Abstract

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. scRNA-seq data that support the findings of this study have been deposited in Arrayexpress with accession code E-MTAB-7606.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

HLA-A2.1 transgenic HHD mice were developed by F. Lemonnier (Pasteur Institute). D227K and Q115E MHC-I constructs were developed and provided by L. Wooldridge (Bristol University). The authors acknowledge the provision of instrumentation, training and technical support by the Monash Biomedical Proteomics Facility. We thank all study participants and organ donors. We are grateful to the coordinators of DonateLife Victoria for obtaining organ tissues, and to L. Mariana and C. Kos (St. Vincent’s Institute) for their assistance. The Australian National Health and Medical Research Council (NHMRC) NHMRC Program Grant (1071916) to K.K. supported this work. M.K. is a recipient of a Melbourne International research scholarship and Melbourne International fee remission scholarship. S.S. is a recipient of a Victoria India doctoral scholarship and Melbourne International fee remission scholarship, University of Melbourne. K.K. is an NHMRC senior research level B fellow (1102792). J.R. is supported by an ARC laureate fellowship. S.G. is a Monash senior research fellow. F.L. and A.C.C. are NHMRC CDF2 fellows. E.B.C. and A.A.E. are NHMRC Peter Doherty fellows. The Melbourne WHO Collaborating Centre for Reference Research on Influenza is supported by the Australian Government Department of Health. A.W.P. is supported by an NHMRC principal research fellowship (1137739) and an NHMRC project grant (1085018) to A.W.P., T.C.K. and N.A.M. D.T. and D.V. are supported by contract HHSN272201400006C from the US National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Department of Health and Human Services. P.T.I. was supported by a NHMRC Peter Doherty fellowship (1072159). P.G.T. is supported by the St. Jude Center of Excellence for Influenza Research and Surveillance (NIAID contract HHSN27220140006C), R01AI107625, R01AI136514, and American Lebanese Syrian Associated Charities. S.I.M. is supported by a JDRF Career Development award (5-CDA2014210-A-N) and NHMRC Project (GNT 1123586).

Author information

Author notes

  1. These authors jointly supervised this work: A. W. Purcell, K. Kedzierska.

Affiliations

  1. Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia

    • Marios Koutsakos
    • , Thi H. O. Nguyen
    • , E. Bridie Clemens
    • , Sneha Sant
    • , Brendon Y. Chua
    • , Chinn Yi Wong
    • , Ludivine Grzelak
    • , Weiguang Zeng
    • , Aeron C. Hurt
    • , Ian Barr
    • , Steve Rockman
    • , David C. Jackson
    • , Linda M. Wakim
    •  & Katherine Kedzierska
  2. Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    • Patricia T. Illing
    • , Nicole A. Mifsud
    • , Jamie Rossjohn
    • , Stephanie Gras
    •  & Anthony W. Purcell
  3. Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA

    • Jeremy Chase Crawford
    • , E. Kaitlynn Allen
    • , Pradyot Dash
    • , David F. Boyd
    •  & Paul G. Thomas
  4. School of Medical Sciences and The Kirby Institute, UNSW, Sydney, New South Wales, Australia

    • Simone Rizzetto
    • , Auda A. Eltahla
    •  & Fabio Luciani
  5. Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan

    • Brendon Y. Chua
    •  & David C. Jackson
  6. Infection and Immunity Program & Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    • Don Teng
    •  & Dhanasekaran Vijaykrishna
  7. Biology Department, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, Cachan, France

    • Ludivine Grzelak
  8. World Health Organization (WHO) Collaborating Centre for Reference and Research on Influenza, at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    • Aeron C. Hurt
    •  & Ian Barr
  9. School of Applied Biomedical Sciences, Federation University, Churchill, Victoria, Australia

    • Ian Barr
  10. Seqirus, Parkville, Victoria, Australia

    • Steve Rockman
  11. Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital, Melbourne, Victoria, Australia

    • Tom C. Kotsimbos
  12. Department of Medicine, Monash University, Central Clinical School, The Alfred Hospital, Melbourne, Victoria, Australia

    • Tom C. Kotsimbos
  13. School of Public Health and Preventive Medicine, Monash University, Melbourne, Victoria, Australia

    • Allen C. Cheng
  14. Infection Prevention and Healthcare Epidemiology Unit, Alfred Health, Melbourne, Victoria, Australia

    • Allen C. Cheng
  15. Victorian Infectious Diseases Service, The Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia

    • Michael Richards
  16. Lung Transplant Unit, Alfred Hospital, Melbourne, Victoria, Australia

    • Glen P. Westall
    •  & Stuart I. Mannering
  17. Immunology and Diabetes Unit, St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

    • Thomas Loudovaris
  18. Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

    • Michael Elliott
  19. Chris O’Brien Lifehouse Cancer Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia

    • Michael Elliott
  20. Immunology Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia

    • Stuart G. Tangye
  21. St. Vincent’s Clinical School, University of New South Wales, Sydney, New South Wales, Australia

    • Stuart G. Tangye
  22. Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

    • Jamie Rossjohn
    •  & Stephanie Gras
  23. Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, UK

    • Jamie Rossjohn

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Contributions

M.K., K.K., T.H.O.N., P.T.I., N.A.M., A.W.P., S.G., D.V., F.L. and P.G.T. designed experiments. M.K., P.I., T.H.O.N., N.A.M., A.A.E., E.B.C., S.S., C.Y.W., B.Y.C., E.K.A., P.D., L.G., W.Z., D.F.B. and S.G. performed experiments. A.C.H., I.B., D.C.J., T.C.K., A.C.C., M.R., G.P.W., L.M.W., S.G.T., S.I.M., T.L., S.Rockman, M.E. and P.G.T. provided reagents and/or samples. M.K., P.I., T.H.O.N., J.C.C., S.S., S.Rizzetto, D.T., D.V., F.L. S.G., J.R., P.G.T., A.W.P. and K.K. analyzed data. M.K., T.H.O.N. and K.K. wrote the manuscript. All authors read and approved the manuscript.

Competing interests

S. Rockman is an employee of Seqirus but has no conflict of interest in the material presented. M.K., K.K. and E.B.C. are named as co-inventors in a patent application filed by the University of Melbourne (AU2017903652) covering the use of certain peptides described in the publication as part of vaccine formulation.

Corresponding author

Correspondence to Katherine Kedzierska.

Integrated supplementary information

  1. Supplementary Figure 1 Gating strategy for human CD8+ T cells for ICS assay.

    CD8+ T cells were gated as CD8+CD3+CD14CD19 live single lymphocytes.

  2. Supplementary Figure 2 Immunopeptidomic analysis of human and viral peptides.

    (a) Infection and viability rates for the immunopeptidomics experiments. (b) Experimental outline for immunopeptidomics analysis of IBV-infected C1R cells. Lysates were generated from C1R parental or C1R.A*02:01 cells and the transfected HLA-A*02:01, endogenous (parental) class I and class II molecules immunoaffinity purified sequentially using BB7.2 (HLA-A2 specific), w6/32 (pan class I) and a mixture of LB3.1 (HLA-DR), SPV-L3 (HLA-DQ) and B721 (HLA-DP). Peptides were eluted from the purified HLA and interrogated by LC–MS/MS using an information dependent acquisition (IDA) strategy and preliminary lists of HLA ligands generated for HLA-A*02:01, parental class I and class II. HLA-A*02:01 ligands were further refined by filtering for sequences in C1R parental purifications from all antibodies (represent non-specific pull down by BB7.2, and contamination with endogenous HLA molecules) as well as sequences in C1R.A*02:01 LB3.1/SPV-L3/B721 purifications (represent contamination with endogenous class II molecules). Peptides from C1R.A*02:01 w6/32 purifications were not included in the contaminant list due to the potential for excess HLA-A*02:01 not bound to the preceding BB7.2 resin to be isolated by w6/32. (c) Number of peptides identified per condition and experiment. (d) Distribution of IBV-derived HLA ligands across the B/Malaysia proteome, distinguished as likely parental class I (HLA-B*35:03 and HLA-C*04:01) or HLA class II ligands. All immunopeptidomic data are from two independent experiments.

  3. Supplementary Figure 3 Dissection of peptide pools in human CD8+ T cell lines.

    Frequency of IFN-γ+TNF+CD8+ T cells for individual peptides for each pool. Dots indicate individual donors and bars indicate the mean (n = 2–5). Red boxes indicate immunogenic peptides. (b) Frequency of responding donors for each immunogenic peptide (n = 6). (c, d) conservation of IBV peptides identified by immunopepidomics. (c) Conservation (average amino acid identity) of novel IBV peptides across IBV viruses in each lineage between 1940 and 2017. (d) Conservation (average amino acid identity) of novel IBV peptides in IAV and ICV. The bars indicate the percent conservation (average amino acid identity) of each peptide across the three types of viruses.

  4. Supplementary Figure 4 Gating strategy for TAME and phenotyping of tetramer+ CD8+ T cells.

    (a) Tetramer+ CD8+ T cells were identified within CD8+CD3+CD14CD19 live single lymphocytes. (b) Representative FACS plots for each marker analyzed on tetramer+ cells. Total CD8+ cells are shown for comparison. (c) Representative FACS plots for Ki-67 staining on tetramer+ and total CD8+ cells along with an isotype control representative of one experiment.

  5. Supplementary Figure 5 Phenotype of tetramer+ CD8+ T cells in healthy donors and during infection with influenza virus.

    Expression of single markers on tetramer+ CD8+ T cells in healthy control (HC) and influenza-infected (IAV or IBV) patients or influenza-negative ILI patients (n = 3 for HC M158, 4 for PB1413 and BHA543 HC, 16 for IAV, 6 IBV, 7 Flu-neg ILI assessed over at least two independent experiments). Median and IQR are shown. A two tailed Mann-Whitney test was used to assess statistical significance. *p < 0.05, **p < 0.005.

  6. Supplementary Figure 6 IBV infection of donor MK34 and expression of selected genes in tetramer+ CD8+ T cells from scRNA-seq.

    (a) Phylogenetic analysis of PCR product from nasal swab. (b) Serological analysis before (~3 months prior) and after (~d14) IBV infection. Antibody titers against the two IBV lineages and IAV were assessed by haemagglutination inhibition assay. (c) Genes relating to T cell differentiation, activation and cytotoxicity or effector function were analyzed. Cells grouped by epitope and time point. (d) Validation of T cell activation genes from scRNA-seq by FACS. Violin plots show gene expression as determined by scRNA-seq and histograms show protein expression as determine by flow cytometry, for selected genes in available samples.

  7. Supplementary Figure 7 Recall CD8+ T cell responses in HHD-A2 mice.

    (a) Experimental outline for secondary infection with IBV. (b) Gating strategy. (c) Number of IFN-γ/TNF-producing CD8+ T cells specific for A2/BHA543 during 1o and 2o IBV infections (n = 5 mice for 1o and n = 4 for 2o). Mean and SEM are shown. (d) Polyfunctionality of BHA543 and BNS1266 during 1o and 2o IBV infections. % of triple (IFN-γ+IL-2+TNF+) CD8+ T cells of the two specificities during 1o and 2o IBV infections (n = 5 mice for 1o and n = 4 for 2o). Mean and SEM are shown. Statistical significance was determine using an unpaired two-tailed t-test, *p < 0.05, **p < 0.005.

  8. Supplementary Figure 8 Lack of A2/PB1413 CD8+ T cells in HHD-A2 mice after infection with influenza virus or PB1413 vaccination.

    Tetramer staining for A2/PB1413 after (a) primary, (b) secondary or (c) tertiary heterotypic infections. Response shown are from BAL, with similar results obtained from the spleen. A2/M158 and A2/BHA543 tetramer staining were used as positive controls. (d) Lack of A2/PB1413+CD8+ T cell responses after Pam2Cys lipopeptide vaccination. Splenocytes from vaccinated mice were enriched by TAME. An additional group of mice was vaccinated with an M158 lipopetide as a positive control. Representative FACS plots from a spleen are shown, with similar results obtained from the BAL. (e) Human PBMC stimulation with PB1413 lipopeptide to confirm its immunogenicity. (f) Lack of A2/PB1413 responses after peptide vaccination and IBV infection. Mice were vaccinated as in Supplementary Figure 7a. A2/BHA543 vaccination and tetramer staining were used as positive controls. (g) Lack of A2/PB1413-specific CD8+ T cells in naïve mice. Spleen and major lymph nodes were harvested and enriched by TAME. A C57/B6 mouse (A2) was used as a control or background tetramer staining. TAME for A2/WT1A-specific CD8+ T cells was used as a positive control for naïve TAME. Data from one experiment.

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https://doi.org/10.1038/s41590-019-0320-6