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Pulmonary immunization with a recombinant influenza A virus vaccine induces lung-resident CD4+ memory T cells that are associated with protection against tuberculosis

Mucosal Immunologyvolume 11pages17431752 (2018) | Download Citation



The lung is the primary site of infection with the major human pathogen, Mycobacterium tuberculosis. Effective vaccines against M. tuberculosis must stimulate memory T cells to provide early protection in the lung. Recently, tissue-resident memory T cells (TRM) were found to be phenotypically and transcriptional distinct from circulating memory T cells. Here, we identified M. tuberculosis-specific CD4+ T cells induced by recombinant influenza A viruses (rIAV) vaccines expressing M. tuberculosis peptides that persisted in the lung parenchyma with the phenotypic and transcriptional characteristics of TRMs. To determine if these rIAV-induced CD4+ TRM were protective independent of circulating memory T cells, mice previously immunized with the rIAV vaccine were treated with the sphingosine-1-phosphate receptor modulator, FTY720, prior to and during the first 17 days of M. tuberculosis challenge. This markedly reduced circulating T cells, but had no effect on the frequency of M. tuberculosis-specific CD4+ TRMs in the lung parenchyma or their cytokine response to infection. Importantly, mice immunized with the rIAV vaccine were protected against M. tuberculosis infection even when circulating T cells were profoundly depleted by the treatment. Therefore, pulmonary immunization with the rIAV vaccine stimulates lung-resident CD4+ memory T cells that are associated with early protection against tuberculosis infection.

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  1. 1.

    World Health Organization, Geneva, Switzerland. Global Tuberculosis Report. (2017).

  2. 2.

    Fine, P. E. BCG: the challenge continues. Scand. J. Infect. Dis. 33, 243–245 (2001).

  3. 3.

    Reiley, W. W. et al. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection. Proc. Natl Acad. Sci. U.S.A. 107, 19408–19413 (2010).

  4. 4.

    Pawlowski, A., Jansson, M., Skold, M., Rottenberg, M. E. & Kallenius, G. Tuberculosis and HIV co-infection. PLoS Pathog. 8, e1002464 (2012).

  5. 5.

    Tameris, M. D. et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet (2013).

  6. 6.

    Masopust, D., Vezys, V., Marzo, A. L. & Lefrancois, L. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413–2417 (2001).

  7. 7.

    Steinert, E. M. et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 161, 737–749 (2015).

  8. 8.

    Schenkel, J. M. & Masopust, D. Tissue-resident memory T cells. Immunity 41, 886–897 (2014).

  9. 9.

    Allende, M. L., Dreier, J. L., Mandala, S. & Proia, R. L. Expression of the sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration. J. Biol. Chem. 279, 15396–15401 (2004).

  10. 10.

    Carlson, C. M. et al. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature 442, 299–302 (2006).

  11. 11.

    Mackay, L. K. et al. Cutting edge: CD69 interference with sphingosine-1-phosphate receptor function regulates peripheral T cell retention. J. Immunol. 194, 2059–2063 (2015).

  12. 12.

    Wu, T. et al. Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-protection against pulmonary virus infection. J. Leukoc. Biol. 95, 215–224 (2014).

  13. 13.

    Gebhardt, T. et al. Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus. Nat. Immunol. 10, 524–530 (2009).

  14. 14.

    Thom, J. T., Weber, T. C., Walton, S. M., Torti, N. & Oxenius, A. The salivary gland acts as a sink for tissue-resident memory CD8(+) T cells, facilitating protection from local cytomegalovirus infection. Cell Rep. 13, 1125–1136 (2015).

  15. 15.

    Sheridan, B. S. et al. Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function. Immunity 40, 747–757 (2014).

  16. 16.

    Fernandez-Ruiz, D. et al. Liver-resident memory CD8+T cells form a front-line defense against malaria liver-stage infection. Immunity 45, 889–902 (2016).

  17. 17.

    Turner, D. L. et al. Lung niches for the generation and maintenance of tissue-resident memory T cells. . Mucosal Immunol. 7, 501–510 (2014).

  18. 18.

    Strutt, T. M. et al. IL-15 supports the generation of protective lung-resident memory CD4 T cells. Mucosal Immunol. (2017).

  19. 19.

    Oja, A. E. et al. Trigger-happy resident memory CD4(+) T cells inhabit the human lungs. Mucosal Immunol. (2017).

  20. 20.

    Florido, M. et al. Epitope-specific CD4+, but not CD8+, T-cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection. Eur. J. Immunol. 45, 780–793 (2015).

  21. 21.

    Anderson, K. G. et al. Intravascular staining for discrimination of vascular and tissue leukocytes. Nat. Protoc. 9, 209–222 (2014).

  22. 22.

    Kerdiles, Y. M. et al. Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. Nat. Immunol. 10, 176–184 (2009).

  23. 23.

    Chiba, K. et al. Fingolimod (FTY720), sphingosine 1-phosphate receptor modulator, shows superior efficacy as compared with interferon-beta in mouse experimental autoimmune encephalomyelitis. Int. Immunopharmacol. 11, 366–372 (2011).

  24. 24.

    Morris, M. A. et al. Transient T cell accumulation in lymph nodes and sustained lymphopenia in mice treated with FTY720. Eur. J. Immunol. 35, 3570–3580 (2005).

  25. 25.

    Redford, P. S. et al. Influenza A virus impairs control of Mycobacterium tuberculosis coinfection through a type I interferon receptor-dependent pathway. J. Infect. Dis. 209, 270–274 (2014).

  26. 26.

    Muflihah, H. et al. Sequential pulmonary immunization with heterologous recombinant influenza A virus tuberculosis vaccines protects against murine M. tuberculosis infection. Vaccine 36, 2462–2470 (2018).

  27. 27.

    Srivastava, S. & Ernst, J. D. Cutting edge: Direct recognition of infected cells by CD4 T cells is required for control of intracellular Mycobacterium tuberculosis in vivo. J. Immunol. 191, 1016–1020 (2013).

  28. 28.

    Sakai, S. et al. Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells. J. Immunol. 192, 2965–2969 (2014).

  29. 29.

    Connor, L. M. et al. A key role for lung-resident memory lymphocytes in protective immune responses after BCG vaccination. Eur. J. Immunol. 40, 2482–2492 (2010).

  30. 30.

    Perdomo, C. et al. Mucosal BCG vaccination induces protective lung-resident memory T cell populations against tuberculosis. MBio 7, (2016).

  31. 31.

    Turner, D. L. & Farber, D. L. Mucosal resident memory CD4 T cells in protection and immunopathology. Front. Immunol. 5, 331 (2014).

  32. 32.

    Horvath, C. N., Shaler, C. R., Jeyanathan, M., Zganiacz, A. & Xing, Z. Mechanisms of delayed anti-tuberculosis protection in the lung of parenteral BCG-vaccinated hosts: a critical role of airway luminal T cells. Mucosal Immunol. 5, 420–431 (2012).

  33. 33.

    Casey, K. A. et al. Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. J. Immunol. 188, 4866–4875 (2012).

  34. 34.

    Teijaro, J. R. et al. Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virus infection. J. Immunol. 187, 5510–5514 (2011).

  35. 35.

    Mackay, L. K. et al. The developmental pathway for CD103(+)CD8+tissue-resident memory T cells of skin. Nat. Immunol. 14, 1294–1301 (2013).

  36. 36.

    Skon, C. N. et al. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+T cells. Nat. Immunol. 14, 1285–1293 (2013).

  37. 37.

    Kumar, B. V. et al. Human tissue-resident memory T cells are defined by core transcriptional and functional signatures in lymphoid and mucosal sites. Cell Rep. 20, 2921–2934 (2017).

  38. 38.

    Huang, H. & Tindall, D. J. Regulation of FOXO protein stability via ubiquitination and proteasome degradation. Biochim. Biophys. Acta 1813, 1961–1964 (2011).

  39. 39.

    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, (2016).

  40. 40.

    Walker, K. B. et al. Novel approaches to preclinical research and TB vaccine development. Tuberc. (Edinb.) 99(Suppl 1), S12–S15 (2016).

  41. 41.

    Hoffmann, E., Krauss, S., Perez, D., Webby, R. & Webster, R. G. Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 20, 3165–3170 (2002).

  42. 42.

    Cukalac, T. et al. Narrowed TCR diversity for immunised mice challenged with recombinant influenza A-HIV Env(311-320) virus. Vaccine 27, 6755–6761 (2009).

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This work was supported by the National and Medical Research Council of Australia through Project Grant APP1044343, and the Center of Research Excellence in TB Control (APP1043225), and the NSW Government through its infrastructure grant to the Centenary Institute. H.M. was a recipient of Australia Award Scholarship from the Australian Department of Foreign Affairs and Trade.

Author information


  1. Tuberculosis Research Program, Centenary Institute, The University of Sydney, Newtown, NSW, Australia

    • Manuela Flórido
    • , Heni Muflihah
    • , Leon C. W. Lin
    • , Mainthan Palendira
    • , Carl G. Feng
    • , James A. Triccas
    •  & Warwick. J. Britton
  2. School of Medicine, Deakin University, Geelong, VIC, Australia

    • Yingju Xia
    •  & John Stambas
  3. Liver Immunology Program, Centenary Institute, The University of Sydney, Newtown, NSW, Australia

    • Frederic Sierro
    •  & Patrick Bertolino
  4. Department of Pathology, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia

    • Frederic Sierro
  5. Department of Infectious Diseases and Immunology, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia

    • Mainthan Palendira
    • , Carl G. Feng
    • , James A. Triccas
    •  & Warwick. J. Britton
  6. AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia

    • Patrick Bertolino
  7. Department of Medicine, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia

    • Warwick. J. Britton


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M.F., H.M. and L.L designed and performed experiments, and M.F, wrote the manuscript. Y.X. developed the rIAV vaccines under supervision of J.S. F.S. assisted with 2-photon microscopy experiments. M.P., C.F., P.B. and J.A.T. contributed to the experimental design and provided intellectual input. W.B. was responsible for study design, data interpretation, and study supervision. All the authors contributed to the editing of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Warwick. J. Britton.

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