Af-CAR drives a unique phenotype in NK cells boosting their antifungal activity against <em>Aspergillus fumigatus </em> — ASN Events
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  3. Af-CAR drives a unique phenotype in NK cells boosting their antifungal activity against Aspergillus fumigatus

Af-CAR drives a unique phenotype in NK cells boosting their antifungal activity against Aspergillus fumigatus (#228)

Beeke Tappe 1 , Jeany Söhnlein 1 , Eslam S. Ibrahim 2 , Maximilian Bauser 1 , Marina M. Bellet 3 , Nora Trinks 4 , Nicole Seifert 4 , Marilena Pariano 3 , Sarah Balucchi 3 , Wibke Krüger 5 , Knut Ohlsen 2 , Luigina Romani 3 , Markus Sauer 4 , Ilse D. Jacobsen 5 , Hermann Einsele 1 , Jürgen Löffler 1 , Michael Hudecek 1 , Michelle Seif 1
  1. Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, Würzburg, Germany
  2. Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany
  3. Dipartimento di Medicina e Chirurgia, Università degli Studi di Perugia, Perugia, Italy
  4. Lehrstuhl für Biotechnologie und Biophysik, Biozentrum und RVZ - Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
  5. Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany

Invasive pulmonary aspergillosis (IPA) poses an escalating threat to immunocompromised patients, driven by increasing risk factors and growing resistance to existing antifungal treatments. To address this challenge, we recently developed a Chimeric Antigen Receptor targeting Aspergillus fumigatus (Af-CAR). Af-CAR successfully redirected the specificity of T cells toward A. fumigatus, inducing potent anti-fungal responses, particularly in CD8+ T cells. Building upon this, we explored the use of CAR NK cells as an attractive "off-the-shelf" alternative, leveraging their intrinsic anti-fungal activity and low risk of cytokine release syndrome.

We engineered NK92 cells to express the Af-CAR using the Sleeping Beauty transposon system and assessed their antifungal efficacy in pre-clinical IPA models. In co-culture with A. fumigatus, the Af-CAR induced a mixed pro-inflammatory and anti-inflammatory phenotype in NK cells and enhanced their capacity to inhibit hyphal growth. In a murine IPA model, Af-CAR NK cells recruited mononuclear cells while reducing neutrophil infiltration, thus lowering fungal burden and alleviating lung tissue damage. To uncover the molecular mechanisms underlying this anti-fungal activity, we performed single-cell RNA sequencing (scRNA-seq). The analysis identified that upon stimulation with A. fumigatus hyphae, small airway epithelial cells and macrophages secreted CXCL-10, CCL-3, and IL-15, which recruited and activated Af-CAR NK cells. Furthermore, CCL-3 and IFN-γ released by Af-CAR NK cells enhanced the anti-fungal function of macrophages. Overexpression of IL-15 further boosted NK cell persistence and cytokine production. Notably, adoptive transfer of Af-CAR NK cells improved survival in neutropenic IPA mouse models.

In conclusion, the Af-CAR induces a distinct phenotype in NK cells, characterized by the expression of IFN-γ, IL-10, and CCL-3, which enhances their direct anti-fungal activity and promotes a collaborative immune response with macrophages. These findings highlight the potential of Af-CAR NK cells as a promising therapeutic approach for IPA.