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Human lung organoid modelling of tissue-resident antiviral T cell responses

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Data availability

scRNA-seq datasets have been deposited in Gene Expression Omnibus (GEO) with the accession codes GSE216049 and GSE344986. This includes raw, processed and final Seurat objects with all annotations. Bulk RNA-sequencing datasets (raw and processed files) have been deposited in GEO with the accession code GSE230398.

References

  1. Zheng, M. Z. M. & Wakim, L. M. Tissue resident memory T cells in the respiratory tract. Mucosal Immunol. 15, 379–388 (2022).

    Article  CAS  PubMed  Google Scholar 

  2. 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 

  3. Tang, J. & Sun, J. Lung tissue-resident memory T cells: the gatekeeper to respiratory viral (re)-infection. Curr. Opin. Immunol. 80, 102278 (2023).

    Article  CAS  PubMed  Google Scholar 

  4. Lam, N. et al. Asynchronous aging and turnover of human circulating and tissue-resident memory T cells across sites. Immunity 58, 2271–2288.e2276 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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).

    Article  PubMed  Google Scholar 

  6. Slütter, B. et al. Dynamics of influenza-induced lung-resident memory T cells underlie waning heterosubtypic immunity. Sci. Immunol. 2, eaag2031 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Masopust, D. & Soerens, A. G. Tissue-resident T cells and other resident leukocytes. Annu. Rev. Immunol. 37, 521–546 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Poon, M. M. L. et al. SARS-CoV-2 infection generates tissue-localized immunological memory in humans. Sci. Immunol. 6, eabl9105 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Carbone, F. R. Unique properties of tissue-resident memory T cells in the lungs: implications for COVID-19 and other respiratory diseases. Nat. Rev. Immunol. 23, 329–335 (2023).

    Article  CAS  PubMed  Google Scholar 

  10. Szabo, P. A., Miron, M. & Farber, D. L. Location, location, location: tissue resident memory T cells in mice and humans. Sci. Immunol. 4, eaas9673 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yang, A. Y. et al. Lung tissue–resident memory T cells optimize protection by IL-10 regulation of innate immunity. J. Exp. Med. 223, e20242307 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Stolley, J. M. et al. Retrograde migration supplies resident memory T cells to lung-draining LN after influenza infection. J. Exp. Med. 217, e20192197 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rydyznski Moderbacher, C. et al. Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity. Cell 183, 996–1012.e1019 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Weiskopf, D. et al. Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome. Sci. Immunol. 5, eabd2071 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tarke, A. et al. SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron. Cell 185, 847–859.e811 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sette, A., Sidney, J. & Crotty, S. T cell responses to SARS-CoV-2. Annu. Rev. Immunol. 41, 343–373 (2023).

    Article  CAS  PubMed  Google Scholar 

  17. Grifoni, A. et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell 181, 1489–1501.e1415 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Swadling, L., Fumagalli, V., Maini, M. K. & Iannacone, M. Abortive infection: T cells as early, antibody-independent defenders. Nat. Rev. Immunol. 26, 327–329 (2026).

    Article  CAS  PubMed  Google Scholar 

  19. Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Salahudeen, A. A. et al. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature 588, 670–675 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tiwari, S. K., Wang, S., Smith, D., Carlin, A. F. & Rana, T. M. Revealing tissue-specific SARS-CoV-2 infection and host responses using human stem cell-derived lung and cerebral organoids. Stem Cell Rep. 16, 437–445 (2021).

    Article  CAS  Google Scholar 

  22. Lamers, M. M. et al. An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells. EMBO J. 40, e105912 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Han, Y. et al. Identification of SARS-CoV-2 inhibitors using lung and colonic organoids. Nature 589, 270–275 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  24. Youk, J. et al. Three-dimensional human alveolar stem cell culture models reveal infection response to SARS-CoV-2. Cell Stem Cell 27, 905–919.e910 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jung, J. H. et al. SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells. Nat. Commun. 12, 4043 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lipsitch, M., Grad, Y. H., Sette, A. & Crotty, S. Cross-reactive memory T cells and herd immunity to SARS-CoV-2. Nat. Rev. Immunol. 20, 709–713 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dan, J. M. et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 371, eabf4063 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schaller, M. A. et al. Ex vivo SARS-CoV-2 infection of human lung reveals heterogeneous host defense and therapeutic responses. JCI Insight 6, e148003 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lee-Ferris, R. E. et al. Prolonged airway explant culture enables study of health, disease, and viral pathogenesis. Sci. Adv. 11, eadp0451 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Neal, J. T. et al. Organoid modeling of the tumor immune microenvironment. Cell 175, 1972–1988.e1916 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Santos, A. J. M. et al. A human autoimmune organoid model reveals IL-7 function in coeliac disease. Nature 632, 401–410 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mulay, A. et al. SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery. Cell Rep. 35, 109055 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hou, Y. J. et al. SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell 182, 429–446.e414 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Beers, M. F. & Moodley, Y. When is an alveolar type 2 cell an alveolar type 2 cell? A conundrum for lung stem cell biology and regenerative medicine. Am. J. Respir. Cell Mol. Biol. 57, 18–27 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shenoy, A. T. et al. Antigen presentation by lung epithelial cells directs CD4. Nat. Commun. 12, 5834 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wosen, J. E., Mukhopadhyay, D., Macaubas, C. & Mellins, E. D. Epithelial MHC class II expression and its role in antigen presentation in the gastrointestinal and respiratory tracts. Front. Immunol. 9, 2144 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e278 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Co, J. Y. et al. Controlling epithelial polarity: a human enteroid model for host–pathogen interactions. Cell Rep. 26, 2509–2520.e2504 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ziegler, C. G. K. et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell 181, 1016–1035.e1019 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Buchrieser, J. et al. Syncytia formation by SARS-CoV-2-infected cells. EMBO J. 39, e106267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhou, X. et al. Interferon induced IFIT family genes in host antiviral defense. Int. J. Biol. Sci. 9, 200–208 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Verhelst, J., Hulpiau, P. & Saelens, X. Mx proteins: antiviral gatekeepers that restrain the uninvited. Microbiol. Mol. Biol. Rev. 77, 551–566 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Bhaskar, S. et al. Cytokine storm in COVID-19-immunopathological mechanisms, clinical considerations, and therapeutic approaches: the REPROGRAM Consortium position paper. Front. Immunol. 11, 1648 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Tay, M. Z., Poh, C. M., Rénia, L., MacAry, P. A. & Ng, L. F. P. The trinity of COVID-19: immunity, inflammation and intervention. Nat. Rev. Immunol. 20, 363–374 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Islam, H., Chamberlain, T. C., Mui, A. L. & Little, J. P. Elevated interleukin-10 levels in COVID-19: potentiation of pro-inflammatory responses or impaired anti-inflammatory action? Front. Immunol. 12, 677008 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kyriazopoulou, E. et al. Effect of anakinra on mortality in patients with COVID-19: a systematic review and patient-level meta-analysis. Lancet Rheumatol. 3, e690–e697 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457–462 (2020).

    Article  ADS  PubMed  Google Scholar 

  49. Mallajosyula, V. et al. CD8+ T cells specific for conserved coronavirus epitopes correlate with milder disease in patients with COVID-19. Sci. Immunol. 6, eabg5669 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Gao, F. et al. Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection. Immunity 56, 864–878.e864 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. da Silva Antunes, R. et al. The MegaPool approach to characterize adaptive CD4+ and CD8+ T cell responses. Curr. Protoc. 3, e934 (2023).

    Article  Google Scholar 

  52. Dijkstra, K. K. et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174, 1586–1598.e1512 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kwon, D. -i et al. Mucosal unadjuvanted booster vaccines elicit local IgA responses by conversion of pre-existing immunity in mice. Nat. Immunol. 26, 908–919 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Paik, D. H. & Farber, D. L. Influenza infection fortifies local lymph nodes to promote lung-resident heterosubtypic immunity. J. Exp. Med. 218, e20200218 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Low, J. S. et al. Tissue-resident memory T cell reactivation by diverse antigen-presenting cells imparts distinct functional responses. J. Exp. Med. 217, e20192291 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Allie, S. R. et al. The establishment of resident memory B cells in the lung requires local antigen encounter. Nat. Immunol. 20, 97–108 (2019).

    Article  CAS  PubMed  Google Scholar 

  57. Yost, K. E. et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wagar, L. E. et al. Modeling human adaptive immune responses with tonsil organoids. Nat. Med. 27, 125–135 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Nolan, S. et al. A large-scale database of T-cell receptor beta sequences and binding associations from natural and synthetic exposure to SARS-CoV-2. Front. Immunol. https://doi.org/10.3389/fimmu.2025.1488851 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Shugay, M. et al. VDJdb: a curated database of T-cell receptor sequences with known antigen specificity. Nucleic Acids Res. 46, D419–D427 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Greene, F. L. et al. (eds). AJCC Cancer Staging Manual 6th edn (Springer, 2002).

  62. Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587.e3529 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. ImmunoMind Team. immunarch: an R package for painless analysis of large-scale immune repertoire data. Zenodo https://doi.org/10.5281/zenodo.3367201 (2019).

  64. Russell, J. N., Clements, J. E. & Gama, L. Quantitation of gene expression in formaldehyde-fixed and fluorescence-activated sorted cells. PLoS One 8, e73849 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Kuo, Davis, Blish, and Baric laboratories for insightful discussions; the Stanford Tissue Bank and Donor Network West for assistance with tissue acquisition; P. Chu for histology; J. Garhyan and A. Pohane for management of the Stanford BSL3 facility; the Stanford Human Immune Monitoring Center; and the Stanford Stem Cell FACS Facility. SARS-Cov-2 isolate USA-WA1/2020, NR-52281 was deposited by the CDC and obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID),  US National Institutes of Health (NIH).

Funding

Generous support was provided by NIH National Heart, Lung, and Blood Institute 5T32HL129970-10 (J.K.R.), Stanford Medical Scientist Training Program grant T32GM007365 (S.S.C.), Netherlands Organization for Scientific Research Rubicon Grant 452181214 (V.v.U.), NIH NIAID T32AI007502-23 and K08AI163369-01A1 (A.R), Bill and Melinda Gates Foundation OPP1113682 (C.J.K., M.M.D. and C.A.B.), Center for Human Systems Immunology (V.M.), Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease 1016687 (C.A.B.), Fast Grant from the Mercatus Center (C.A.B.), Chan Zuckerberg Biohub (C.A.B.), NIH NIAID 2U19057229 (M.M.D.), NIH R01AI127877 (S.D.B), R01AI130398 (S.D.B.), NIAID Biomimetic Consortium U19AI116484 (C.J.K., R.S.B., C.A.B.), R01DK130414 (C.J.K.), the PROMINENT team supported by the Cancer Grand Challenges partnership, Cancer Research UK CGCATF-2021/100010 and NIH OT2CA278713 (C.J.K.) and Arc Institute (C.J.K.). Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the NIH under award number UM1TR004921.

Author information

Author notes

  1. These authors contributed equally: Joseph K. Rathkey, Shannon S. Choi

Authors and Affiliations

  1. Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Joseph K. Rathkey, Shannon S. Choi, Vincent van Unen, Huimin Zhang, Min Liu, Jie Ding, Samira A. Alwahabi, António J. M. Santos, Joshua E. Chan, Maher M. Elsheikh, Steven M. Chirieleison, Hudson T. Horn, Alexander Guh-Siesel, Zhongqi Lin, Hannah S. Powell & Calvin J. Kuo

  2. Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Joseph K. Rathkey

  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Vincent van Unen, Vamsee Mallajosyula & Mark M. Davis

  4. Stanford Institute of Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA

    Vincent van Unen, Vamsee Mallajosyula, Azam Mohsin, Catherine A. Blish & Mark M. Davis

  5. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Arjun Rustagi

  6. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Brandon Lam, Steven M. Chirieleison, Daniel Solis, Jordan Mah, Katharina Röltgen, Brock A. Martin, Benjamin Pinsky & Scott D. Boyd

  7. Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Bailey Wallen, Winston Trope & Joseph Shrager

  8. Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Ramesh Nair

  9. Donor Network West, San Ramon, CA, USA

    Ahmad Salehi

  10. Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Aimee Beck & Catherine A. Blish

  11. Department of Epidemiology, Microbiology, and Immunology, University of North Carolina Chapel Hill, Chapel Hill, NC, USA

    Caitlin Edwards & Ralph S. Baric

  12. Department of Pediatric Surgery, Stanford University School of Medicine, Stanford, CA, USA

    James C. Y. Dunn

  13. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Catherine A. Blish

  14. Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La Jolla, CA, USA

    Alessandro Sette & Alba Grifoni

  15. Department of Medicine, Division of Infectious Diseases and Global Public Health, University of California, San Diego, La Jolla, CA, USA

    Alessandro Sette & Alba Grifoni

  16. Arc Institute, Palo Alto, CA, USA

    Calvin J. Kuo

Authors

  1. Joseph K. Rathkey
  2. Shannon S. Choi
  3. Vincent van Unen
  4. Huimin Zhang
  5. Min Liu
  6. Jie Ding
  7. Samira A. Alwahabi
  8. António J. M. Santos
  9. Vamsee Mallajosyula
  10. Joshua E. Chan
  11. Azam Mohsin
  12. Maher M. Elsheikh
  13. Arjun Rustagi
  14. Brandon Lam
  15. Steven M. Chirieleison
  16. Bailey Wallen
  17. Daniel Solis
  18. Jordan Mah
  19. Hudson T. Horn
  20. Katharina Röltgen
  21. Ramesh Nair
  22. Winston Trope
  23. Alexander Guh-Siesel
  24. Zhongqi Lin
  25. Hannah S. Powell
  26. Ahmad Salehi
  27. Aimee Beck
  28. Caitlin Edwards
  29. Brock A. Martin
  30. James C. Y. Dunn
  31. Joseph Shrager
  32. Ralph S. Baric
  33. Benjamin Pinsky
  34. Scott D. Boyd
  35. Catherine A. Blish
  36. Alessandro Sette
  37. Alba Grifoni
  38. Mark M. Davis
  39. Calvin J. Kuo

Contributions

J.K.R. and S.S.C. conceived, designed and performed experiments, analysed data and wrote the manuscript. V.v.U. advised on experiments, wrote the manuscript and designed and interpreted scRNA-seq studies. S.A.A., H.Z., M.L., J.D., M.M.E., H.S.P., H.T.H. and S.M.C. performed organoid culture and experiments. A.R. conceived, designed and performed SARS-CoV-2 experiments and provided SARS-CoV-2 viral stocks, with C.A.B. advising. A.B. performed SARS-CoV-2 experiments. A.J.M.S. advised on organoid culture and experiments and performed RNA isolations. A.M., R.N., J.E.C. and A.G.-S. performed scRNA-seq analysis. B.L., D.S. and K.R. performed serologic experiments. A.G. and A. Sette generated MegaPool reagents. J.M., B.P. and S.D.B. analysed serologic data. C.E. designed and performed SARS-CoV-2 experiments and provided SARS-CoV-2 viral stocks with R.S.B. advising. Z.L. performed qPCR. V.M. contributed spheromer reagents. M.M.D. advised on experiments, reagent construction and data analysis. B.A.M. performed histologic analysis. W.T., J.S., B.W., A. Salehi and J.C.Y.D. provided tissue samples. C.J.K. conceived and designed experiments and wrote the manuscript. All authors reviewed the manuscript and provided edits and critical comments.

Corresponding author

Correspondence to Calvin J. Kuo.

Ethics declarations

Competing interests

C.J.K. and S.S.C. are listed as inventors on provisional patent PCT/US2022/029869 describing the methods in this paper. C.J.K. is a founder and scientific advisory board member of Surrozen. C.J.K. and M.M.D. are founders and scientific advisory board members of Mozart Therapeutics and Organomx. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Antonio Bertoletti, Thomas Kupper, Toshiro Sato and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 Characterization of epithelial and endothelial cells in lung ALI organoids.

a, Brightfield, lung ALI organoid culture, day 180, scale bar, 2 mm. Successful organoid culture was defined by enlargement of organoids on brightfield microscopy which was further augmented by histologic analysis via H&E staining and Ki67 positivity. b, H&E of lung ALI organoid, day 8, with alveolar-like airspaces, plasma cells, lymphocytes, and pigment-laden histiocytes (yellow arrows). Scale bar, 1000 µm. c, Representative H&E from n = 3 technical replicates and N = 3 biological replicates demonstrating consistency of lung ALI organoid culture within and between individuals. Scale bar, 100 µm. d, IHC staining of CD31, cytokeratin 5/6 (KRT5/6), and E-cadherin (ECAD) within organoid technical replicates from a single donor. Scale bar = 100 µm. ns = not significant. e, Biological replicates (N = 3) and IHC quantification for organoid E-cadherin (ECAD), cytokeratin-5/6 (KRT5/6), and CD31 IHC at day 0, day 5–7 and day 53–54. Each dot represents an individual organoid (total of n = 5 organoids). Time points were compared by 2-way ANOVA, ***P < 0.001, ****P < 0.0001. Biological replicates from each time point were compared by one-way ANOVA. ns = not significant. *P < 0.05, ***P < 0.001. Scale bars = 100 µm. f, Lung ALI organoid alveolar and bronchiolar elements are distinct and adjacent at early (days 3 and 7, top) but are intermingled at late time points (day 221, bottom). IF, HT1-56 (red), SFTPC (white), KRT5 (green), DAPI (blue). Scale bar, 70 µm. g, Day 60 organoids with HT1-56+ AT1 cells (red), SFTPC+ AT2 cells (green), DAPI (blue). Scale bar, 100 µm. h, Ciliated cells at culture day 101. IF, acetylated tubulin (white), ECAD (red), DAPI (blue). Scale bar, 10 µm. i, Lung organoid vascular networks, IF staining, culture day 40, ECAD+ epithelium (red), CD31+ endothelium (green), DAPI (blue). Scale bar, 100 µm.

Extended Data Fig. 2 Characterization of epithelial and mesenchymal elements of lung ALI organoids.

a, Violin plots of selected genes from day 12 organoid scRNA-seq, CD45− cells, data are integrated from 3 patients from Fig. 1n–p. b-c, Violin plots (b) and feature plots (c) of SFTPB and SCGB3A3 from combined tissue, ALI organoid, and suspension organoid scRNA-seq data in Fig. 2t. d-e, scRNA-seq data from fresh lung tissue from a representative individual as comparator to cognate organoids in Fig. 1m–o. (d) UMAP plot of CD45− cells from fresh lung tissue. (e) Feature plots of genes used to phenotype clusters from fresh tissue.

Extended Data Fig. 3 Characterization of immune cells from lung ALI organoids.

a-b, scRNA-seq feature plots (a) and violin plots (b) of transcripts used for cell-type assignments in Fig. 2g,h scRNA-seq. c, scRNA-seq feature plots of tissue residency markers CD69 and ITGAE (CD103) from organoid UMAP in Fig. 2h. d, Flow cytometry gating strategy of immune cells from day 12 organoids. e, Flow cytometric analysis of CD45+ organoid cells, 3 technical replicate wells from 3 biological replicates. f, scRNA-seq UMAP plot of CD45+ cell types from fresh lung tissue from Fig. 2g,h (corresponding organoid culture is in Fig. 2g,h). g, Feature plots of genes used to phenotype clusters from (f). h, UMAP of scRNA-seq data from Fig. 2t across N = 3 biological replicates (rep. 1 = salmon, rep. 2 = aqua, rep. 3 = olive) from days 80–92, demonstrating consistency of organoid culture between individual organoid lines and maintenance of both non-immune and immune cell populations for extended time periods with IL-2, IL-7 and IL-15 supplementation. i, Flow cytometry of macrophage persistence in ALI lung organoids without cytokine supplementation, days 10–44. The y-axis is the number of SSChi, CD68+, CD11C− cells per 1000 live organoid single cells.

Extended Data Fig. 4 Epithelial cell antigen presentation and T cell activation in lung organoids.

a, Flow cytometry plot of epithelial (EPCAM+) expression of surface HLA-DR in day 12 ALI lung organoids. b, scRNA-seq feature plots of HLA-DRB1 and TAP1 in both day 12 lung ALI organoids (3 individuals for CD45− and 2 individuals for CD45+) as in Fig. 1n and corresponding fresh tissue (one individual) from Extended Data Figs. 2d and 3f. c, DQ-OVA presentation on EPCAM+ cells from organoids, pre-gated on live, single cells. DQ-OVA (20 µg/mL) or PBS control were added to lung ALI organoid cultures for 72 h. CD45+CD3+ T cells and EPCAM+ epithelium were assayed for DQ-OVA uptake by flow cytometry. d, Gating strategy for organoid AIM assay. e-f, CD8+ and CD4+ T cells in lung ALI organoids are activated in response to stimulation with anti-CD3/28/2 for 24 h. Representative flow plots from Fig. 2l,m.

Extended Data Fig. 5 Cryopreservation and cryorecovery of lung ALI organoids.

a, Schematic of lung ALI cryopreservation and cryorecovery. Lung ALI organoids within the collagen matrix were rolled and frozen in cryopreservation solution at -80 °C. Following rapid thawing at 37 °C, lung ALI organoids were replated on a collagen matrix and cultured for 30 days. Created in BioRender; Rathkey, J. https://BioRender.com/lg819su (2026). b-d, Analysis of cryopreserved and cryorecovered lung ALI organoids 30 days post-recovery. (b) H&E, scale bar, 100 µm. Organoid structure was preserved compared with patient-matched control organoids with no significant difference in proliferation after cryorecovery. (c) Ki67 staining quantitation, 3 biological replicates, each data point is an individual organoid. (d) CD8+ T cell activation in cryopreserved lung ALI organoids. Lung ALI organoids were cryopreserved for 72 h followed by cryorecovery. Organoids were stimulated with PBS (mock) or superantigen-like TCR:MHC crosslinking (CytoStim) with CD8 + T cell activation evaluated by flow cytometry AIM assay as in Extended Data Fig. 4d. Cryopreserved and cryorecovered lung ALI CD8+ T cells demonstrated marked activation by TCR-MHC crosslinking versus donor-paired mock controls.

Extended Data Fig. 6 Characterization of suspension lung organoid culture.

a, Schema. Created in BioRender; Rathkey, J. https://BioRender.com/u3k7ahi (2026). b-c, Serial brightfield microscopy (b) and H&E (c) of a single lung ALI organoid after removal from collagen. Scale bars, 500 µm. d-e, IF of apical tight-junction marker ZO-1 (green), ECAD (red), DAPI (blue), on organoids continuously in collagen (d) or in suspension (e) for 4 days. Both panels are cumulative culture day 15. Scale bar, 50 µm. f, HT1-56+ AT1 cells (red), SFTPC+ AT2 cells (green) with DAPI (blue) in suspension lung organoids. Scale bar, 100 µm. g, scRNA-seq UMAP plots of CD45− (left, epithelial/mesenchymal) and CD45+ (right, hematopoietic) cells from suspension organoids, merged from two individual donors, day 12, with the last 5 days in suspension as in Fig. 2t. h-i, Quantification of CD45− epithelial (h) and CD45+ hematopoietic (i) cell types from (g).

Extended Data Fig. 7 SARS-CoV-2 infection of suspension lung ALI organoids.

a, Heatmap of SARS-CoV-2 transcripts detected by Nanostring nCounter in purified lung organoid epithelial cells from three patients, after 7 days of SARS-CoV-2 infection. Color intensity is log2 normalized transcript counts of the SARS-CoV-2 ORFs. b, Bulk RNA-sequencing of SARS-CoV-2 ORFs in lung ALI organoids from a single biological replicate, 3–14 days post-infection, y axis = log2 FPKM. c, VeroE6 cell plaque assay performed with supernatants from SARS-CoV-2-infected suspension lung organoids at 24 and 48 h post-infection, d-e, IF imaging of mock (d) and SARS-CoV-2-infected suspension organoid culture (e). SARS-CoV-2 NP (green), cleaved caspase-3 (red), phalloidin (white), and DAPI (blue), scale bar, 100 µm. f, Immune cell clustering around SARS-CoV-2-infected cells. SARS-CoV-2-infected suspension lung organoids, 48 h.p.i. SARS-CoV-2 nucleocapsid protein (green), ECAD (white), CD45 (red), DAPI (blue), scale bar, 100 µm g, IF imaging of infected organoid macrophage, 7 d.p.i., SARS-CoV-2 NP (green), CD68 (red), DAPI (blue), scale bar, 30 µm. All images are representative of ≥3 biological replicates.

Extended Data Fig. 8 Lung ALI organoids mount innate immune responses to SARS-CoV-2 infection.

a-b, qRT-PCR of type I IFN response and chemotaxis-related gene expression at 3–14 days post-infection (dpi) with SARS-CoV-2 from a single biological replicate. c, Volcano plots of bulk RNA sequencing downregulated (blue) or induced (red) genes, 3–14 days post-infection with SARS-CoV-2 or mock control. Data represents organoids from a single patient. Y-axis represents -log10(p value), x-axis is log2(fold change normalized to mock). d-e, Luminex analysis of supernatants from infected suspension organoids from early (3 days) or late (10 days) infection, two additional biological replicates of Fig. 3q.

Extended Data Fig. 9 Organoid T cell activation in response to SARS-CoV-2 infection.

a, Suspension organoids were stimulated with or without SARS-CoV-2 spike peptide MegaPool for 24 h and analyzed for AIM marker expression on CD4+ T cells. Organoids were derived from N = 16 independent patients. Wilcoxon matched-pairs test. **P < 0.01, ** P < 0.001, ns= not significant. b, Representative flow cytometry AIM plots from (a). c, Flow cytometry gating strategy for identifying EdU+ proliferating T cells in lung ALI organoid cultures from Fig. 4g,h. d, Subgroup analysis of organoid lines from age 60–90 donors in Fig. 5a,b. Identification of human ALI lung organoid SARS-CoV-2-responding CD8+ T cells by flow cytometry AIM assay. Suspension organoids were infected with SARS-CoV-2 virus for 6 days and activated CD8+ T cells were identified based on induction of double-positive pairs of CD25, OX40, 4-1BB, and CD40L. Wilcoxon matched-pairs test, **P < 0.01. e, AIM activation in CD4+ T cells. CD25+OX40+ CD4+ T cells in mock- or SARS-CoV-2-treated lung organoids. Wilcoxon matched-pairs test. ns=not significant.

Extended Data Fig. 10 Peptide:MHC spheromer evaluation of SARS-CoV-2–stimulated, virus-specific organoid T cell activation.

a, Flow cytometry of PBMC from two CMV seropositive donors stained with both HLA-A*02:01 SARS-CoV-2-spike/ORF1ab and HLA-A*02:CMV spheromers. Discrete SARS-CoV-2 versus CMV-specific CD8+ T cells confirmed SARS-CoV-2 spheromer specificity. b-c, Flow cytometry of organoid CD8+ T cells from HLA-A*02+ (b) or HLA-A*02− individuals (c), mock- or SARS-CoV-2-infected, 10 dpi. HLA-A*02:SARS-CoV-2 spike/ORF1ab spheromer-reactive CD8+ T cells were present in HLA-A2+ donors, increased post-SARS-CoV-2 infection and did not cross-react against HLA-A*02:HIV spheromers (b), and were absent in the HLA-A*02− donor (c). d, Organoid SARS-CoV-2-specific versus CMV-specific CD8+ T cells are non-overlapping. Organoids from an HLA-A*02+ but CMV seronegative donor (left) versus two HLA-A*02− (right) donors underwent mock (top) versus SARS-CoV-2 infection (bottom) for 10 d, In the HLA-A*02+ donor, flow cytometry of CD8+ T cells stained with both HLA-A*02:SARS-CoV-2 S/ORF1ab and HLA-A*02:CMV spheromers revealed that SARS-CoV-2 spheromer-reactive T cells did not cross-react with CMV spheromers and were induced by SARS-CoV-2 infection. Organoids from two HLA-A*02− donors lacked SARS-CoV-2 or CMV spheromer staining regardless of SARS-CoV-2 infection. e-f, Effects of SARS-CoV-2 infection on organoid HLA-A*02:SARS-CoV-2 spheromer+CD25+CD8+ T cells, 7–10 d.p.i. Representative flow cytometry (e) and quantification (f), N = 5 biological replicates. Wilcoxon matched-pairs test. ns=not significant. g, Organoid SARS-CoV-2 antigen-specific T cells are activated by SARS-CoV-2 infection. Flow cytometry, mock or SARS-CoV-2 infection, pre-gated on CD8+ T cells, with HLA-A*02:SARS-CoV-2 S/ORF1ab spheromer and intracellular IFNγ staining. h, Viral burden from Fig. 5f, with qRT-PCR determination of viral transcripts/mg organoids (left) or plaque assay determination of PFU/mg organoids (right). Groups analyzed by Friedman test, P < 0.05 followed by analysis by Wilcoxon matched-pairs test. * P < 0.05, ns=not significant. N = 6 biological replicates.

Supplementary information

Supplementary Figure 1 (download JPG )

Proliferation and apoptosis in ALI lung organoids. a, Flow cytometry of EdU+ subsets of organoid day 54 epithelial (EPCAM+), immune (CD45+) and EPCAM−CD45− cells. b, IHC and quantification of cleaved caspase-3 IHC in fresh, day 7 and day 54 organoids, n = 3 biological replicates. ns, not significant

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Rathkey, J.K., Choi, S.S., van Unen, V. et al. Human lung organoid modelling of tissue-resident antiviral T cell responses. Nature (2026). https://doi.org/10.1038/s41586-026-11114-1

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