Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: CD94 expression reveals the heterogeneity of the multifunctional CD56lowCD16low NK cell population

Innate lymphoid cells (ILC) include natural killer (NK) cells, ILC1, ILC2, ILC3 and lymphoid tissue inducer cells (LTi). In addition to the two main CD56high and CD56lowCD16high NK cells, another NK cell subset has been identified in our laboratory, namely the CD56lowCD16low cells, able both to release IFN-γ and to kill tumor cells. To better characterize this population and its developmental relationship with other ILCs, we performed a single-cell RNA sequencing on peripheral blood sorted CD56lowCD16high, CD56negCD16pos/neg and CD56high pooled with CD56lowCD16low cells from four healthy donors. A total of six clusters were identified and based on known gene signatures we found a cluster negative for NK cell-associated gene KLRD1 (CD94) and positive for the stem cell factor receptor CD117 and AHR transcription factor. This population showed only weak expression of other genes associated with NK cells, but we found a high expression levels of IL-1R, IL-18R and IL-7R along with the absence of IL-12Rβ transcript. In line to scRNA-seq data, we found that the expression of CD94 dissects CD56lowCD16low NK cell subset into two distinct populations: CD94neg and CD94pos cells. Based on the linear defining transcription factor (LDTF) profile, the CD94pos cells resemble CD56high cells. Differently, the CD94neg cells are less positive for the conventional NK cell TFs but express high levels of AHR and c-MYC known to characterize NK/ILC precursors. The study of cell metabolism corroborated the similarity between CD94pos and CD56high cells and highlighted peculiar metabolic pathways for CD94neg cells. Indeed, the CD94neg cells show a high expression of the nutrient receptors suggesting that they have an elevated rate of glucose-driven glycolysis, also sustained by the high glucose and fatty acid uptake. This is also supported by presence of high levels of pS6 at steady state, a downstream target of mTORC1 activity, which is essential for attaining an elevated glycolytic state. Moreover, CD94neg cells phenotypically display low levels of the common NK-cell activating receptors but express some receptors for key cytokines involved in NK cell differentiation, proliferation, survival, and activation. Furthermore, these cells are equipped with a large array of inhibitory receptors, and express some specific markers of ILCs (CD200R1, SIGLEC-9, CD9 and HLADR). Regarding the functional capacities, the CD94pos cells can kill and produce cytokines, while similarly to ILC1, the CD94neg cells are only characterized by the ability to secrete pro-inflammatory cytokines. In pathological conditions, we investigated the impact of colorectal cancer (CRC) on CD94neg and CD94pos cell subsets in PB. In CRC patients we found a significant reduction of CD94pos cells alongside an increase of CD94neg cell percentage and we observed an increase of the inhibitory receptors on both CD94neg and CD94pos cells accompanied by a significant impairment of functional ability. Collectively, these results demonstrate the multifunctional properties of CD94pos NK cells and highlight the unique phenotypic and metabolic features of CD94neg population and their ability to produce pro-inflammatory cytokines, suggesting that these cells may represent a precursor and/or an immature stage of NK cell development.