Tracing NK cell subsets in the blood and tumour microenvironment

Natural Killer (NK) cells are key innate lymphocytes involved in tumour surveillance, exerting cytotoxic activity and shaping adaptive immune responses. However, their functional heterogeneity and plasticity within peripheral blood (PB) and the tumour microenvironment (TME) remain incompletely understood. This thesis aimed to elucidate how a hypomethylating agent can modulate NK cell subsets in peripheral blood. In addition, it aimed to characterize tumour-infiltrating NK (Ti-NK) cells across diverse tumour types (ovarian, melanoma, lung, and renal cancers).The first part of the project investigated the impact of Guadecitabine (Gua), a next-generation DNA methyltransferase inhibitor, on human PB-NK cells. Through phenotypic, functional, and single-cell transcriptomic analyses, Gua was shown to promote NK cell maturation, particularly within the CD56^bright subset. Gua treatment induced increased KIR expression and enhanced pro-inflammatory cytokine production, notably TNF-α and IFN-γ, without impairing cytotoxic potential. Single-cell RNA sequencing further revealed transcriptional reprogramming consistent with enhanced functional competence. These findings indicate that Gua preferentially targets less mature NK cells, reinforcing their immunoregulatory properties and accelerating their maturation state.The second part focused on Ti-NK cells across four solid cancer types. Using high-dimensional flow cytometry and ex vivo patient-derived tumour fragment (PDTF) cultures, distinct Ti-NK cell clusters were identified, highlighting substantial heterogeneity within the TME. Functional perturbation with anti-PD-1 and anti-NKG2A monoclonal antibodies demonstrated differential responsiveness of Ti-NK subsets, supporting the rationale for combinatorial immunotherapeutic strategies.Overall, this work provides novel insight into NK cell plasticity and supports epigenetic priming and checkpoint blockade as complementary strategies to enhance NK cell–mediated anti-tumour immunity.