm/Qa-1b, M-SL9/Qa-1b, and control NP366-374/Db tetramers at 37 °C and gated on CD3− CD19− cells to interrogate natural killer (NK) cells. NK cells expressing NKG2A/C/E (the natural receptors for Qdm/Qa-1b) were the only population that stained with Qdm/Qa-1b tetramer, but neither these nor other NK cells stained with M-SL9/Qa-1b tetramer. b, C57Bl/6 mice were intranasally infected with 160 FFU of PR8 and 9 days later lung lymphocytes were stained with anti-NKG2A/C/E and the indicated tetramers at 37 °C. Qdm/Qa-1b tetramer generally stained PR8-induced CD8 T cells in a manner that was dependent on NKG2A/C/E but independent of TCR specificity. Flow plots are representative and show the gating strategy used, and bars show mean +/- s.e.m. for (a) N = 4 mice and (b) N = 5 to 6 mice per group across 2 independent experiments each. P-values are shown from two-way ANOVA with Dunnett’s multiple comparisons test.

Extended Data Fig. 8 Analysis of TCRβ V and J gene usage in sorted CD8 T cell populations.

a-d, CD8 T cell populations were sorted by FACS into three populations: naïve (CD44− CD62L+), M-SL9-specific (CD44+ M-SL9/Qa-1b tetramer+), and NP366-374-specific (CD44+ NP366-374/H2-Db tetramer+). Genomic DNA was isolated, the VDJ region of recombined TCRβ-coding genes was sequenced, and gene usage was analyzed by (a,b) the immunoSEQ Analyzer and (c,d) Immunarch. a-b, The frequencies of the top 10 most-used (a) V genes and (b) J genes, on average across all mice, are shown as stacked bar graphs, where each bar represents one mouse. c, Principal component analysis (PCA) of the Tcrb V and J gene usage showing clustering by T cell population. d, Pearson correlation analysis of M-SL9- and NP366-374-specific T cells showing greater correlation between mice within each T cell specificity rather than between specificities within each mouse. N = 6 mice, half males and half females.

Extended Data Fig. 9 Tracking CD69 and CD103 expression in PR8-infected mice.

a-d, C57Bl/6 mice were intranasally infected with 160 FFU of PR8 and were euthanized at day 6 (N = 7), 9 (N = 5-6), 14, 31 (N = 7), or 56 (N = 9) to collect the indicated tissues/fluids. Uninfected mice were used as day 0 controls (N = 7-11). a, Gating strategy. b, Frequency of all CD3+ T cells in lung and BALF over time, showing the lack of T cell infiltration in uninfected mice (plotted as day 0). Data points were omitted when there were <20 live singlet CD3+ CD8+ T cells collected in total. P-values are calculated from Brown-Forsythe and Welch one-way ANOVA with Dunnett T3 multiple comparisons test comparing each condition to day 0 controls. c, Frequency of CD103+ and CD69+ CD8 T cells (analyzed separately) in lung and BALF starting from the approximate peak of the T cell response on day 9. d, Frequency of CD103+ CD69+ double positive TRM cells in lung at 9 days after PR8 only, X31 only, or PR8 prime and X31 boost. c, d, P-values were calculated by two-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 10 Sequence evolution and variation of the M-SL9 epitope and open reading frame in IAV strains over time since 1980.

a–c, Sequence logo diagrams were produced from M-SL9-homologous sequences from (a) human H1N1 isolates, (b) human H3N2 isolates, and (c) H5N1 isolates from all avian species, downloaded between April and June 2023 for the indicated sample collection time periods. The BV-BRC database was used for sequences from 1980-1999, while the GISAID database was used for all others. Diagrams were created using the ggseqlogo package in R, and the y-axis units are the probability of each amino acid from 0 to 1. H1N1 sequences after 2009 correspond to the swine-origin pandemic H1N1 lineage, while H1N1 sequences prior to 2009 are from the earlier seasonal H1N1 lineage; sequences from 2009 were omitted to avoid ambiguity. Amino acids are numbered so that position 1 corresponds to the initial serine residue of the M-SL9 epitope, and the preceding residue was designated as position −1 and shown to assess the presence of an initiation codon. The two forms of M-SL9 encoded by PR8 isolates are shown at bottom; note that the avian H5N1 consensus sequence exactly matches the M-SL9-P amino acid sequence.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Source data

Source Data Fig. 7

Source data for Fig. 7d (full unprocessed image for IAV matrix RNA primer extension gel).

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Hogan, M.J., Maheshwari, N., Begg, B.E. et al. Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response. Nat Immunol 24, 1933–1946 (2023). https://doi.org/10.1038/s41590-023-01644-5

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