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Clinical-scale production of Aspergillus-specific T cells for the treatment of invasive aspergillosis in the immunocompromised host

Bone Marrow Transplantation (2019) | Download Citation

Abstract

Invasive aspergillosis (IA) represents a leading cause of mortality in immunocompromised patients. Although adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) represents a promising therapeutic approach against IA, the complex and costly production limits its broader application. We generated Asp-STs from a single blood draw of healthy individuals or IA patients in only 10 days, by either Aspergillus fumigatus (AF) lysate or peptide stimulation of mononuclear cells. The cells were phenotypically and functionally characterized, and safety was assessed in xenografts. Healthy donor-derived and lysate- or peptide-pulsed Asp-STs presented comparable fold expansion, immunophenotype, and Th1 responses. Upon cross-stimulation, only the lysate-pulsed Asp-STs were empowered to respond to peptide stimulation, although both cell products induced hyphal damage. Importantly, Asp-STs cross-reacted with other fungal species and did not induce alloreactivity in vivo. IA patient-derived T cells displayed an anergic phenotype that prohibited sufficient expansion and yield of meaningful doses of Asp-STs for autologous immunotherapy. Using a rapid and simple process, we generated, from healthy donors but not IA patients, functionally active Asp-STs of broad specificity and at clinically relevant numbers. Such an approach may form the basis for the effective management of IA in the context of allogeneic hematopoietic cell transplantation.

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References

  1. 1.

    Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, et al. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001-2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database. Clin Infect Dis. 2010;50:1091–100.

  2. 2.

    Shoham S, Marr KA. Invasive fungal infections in solid organ transplant recipients. Future Microbiol. 2012;7:639–55.

  3. 3.

    Kumaresan PR, da Silva TA, Kontoyiannis DP. Methods of controlling invasive fungal infections using CD8+T cells. Front Immunol. 2017;8:1939.

  4. 4.

    Kontoyiannis DP. Antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success. Bone Marrow Transplant. 2011;46:165–73.

  5. 5.

    Marr KA. Fungal infections in hematopoietic stem cell transplant recipients. Med Mycol. 2008;46:293–302.

  6. 6.

    Kim A, Nicolau DP, Kuti JL. Hospital costs and outcomes among intravenous antifungal therapies for patients with invasive aspergillosis in the United States. Mycoses. 2011;54:e301–12.

  7. 7.

    Papadopoulou A, Kaloyannidis P, Yannaki E, Cruz CR. Adoptive transfer of Aspergillus-specific T cells as a novel anti-fungal therapy for hematopoietic stem cell transplant recipients: progress and challenges. Crit Rev Oncol Hematol. 2016;98:62–72.

  8. 8.

    Blyth E, Clancy L, Simms R, Ma CKK, Burgess J, Deo S, et al. Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation. Blood. 2013;121:3745–58.

  9. 9.

    Bollard CM, Heslop HE. T cells for viral infections after allogeneic hematopoietic stem cell transplant. Blood. 2016;127:3331–40.

  10. 10.

    Rooney CM, Smith CA, Ng CY, Loftin S, Li C, Krance RA, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet (Lond, Engl). 1995;345:9–13.

  11. 11.

    Tzannou I, Papadopoulou A, Naik S, Leung K, Martinez CA, Ramos CA, et al. Off-the-shelf virus-specific T cells to treat BK virus, human Herpesvirus 6, cytomegalovirus, Epstein-Barr virus, and adenovirus infections after allogeneic hematopoietic stem-cell transplantation. J Clin Oncol. 2017;35:3547–57.

  12. 12.

    Walter EA, Greenberg PD, Gilbert MJ, Finch RJ, Watanabe KS, Thomas ED, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med. 1995;333:1038–44.

  13. 13.

    Doubrovina E, Oflaz-Sozmen B, Prockop SE, Kernan NA, Abramson S, Teruya-Feldstein J, et al. Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+lymphomas after allogeneic hematopoietic cell transplantation. Blood. 2012;119:2644–56.

  14. 14.

    Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S, Löffler J, et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood. 2002;99:3916–22.

  15. 15.

    Feuchtinger T, Matthes-Martin S, Richard C, Lion T, Fuhrer M, Hamprecht K, et al. Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation. Br J Haematol. 2006;134:64–76.

  16. 16.

    Gerdemann U, Katari UL, Papadopoulou A, Keirnan JM, Craddock JA, Liu H, et al. Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant. Mol Ther. 2013;21:2113–21.

  17. 17.

    Kaloyannidis P, Leen AM, Papadopoulou A. T-cell therapy: a powerful tool for the management of viral infections and relapse post hematopoietic stem cell transplantation. Expert Rev Hematol. 2012;5:471–3.

  18. 18.

    Leen AM, Myers GD, Sili U, Huls MH, Weiss H, Leung KS, et al. Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med. 2006;12:1160–6.

  19. 19.

    Papadopoulou A, Gerdemann U, Katari UL, Tzannou I, Liu H, Martinez C et al. Activity of broad-spectrum T cells as treatment for AdV, EBV, CMV, BKV, and HHV6 infections after HSCT. Sci Transl Med. 2014;6:242ra83.

  20. 20.

    Peggs KS, Verfuerth S, Pizzey A, Khan N, Guiver M, Moss PA, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet. 2003;362:1375–7.

  21. 21.

    Perruccio K, Tosti A, Burchielli E, Topini F, Ruggeri L, Carotti A, et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood. 2005;106:4397–406.

  22. 22.

    Gerdemann U, Keirnan JM, Katari UL, Yanagisawa R, Christin AS, Huye LE, et al. Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections. Mol Ther. 2012;20:1622–32.

  23. 23.

    De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis. 2008;46:1813–21.

  24. 24.

    Kumaresan PR, Manuri PR, Albert ND, Maiti S, Singh H, Mi T, et al. Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection. Proc Natl Acad Sci USA. 2014;111:10660–5.

  25. 25.

    Charan J, Kantharia N. How to calculate sample size in animal studies? J Pharmacol Pharmacother. 2013;4:303.

  26. 26.

    de Winter JCF. ERIC - using the Student’s ‘t’-test with extremely small sample sizes.Pract Assess Res Eval. v18 n10. 2013. https://eric.ed.gov/?id=EJ1015748.

  27. 27.

    Khanna N, Stuehler C, Conrad B, Lurati S, Krappmann S, Einsele H, et al. Generation of a multipathogen-specific T-cell product for adoptive immunotherapy based on activation-dependent expression of CD154. Blood. 2011;118:1121–31.

  28. 28.

    Foster AE, Marangolo M, Sartor MM, Alexander SI, Hu M, Bradstock KF, et al. Human CD62L- memory T cells are less responsive to alloantigen stimulation than CD62L+naive T cells: potential for adoptive immunotherapy and allodepletion. Blood. 2004;104:2403–9.

  29. 29.

    Corzo-León DE, Satlin MJ, Soave R, Shore TB, Schuetz AN, Jacobs SE, et al. Epidemiology and outcomes of invasive fungal infections in allogeneic haematopoietic stem cell transplant recipients in the era of antifungal prophylaxis: a single-centre study with focus on emerging pathogens. Mycoses. 2015;58:325–36.

  30. 30.

    Wurster S, Weis P, Page L, Helm J, Lazariotou M, Einsele H, et al. Intra- and inter-individual variability of Aspergillus fumigatus reactive T-cell frequencies in healthy volunteers in dependency of mould exposure in residential and working environment. Mycoses. 2017;60:668–75.

  31. 31.

    van de Veerdonk FL, Gresnigt MS, Romani L, Netea MG, Latgé J-P. Aspergillus fumigatus morphology and dynamic host interactions. Nat Rev Microbiol. 2017;15:661–74.

  32. 32.

    Stephen-Victor E, Karnam A, Fontaine T, Beauvais A, Das M, Hegde P, et al. Aspergillus fumigatus cell wall α-(1,3)-glucan stimulates regulatory T-cell polarization by inducing PD-L1 expression on human dendritic cells. J Infect Dis. 2017;216:1281–94.

  33. 33.

    Cenci E, Mencacci A, Bacci A, Bistoni F, Kurup VP, Romani L. T cell vaccination in mice with invasive pulmonary aspergillosis. J Immunol. 2000;165:381–8.

  34. 34.

    Bacher P, Jochheim-Richter A, Mockel-Tenbrink N, Kniemeyer O, Wingenfeld E, Alex R, et al. Clinical-scale isolation of the total Aspergillus fumigatus-reactive T-helper cell repertoire for adoptive transfer. Cytotherapy. 2015;17:1396–405.

  35. 35.

    Deo SS, Virassamy B, Halliday C, Clancy L, Chen S, Meyer W, et al. Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells. Cytotherapy. 2016;18:65–79.

  36. 36.

    Gaundar SS, Clancy L, Blyth E, Meyer W, Gottlieb DJ. Robust polyfunctional T-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of Aspergillus fumigatus. Cytotherapy. 2012;14:1119–30.

  37. 37.

    Tramsen L, Schmidt S, Boenig H, Latgé J-P, Lass-Flörl C, Roeger F, et al. Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida, Aspergillus and mucormycetes. Cytotherapy. 2013;15:344–51.

  38. 38.

    Zhu F, Ramadan G, Davies B, Margolis DA, Keever-Taylor CA. Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses. Clin Exp Immunol. 2008;151:284–96.

  39. 39.

    Ramadan G, Davies B, Kurup VP, Keever-Taylor CA. Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen. Clin Exp Immunol. 2005;140:81–91.

  40. 40.

    Stuehler C, Nowakowska J, Bernardini C, Topp MS, Battegay M, Passweg J, et al. Multispecific Aspergillus T cells selected by CD137 or CD154 induce protective immune responses against the most relevant mold infections. J Infect Dis. 2015;211:1251–61. https://doi.org/10.1093/infdis/jiu607.

  41. 41.

    Bozza S, Clavaud C, Giovannini G, Fontaine T, Beauvais A, Sarfati J, et al. Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination. J Immunol. 2009;183:2407–14.

  42. 42.

    Bacher P, Kniemeyer O, Teutschbein J, Thön M, Vödisch M, Wartenberg D, et al. Identification of immunogenic antigens from Aspergillus fumigatus by direct multiparameter characterization of specific conventional and regulatory CD4+T cells. J Immunol. 2014;193:3332–43.

  43. 43.

    Beck O, Topp MS, Koehl U, Roilides E, Simitsopoulou M, Hanisch M, et al. Generation of highly purified and functionally active human TH1 cells against Aspergillus fumigatus. Blood. 2006;107:2562–9.

  44. 44.

    Tramsen L, Koehl U, Tonn T, Latgé J-P, Schuster FR, Borkhardt A, et al. Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy. Bone Marrow Transplant. 2009;43:13–9.

  45. 45.

    Romani L. Immunity to fungal infections. Nat Rev Immunol. 2011;11:275–88.

  46. 46.

    Nanjappa SG, Heninger E, Wüthrich M, Sullivan T, Klein B. Protective antifungal memory CD8(+) T cells are maintained in the absence of CD4( + ) T cell help and cognate antigen in mice. J Clin Invest. 2012;122:987–99.

  47. 47.

    Carvalho A, De Luca A, Bozza S, Cunha C, D’Angelo C, Moretti S, et al. TLR3 essentially promotes protective class I-restricted memory CD8+ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients. Blood. 2012;119:967–77.

  48. 48.

    Potenza L, Vallerini D, Barozzi P, Riva G, Forghieri F, Beauvais A, et al. Characterization of specific immune responses to different Aspergillus antigens during the course of invasive Aspergillosis in hematologic patients. PLoS ONE 2013;8:e74326.

  49. 49.

    Smith C, Beagley L, Rehan S, Neller MA, Crooks P, Solomon M, et al. Autologous adoptive T-cell therapy for recurrent or drug-resistant cytomegalovirus complications in solid organ transplant recipients: a single-arm open-label phase I clinical trial. Clin Infect Dis. 2019;68:632–40. https://doi.org/10.1093/cid/ciy549.

  50. 50.

    Stanzani M, Orciuolo E, Lewis R, Kontoyiannis DP, Martins SLR, St, John LS, et al. Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood. 2005;105:2258–65.

  51. 51.

    Campanelli AP, Martins GA, Souto JT, Pereira MSF, Livonesi MC, Martinez R, et al. Fas‐Fas ligand (CD95‐CD95L) and cytotoxic T lymphocyte antigen–4 engagement mediate T cell unresponsiveness in patients with paracoccidioidomycosis. J Infect Dis. 2003;187:1496–505.

  52. 52.

    Chang KC, Burnham C-A, Compton SM, Rasche DP, Mazuski R, SMcDonough J, et al. Blockade ofthe negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis. Crit Care. 2013;17:R85.

  53. 53.

    Daver N, Kontoyiannis DP. Checkpoint inhibitors and aspergillosis in AML: the double hit hypothesis. Lancet Oncol. 2017;18:1571–3.

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Acknowledgements

This work was supported by the Greek State Scholarships Foundation, IKY fellowships of excellence for postdoctoral studies in Greece-SIEMENS program.

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Affiliations

  1. Hematology Department-Hematopoietic Cell Transplantation Unit, Gene and Cell Therapy Center, “George Papanikolaou” Hospital, Thessaloniki, 57010, Greece

    • Anastasia Papadopoulou
    • , Maria Alvanou
    • , Kiriakos Koukoulias
    • , Evangelia Athanasiou
    • , Andriana Lazaridou
    • , Nikolaos Savvopoulos
    • , Achilles Anagnostopoulos
    •  & Evangelia Yannaki
  2. Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece

    • Maria Alvanou
    • , Kiriakos Koukoulias
    • , Nikolaos Savvopoulos
    •  & Minas Yiangou
  3. Adult Hematology and Stem Cell Transplant, King Fahad Specialist Hospital Dammam, Dammam, 32253, Saudi Arabia

    • Panayotis Kaloyannidis
  4. 1st Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece

    • Anthi-Marina Markantonatou
    •  & Timoleon-Achilleas Vyzantiadis
  5. Department of Medicine, University of Washington, Seattle, WA, 98195, USA

    • Evangelia Yannaki

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Correspondence to Anastasia Papadopoulou.

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https://doi.org/10.1038/s41409-019-0501-9