Investigating NK cell diversity in colorectal cancer: a role for adaptive NK cells in immunotherapeutic strategies

Natural killer (NK) cells are cytotoxic innate lymphocytes that play a central role in immune surveillance against malignant transformation through the integration of activating and inhibitory receptor signals. Their phenotype and function can be reshaped by inflammatory conditions, viral exposure, and tissue-specific microenvironments. Adaptive NK cells, arising upon cytomegalovirus (CMV) infection, exemplify this concept and have attracted attention for their enhanced cytotoxic potential and long-term persistence, making them promising candidates for cancer immunotherapy. Indeed, in cancer, such plasticity drives the selection and remodeling of NK cell subsets with important implications for antitumor immunity and NK cell-based immunotherapeutic strategies. Colorectal cancer (CRC), the second cause of cancer-related mortality worldwide, remains a major clinical challenge, highlighting the need for more effective therapies. However, the composition and functional state of tumor-associated NK cell subsets in CRC remain incompletely defined. This PhD thesis investigates the NK cell diversity in CRC, through extensive phenotypic analyses with potential translational applications. Tumor-associated NK cells from 80 CRC patients were characterized by multiparametric flow cytometry and compared with matched tumor-free tissue (TFT) and peripheral blood (PB), with sample stratification according to their microsatellite status (MSI: microsatellite instability; MSS: microsatellite stability). Tumor-infiltrating NK cells were significantly enriched in immune checkpoint (IC) expression (i.e. KIRs, NKG2A, and TIM-3) relative to TFT. Notably, PD-1+ NK cells were increased in tumors compared to both PB and TFT and were preferentially associated with MSI CRC, indicating a tumor microenvironment-induced modulation. Moreover, tumor-associated NK cells displayed tissue-residency markers (CD103 and/or CD49a), including PD-1+ NK cells in MSI CRC, consistent with local retention. Despite the IC enrichment, tissue-resident NK cells expressed activating receptors and cytotoxic molecules and preserved at least in part their functional competence. Thus, these findings will support the design of patient-tailored immunotherapies exploiting both circulating and resident NK cells. In line with this concept, a clinical case analysis of two MSI metastatic CRC patients, expressing PD-L1 and lacking HLA class I (HLA-I) on tumor cells, treated with pembrolizumab (anti-PD-1), showed favorable clinical outcomes despite poor prognostic factors, suggesting a contribution of PD-1+ NK cells in PD-L1+ HLA-I- tumor eradication upon PD-1 blockade. Moreover, emerging NK cell-based immunotherapies rely on combinations of multiple IC inhibitors, as well as on the targeted engagement of activating receptors. In this framework, a subset of NK cells expressing the activating receptor NKG2C was detected within CRC specimens. Since NKG2C is a hallmark receptor of circulating adaptive NK cells, its presence in tumors raises relevant biological questions. However, the relationship between CRC-associated NKG2C+ NK cells and canonical circulating adaptive NK cells remains to be defined. In parallel, to gain further insights in this clinically relevant NK subset, canonical and adaptive NK cells from the PB of healthy individuals were characterized by proteomic analysis integrated with publicly available CITE-seq data. Adaptive NK cells displayed increased expression of T-cell-associated receptors autophagy-related proteins and anti-apoptotic molecules, consistent with long-term persistence and specialized memory competence. Then, an in vitro expansion protocol was established to selectively expand adaptive NK cells starting from NK-enriched PB of CMV-seropositive healthy donors and using an anti-CD94 monoclonal antibody. Expanded adaptive NK cells retained their molecular signature and strong cytotoxic activity against multiple tumor targets. Importantly, preliminary data demonstrated the feasibility of selectively expanding adaptive NK cells also from the PB of CRC patients, providing proof-of-concept data for patient-derived NK cell products. Collectively, this work delineates NK cell remodeling in CRC, highlights IC-modulated and tissue-resident NK subsets, identifies CRC-associated NKG2C+ NK cells as a relevant population warranting further investigations, and establishes a scalable and translational strategy for generating adaptive NK cells for future NK cell-based immunotherapeutic applications.