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Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell … – Nature.com

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Nature Immunology (2023)
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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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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 supporting analysis of TCR sequences and M-SL9 variant sequences is deposited in GitHub at the URL https://github.com/mhogan240/NatImmuno2023.
Hansen, S. G. et al. Immune clearance of highly pathogenic SIV infection. Nature 502, 100–104 (2013).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Hansen, S. G. et al. A live-attenuated RhCMV/SIV vaccine shows long-term efficacy against heterologous SIV challenge. Sci. Transl. Med. 11, eaaw2607 (2019).
Article  PubMed  PubMed Central  Google Scholar 
Malouli, D. et al. Cytomegaloviral determinants of CD8+ T cell programming and RhCMV/SIV vaccine efficacy. Sci. Immunol. 6, eabg5413 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Hansen, S. G. et al. Myeloid cell tropism enables MHC-E–restricted CD8+ T cell priming and vaccine efficacy by the RhCMV/SIV vaccine. Sci. Immunol. 7, eabn9301 (2022).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Anderson, C. K., Reilly, E. C., Lee, A. Y. & Brossay, L. Qa-1-restricted CD8+ T cells can compensate for the absence of conventional T cells during viral infection. Cell Rep. 27, 537–548 (2019).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Chen, L., Jay, D. C., Fairbanks, J. D., He, X. & Jensen, P. E. An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus. J. Immunol. 187, 6463–6472 (2011).
Article  CAS  PubMed  Google Scholar 
Hansen, S. G. et al. Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine. Nat. Med. 24, 130–143 (2018).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Rodgers, J. R. & Cook, R. G. MHC class Ib molecules bridge innate and acquired immunity. Nat. Rev. Immunol. 5, 459–471 (2005).
Article  CAS  PubMed  Google Scholar 
Braud, V. M. et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391, 795–799 (1998).
Article  CAS  PubMed  Google Scholar 
Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E. & Raulet, D. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical MHC class I molecule Qa-1b. J. Exp. Med. 188, 1841–1848 (1998).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Anderson, C. K. & Brossay, L. The role of MHC class Ib-restricted T cells during infection. Immunogenetics 68, 677–691 (2016).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Chen, X.-R. et al. A signal peptide derived from Hsp60 induces protective cytotoxic T lymphocyte immunity against lymphoid malignancies independently of TAP and classical MHC-I. Cancer Lett. 494, 47–57 (2020).
Article  CAS  PubMed  Google Scholar 
Malouli, D. et al. Cytomegalovirus-vaccine-induced unconventional T cell priming and control of SIV replication is conserved between primate species. Cell Host Microbe 30, 1207–1218.e7 (2022).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Grifoni, A. et al. SARS-CoV-2 human T cell epitopes: adaptive immune response against COVID-19. Cell Host Microbe 29, 1076–1092 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Mahajan, S. et al. Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals. Sci. Rep. 11, 13164 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Saini, S. K. et al. SARS-CoV-2 genome-wide T cell epitope mapping reveals immunodominance and substantial CD8+ T cell activation in COVID-19 patients. Sci. Immunol. 6, eabf7550 (2021).
Article  PubMed  PubMed Central  Google Scholar 
Bullock, T. N. & Eisenlohr, L. C. Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames. J. Exp. Med. 184, 1319–1329 (1996).
Article  CAS  PubMed  Google Scholar 
Bullock, T. N. J., Patterson, A. E., Franlin, L. L., Notidis, E. & Eisenlohr, L. C. Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I–restricted cryptic epitope expression. J. Exp. Med. 186, 1051–1058 (1997).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Schwab, S. R., Li, K. C., Kang, C. & Shastri, N. Constitutive display of cryptic translation products by MHC class I molecules. Science 301, 1367–1371 (2003).
Article  CAS  PubMed  Google Scholar 
Zook, M. B., Howard, M. T., Sinnathamby, G., Atkins, J. F. & Eisenlohr, L. C. Epitopes derived by incidental translational frameshifting give rise to a protective CTL response. J. Immunol. 176, 6928–6934 (2006).
Article  CAS  PubMed  Google Scholar 
Starck, S. R. et al. Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Science 336, 1719–1723 (2012).
Article  CAS  PubMed  Google Scholar 
Apcher, S. et al. Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway. Proc. Natl Acad. Sci. USA 110, 17951–17956 (2013).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Goodenough, E. et al. Cryptic MHC class I-binding peptides are revealed by aminoglycoside-induced stop codon read-through into the 3′ UTR. Proc. Natl Acad. Sci. USA 111, 5670–5675 (2014).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Yang, N. et al. Defining viral defective ribosomal products: standard and alternative translation initiation events generate a common peptide from influenza A virus M2 and M1 mRNAs. J. Immunol. 196, 3608–3617 (2016).
Article  CAS  PubMed  Google Scholar 
Sanz, M. A., Almela, E. G., García-Moreno, M., Marina, A. I. & Carrasco, L. A viral RNA motif involved in signaling the initiation of translation on non-AUG codons. RNA 25, 431–452 (2019).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Zanker, D. J. et al. Influenza A virus infection induces viral and cellular defective ribosomal products encoded by alternative reading frames. J. Immunol. 202, 3370–3380 (2019).
Article  CAS  PubMed  Google Scholar 
Hanada, K., Yewdell, J. W. & Yang, J. C. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427, 252–256 (2004).
Article  CAS  PubMed  Google Scholar 
Delong, T. et al. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science 351, 711–714 (2016).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Paes, W. et al. Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8+ T cells in HIV-1 infection. Proc. Natl Acad. Sci. USA 116, 24748–24759 (2019).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Tran, M. T. et al. T cell receptor recognition of hybrid insulin peptides bound to HLA-DQ8. Nat. Commun. 12, 5110 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Purcell, A. W. Is the immunopeptidome getting darker?: a commentary on the discussion around Mishto et al., 2019. Front. Immunol. 12, 720811 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Miller, M. A., Ganesan, A. P. V., Luckashenak, N., Mendonca, M. & Eisenlohr, L. C. Endogenous antigen processing drives the primary CD4+ T cell response to influenza. Nat. Med. 21, 1216–1222 (2015).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Tewari, M. K., Sinnathamby, G., Rajagopal, D. & Eisenlohr, L. C. A cytosolic pathway for MHC class II-restricted antigen processing that is proteasome and TAP dependent. Nat. Immunol. 6, 287–294 (2005).
Article  CAS  PubMed  Google Scholar 
Reynisson, B. et al. Improved prediction of MHC II antigen presentation through integration and motif deconvolution of mass spectrometry MHC eluted ligand data. J. Proteome Res. 19, 2304–2315 (2020).
Article  CAS  PubMed  Google Scholar 
Parker, R. et al. Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells. Cell Rep. 35, 109179 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Partridge, T. et al. Discrimination between human leukocyte antigen class I-bound and co-purified HIV-derived peptides in immunopeptidomics workflows. Front. Immunol. 9, 912 (2018).
Article  PubMed  PubMed Central  Google Scholar 
Martínez-Sobrido, L. & García-Sastre, A. Generation of recombinant influenza virus from plasmid DNA. J. Vis. Exp. https://doi.org/10.3791/2057 (2010).
Ljunggren, H.-G. et al. Empty MHC class I molecules come out in the cold. Nature 346, 476–480 (1990).
Article  CAS  PubMed  Google Scholar 
Kraft, J. R. et al. Analysis of Qa-1b peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1–peptide complexes. J. Exp. Med. 192, 613–624 (2000).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Ying, G., Wang, J., Kumar, V. & Zajonc, D. M. Crystal structure of Qa-1a with bound Qa-1 determinant modifier peptide. PLoS ONE 12, e0182296 (2017).
Article  PubMed  PubMed Central  Google Scholar 
Davies, A. et al. Infection-induced expansion of a MHC class Ib-dependent intestinal intraepithelial γδ T cell subset. J. Immunol. 172, 6828–6837 (2004).
Article  CAS  PubMed  Google Scholar 
Tang, X. et al. Regulation of immunity by a novel population of Qa-1-restricted CD8αα+ TCRαβ+ T cells. J. Immunol. 177, 7645–7655 (2006).
Article  CAS  PubMed  Google Scholar 
Niederlova, V., Tsyklauri, O., Chadimova, T. & Stepanek, O. CD8+ Tregs revisited: a heterogeneous population with different phenotypes and properties. Eur. J. Immunol. 51, 512–530 (2021).
Article  CAS  PubMed  Google Scholar 
Miller, J. D. et al. CD94/NKG2 expression does not inhibit cytotoxic function of lymphocytic choriomeningitis virus-specific CD8+ T cells. J. Immunol. 169, 693–701 (2002).
Article  CAS  PubMed  Google Scholar 
Borst, L. et al. NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division. Int. J. Cancer 150, 688–704 (2022).
Article  CAS  PubMed  Google Scholar 
Van Montfoort, N. et al. NKG2A blockade potentiates CD8 T cell immunity induced by cancer vaccines. Cell 175, 1744–1755.e15 (2018).
Article  PubMed  PubMed Central  Google Scholar 
Borst, L., Van Der Burg, S. H. & Van Hall, T. The NKG2A–HLA-E axis as a novel checkpoint in the tumor microenvironment. Clin. Cancer Res. 26, 5549–5556 (2020).
Article  CAS  PubMed  Google Scholar 
Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire. Nat. Commun. 5, 3866 (2014).
Article  PubMed  Google Scholar 
Yuan, R. et al. The roles of tissue-resident memory T cells in lung diseases. Front. Immunol. 12, 710375 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Schön, M. P. et al. Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice. J. Immunol. 162, 6641–6649 (1999).
Article  PubMed  Google Scholar 
Takamura, S. et al. Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance. J. Exp. Med. 213, 3057–3073 (2016).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Walsh, D. A. et al. The functional requirement for CD69 in establishment of resident memory CD8+ T cells varies with tissue location. J. Immunol. 203, 946–955 (2019).
Article  CAS  PubMed  Google Scholar 
Laidlaw, B. J. et al. CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection. Immunity 41, 633–645 (2014).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Zens, K. D., Chen, J. K. & Farber, D. L. Vaccine-generated lung tissue-resident memory T cells provide heterosubtypic protection to influenza infection. JCI Insight 1, e85832 (2016).
Article  PubMed  PubMed Central  Google Scholar 
Belz, G. T., Xie, W., Altman, J. D. & Doherty, P. C. A previously unrecognized H-2Db-restricted peptide prominent in the primary influenza A virus-specific CD8+ T-cell response is much less apparent following secondary challenge. J. Virol. 74, 3486–3493 (2000).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Machkovech, H. M., Bloom, J. D. & Subramaniam, A. R. Comprehensive profiling of translation initiation in influenza virus infected cells. PLoS Pathog. 15, e1007518 (2019).
Article  PubMed  PubMed Central  Google Scholar 
Wise, H. M. et al. Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain. PLoS Pathog. 8, e1002998 (2012).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Pardi, N., Hogan, M. J. & Weissman, D. Recent advances in mRNA vaccine technology. Curr. Opin. Immunol. 65, 14–20 (2020).
Article  CAS  PubMed  Google Scholar 
Liu, X. et al. MARCH8 inhibits influenza A virus infection by targeting viral M2 protein for ubiquitination-dependent degradation in lysosomes. Nat. Commun. 12, 4427 (2021).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Kozak, M. Adherence to the first-AUG rule when a second AUG codon follows closely upon the first. Proc. Natl Acad. Sci. USA 92, 2662–2666 (1995).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Hogan, M. J. & Pardi, N. mRNA vaccines in the COVID-19 pandemic and beyond. Annu. Rev. Med. 73, 17–39 (2022).
Article  PubMed  Google Scholar 
Knudson, C. J., Hartwig, S. M., Meyerholz, D. K. & Varga, S. M. RSV vaccine-enhanced disease is orchestrated by the combined actions of distinct CD4 T cell subsets. PLoS Pathog. 11, 1–23 (2015).
Article  Google Scholar 
Wei, J. & Yewdell, J. W. Flu DRiPs in MHC class I immunosurveillance. Virol. Sin. 34, 162–167 (2019).
Article  PubMed  Google Scholar 
Lodha, M., Erhard, F., Dölken, L. & Prusty, B. K. The hidden enemy within: non-canonical peptides in virus-induced autoimmunity. Front. Microbiol. 13, 840911 (2022).
Article  PubMed  PubMed Central  Google Scholar 
Kracht, M. J. L. et al. Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes. Nat. Med. 23, 501–507 (2017).
Article  CAS  PubMed  Google Scholar 
Marcu, A. et al. Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors. J. Immunother. Cancer 9, e003404 (2021).
Article  PubMed  PubMed Central  Google Scholar 
Chong, C. et al. Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes. Nat. Commun. 11, 1293 (2020).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Ruiz Cuevas, M. V. et al. Most non-canonical proteins uniquely populate the proteome or immunopeptidome. Cell Rep. 34, 108815 (2021).
Article  CAS  PubMed  Google Scholar 
Croft, N. P. et al. Kinetics of antigen expression and epitope presentation during virus infection. PLoS Pathog. 9, e1003129 (2013).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Wu, T. et al. Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses. Nat. Commun. 10, 2846 (2019).
Article  PubMed  PubMed Central  Google Scholar 
Yewdell, J. W., Dersh, D. & Fåhraeus, R. Peptide channeling: the key to MHC class I immunosurveillance? Trends Cell Biol. 29, 929–939 (2019).
Article  CAS  PubMed  Google Scholar 
Rutigliano, J. A. et al. Highly pathological influenza A virus infection is associated with augmented expression of PD-1 by functionally compromised virus-specific CD8+ T cells. J. Virol. 88, 1636–1651 (2014).
Article  PubMed  PubMed Central  Google Scholar 
Vogel, A. J., Harris, S., Marsteller, N., Condon, S. A. & Brown, D. M. Early cytokine dysregulation and viral replication are associated with mortality during lethal influenza infection. Viral Immunol. 27, 214–224 (2014).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Seaman, M. S., Wang, C.-R. & Forman, J. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J. Immunol. 165, 5192–5201 (2000).
Article  CAS  PubMed  Google Scholar 
Laidlaw, B. J. et al. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity. PLoS Pathog. 9, e1003207 (2013).
Article  CAS  PubMed  PubMed Central  Google Scholar 
LaMere, M. W. et al. Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus. J. Immunol. 186, 4331–4339 (2011).
Article  CAS  PubMed  Google Scholar 
Kanevskiy, L. et al. Dimorphism of HLA-E and its disease association. Int. J. Mol. Sci. 20, 5496 (2019).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Sahin, U. et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222–226 (2017).
Article  CAS  PubMed  Google Scholar 
Rojas, L. A. et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature 618, 144–150 (2023).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Voogd, L., Ruibal, P., Ottenhoff, T. H. M. & Joosten, S. A. Antigen presentation by MHC-E: a putative target for vaccination? Trends Immunol. 43, 355–365 (2022).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Sinnathamby, G., Maric, M., Cresswell, P. & Eisenlohr, L. C. Differential requirements for endosomal reduction in the presentation of two H2-Ed-restricted epitopes from influenza hemagglutinin. J. Immunol. 172, 6607–6614 (2004).
Article  CAS  PubMed  Google Scholar 
Sanderson, S. & Shastri, N. LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369–376 (1994).
Article  CAS  PubMed  Google Scholar 
Chen, L. et al. Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity. J. Immunol. 193, 1427–1439 (2014).
Article  CAS  PubMed  Google Scholar 
Purcell, A. W., Ramarathinam, S. H. & Ternette, N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat. Protoc. 14, 1687–1707 (2019).
Article  CAS  PubMed  Google Scholar 
Pardi, N., Muramatsu, H., Weissman, D. & Karikó, K. In vitro transcription of long RNA containing modified nucleosides. Methods Mol. Biol. 969, 29–42 (2013).
Article  CAS  PubMed  Google Scholar 
Laczkó, D. et al. A single immunization with nucleoside-modified mRNA vaccines elicits strong cellular and humoral immune responses against SARS-CoV-2 in mice. Immunity 53, 724–732.e7 (2020).
Article  PubMed  PubMed Central  Google Scholar 
Baiersdörfer, M. et al. A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA. Mol. Ther. Nucleic Acids 15, 26–35 (2019).
Article  PubMed  PubMed Central  Google Scholar 
Freyn, A. W. et al. A multi-targeting, nucleoside-modified mRNA influenza virus vaccine provides broad protection in mice. Mol. Ther. 28, 1569–1584 (2020).
Article  CAS  PubMed  PubMed Central  Google Scholar 
Thompson, M. G. et al. Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing. Nat. Commun. 9, 2407 (2018).
Article  PubMed  PubMed Central  Google Scholar 
Shu, Y. & McCauley, J. GISAID: global initiative on sharing all influenza data—from vision to reality. Eurosurveillance 22, 30494 (2017).
Article  PubMed  PubMed Central  Google Scholar 
Wagih, O. ggseqlogo: a versatile R package for drawing sequence logos. Bioinformatics 33, 3645–3647 (2017).
Article  CAS  PubMed  Google Scholar 
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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.
Nikita Maheshwari
Present address: Department of Pathology, University of Chicago, Chicago, IL, USA
Michael A. Miller
Present address: Century Therapeutics, Philadelphia, PA, USA
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
School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA
Nikita Maheshwari
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Bridget E. Begg & Kristen W. Lynch
The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK
Annalisa Nicastri & Nicola Ternette
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Hiromi Muramatsu & Norbert Pardi
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA
Michael A. Miller
Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA
Shanelle P. Reilly & Laurent Brossay
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Laurence C. Eisenlohr
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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.
Correspondence to Michael J. Hogan or Laurence C. Eisenlohr.
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.
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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a, b, Histograms of peptide lengths of unique peptide species identified from (a) any origin and (b) IAV origin. cf, 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.
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.
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.
ad, Representative gating strategy for intracellular cytokine and cytolytic marker staining (lower boxes from two different experiments/stains). be, 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. bd, 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.
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.
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.
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.
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.
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.
ac, 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 Tables 1 and 2.
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 (2023). https://doi.org/10.1038/s41590-023-01644-5
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DOI: https://doi.org/10.1038/s41590-023-01644-5
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