A Novel CD94+ Immature NK Cell Population Based on NKp80 Expression with ILC3-like Features in Human Secondary Lymphoid Tissue

Natural killer (NK) cells are important for their innate ability to identify and kill cancer cells without previous stimulation. However, many of the mechanisms concerning their differentiation from immature precursor cells are still unknown. Elucidation of these pathways are critical to improving targeted therapies that can both activate as well as overcome inhibitory pathways that prevent an effective response. NK cells are thought to develop in secondary lymphoid tissue (SLT) through five stages distinguished by the expression of CD34, CD117, CD94, and CD16. While virtually all NK cells in blood express the pan-NK cell activating receptor, NKp80, we have discovered a distinct Lin CD94+NKp80-CD16- population that is selectively enriched in SLT. The NKp80- population appears to comprise those cells “in transit” between stage 3 (CD117+CD94-) and stage 4 (CD117loCD94+) when evaluating CD94 and CD117 expression among Lin-CD34- cells by flow cytometry. These ex vivo data suggest that this NKp80- population may represent a novel NK cell developmental intermediate (NKDI). SLT-derived NKp80- cells lack cytolytic granules, show minimal cytotoxicity, and produce negligible amounts of interferon-gamma when compared to NKp80+ NK cells in SLT and blood. In addition, NKp80- cells have lower mRNA and protein expression of TBET and EOMES compared to NKp80+ NK, while expressing these factors at higher levels compared to stage 3 cells. Unexpectedly, the NKp80- population also demonstrates Group 3 innate lymphoid cell (ILC3)-like features ex vivo with expression of surface interleukin-1 (IL-1) receptor 1 and CD127 and mRNA and protein expression of the ILC3-associated transcription factors, AHR and RORC. Moreover, following overnight stimulation with IL-1-beta and IL-23, NKp80- cells produce IL-22, albeit at lower concentrations compared to stage 3 cells. Collectively, these data identify NKp80 as a surrogate marker of functional competence during in vivo human NK cell development and provide evidence for the existence of at least two distinct maturation steps within the previously described stage 4 NK cell population in SLT. Since CD94 expression is currently considered to be NK-restricted, these findings raise new questions regarding the developmental relationship between NK cells and ILC3 in humans. In addition, as ILC3s are primarily used to stimulate an anti-microbial defense while NK cells are functional anti-cancer effector cells, defining the differentiation patterns of these cells can have impacts in both of these areas of research. Studies are ongoing to investigate the in vitro differentiation potential(s) of this novel SLT-derived Lin-CD94+NKp80-CD16- population.