Ralph J. Maas ¹ Janneke S. Hoogstad-van Evert ^1,2 Iris M. Hagemans ¹ Jolanda Brummelman ¹ Diede Van Ens ¹ Paul K. De Jonge ¹ Laura Hooijmaijers ¹ Shweta Mahajan ¹ Anniek B. Van Der Waart ¹ Charlotte K. Hermans ¹ Janne De Klein ¹ Rob Woestenenk ¹ Antonius E. Van Herwaarden ¹ Nicolaas P. Schaap ¹ Somayeh Rezaeifard ¹ Daniele V. Tauriello ¹ Petra L. Zusterzeel ¹ Nelleke Ottevanger ¹ Joop H. Jansen ¹ Willemijn Hobo ¹ Harry Dolstra ^1*

• ¹ Radboud University Medical Centre, Nijmegen, Netherlands
• ² Amphia Ziekenhuis, Breda, Netherlands

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signalling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signalling could increase NK cell immune responses in high-grade EOC patients.