Aspergillus fumigatus-specific CAR T cells are effective at treating invasive pulmonary aspergillosis
SEIF M. 1, KAKOSCHKE T. 2, EBEL F. 2, BELLET M. 3, TRINKS N. 7, ROMANI L. 3, TAPPE B. 1, ESPIE D. 6, DONNADIEU E. 6, SAUER M. 7, HÜNNIGER K. 5, KURZAI O. 4,5, EINSELE H. 1, HUDECEK M. 1, LÖFFLER J. 1
1 Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany; 2 Institut für Infektionsmedizin und Zoonosen, Medizinische Fakultät, LMU, München, Germany; 3 Dipartimento di Medicina e Chirurgia, Università degli Studi di Perugia, Perugia, Italy; 4 Institut für Hygiene und Mikrobiologie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany; 5 Fungal Septomics Research Group, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie – Hans-Knöll-Institut (HKI), Jena, Germany; 6 Université de Paris, Institut Cochin, INSERM, CNRS, Paris, France; 7 Biozentrum und RVZ - Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
Invasive pulmonary aspergillosis (IPA), mainly caused by Aspergillus fumigatus (A. fumigatus), is a leading cause of mortality in immunocompromised patients. Aspergillus-specific T cells are essential for fungal clearance. Such T cells are infrequent in the endogenous repertoire and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and explored their potential in IPA therapy.
We expressed the Af-CAR in T cells using non-viral sleeping beauty transposon system. We verified the CAR expression and specificity by flow cytometry, microscopy, calcium flux, and IFN-? secretion. In co-cultures with A. fumigatus hyphae, we analyzed the antifungal activity of Af-CAR T cells alone or together with innate immune cells, we measured cytokine secretion, ROS production, and hyphal damage. Finally, we analyzed Af-CAR T cells therapeutic potential in vivo.
T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and showed no off-targets in the human lung or cross-reaction with other fungi. CD8+ Af-CAR T cells released perforin and granzyme B and exerted a direct anti-fungal effect against A. fumigatus hyphae. Furthermore, CD4+ and CD8+ Af-CAR T cells produced mainly Th1 cytokines that activated macrophages and neutrophils to potentiate the anti-fungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T showed a higher potential to reduce fungal burden than CD4+ T cells. They controlled the local inflammation in the lung by recruiting mainly mononuclear cells and reducing the damage induced to the lung. Adoptive transfer of CD8+ Af-CAR T cells in a neutropenic IPA mouse model improved overall survival.
Taken together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.