Assessment of NK Cell Function in the Immune Landscape of Endometriosis

Abstract

Endometriosis (EN) is a chronic inflammatory disease characterised by the growth of endometrium-like tissue outside the uterus. Altered immune responses, including impaired natural killer (NK) cell cytotoxicity, have been proposed to contribute to disease development. The aim of this study was therefore to assess NK cell function in EN and identify what NK cell receptor interactions contribute to NK cell degranulation and cytotoxic responses towards EN-derived cells. In parallel, protocols for immune cell isolation from EN samples were developed and optimised to support future phenotypic and functional characterisation. Healthy donor (HD) NK cells were co-cultured with EN-derived cultured cells (ENCCs) and their response assessed using a flow cytometry-based combined degranulation and cytotoxicity assay. Addition of antibodies targeting specific NK cell receptors or their ligands enabled the identification of key NK cell receptors involved in EN-induced NK cell activation. Complementary ligand expression on ENCCs was analysed using flow cytometry. Our studies revealed ENCC-induced NK cell degranulation, demonstrating their ability to activate NK cells. Moreover, receptor-blocking experiments identified DNAM-1, NKG2D, and NKp46 as important contributors to this response. Ligand expression analyses supported the involvement of these activating receptor-ligand interactions, showing high expression of the PVR, Nectin-2, ULBP-2/5/6, and TRAIL R2. High expression of HLA-ABC, which mainly mediates inhibitory signalling, was also observed on ENCCs. In conclusion, this study demonstrates that ENCCs can be recognised by NK cells through multiple activating receptor pathways and provides further insight into NK cell interactions in EN. The developed protocols for processing EN samples, furthermore, form a foundation for future studies within the EndoMIC project.