Molecular Signatures And Transcriptional Regulatory Networks Of Immature And Mature Human NK Cells

Natural killer (NK) cells are one of the most important components of innate immunity and also participate in adaptive immunity. They play key roles in early host defense against viruses and other pathogenic infections as well as in killing tumor cells by releasing cytokines and/or chemokines and by mediating cytotoxicity. Human NK cells originate from CD34+hematopoietic stem cells (HSCs) mainly present in the bone marrow (BM). When properly cultured in vitro, CD34+HSCs isolated from various sites, including cord blood, peripheral blood, thymus, secondary lymphoid organs, liver, and decidua, give rise to mature NK cells, indicating that NK cells could, at least in part, undergo terminal differentiation in situ. The maturation of NK cells needs discrete stages, but the molecular signatures and transcriptional regulatory differences between immature and mature NK cells are mostly unknown. As the molecular definition of NK cells in mice has been well described, the comprehensive understanding of human NK cells has not yet been achieved.NK cells in the peripheral blood account for a small fraction of total lymphocytes (-10%) and a approximately95%of peripheral NK cells (pNK) were of the CD56dim CD16+mature subset. In contrast, NK cells are the dominant lymphocyte in the decidua during normal pregnancy, comprising up to～70%of the total lymphocytes and approximately90%of decidual NK cells (dNK) were of the CD56bright CD16-immature phenotype. To further confirm that dNK cells are more immature than pNK cells, we analyzed T-bet and ID2expression. Interestingly, we found that dNK cells were mostly T-bet-, while＞90%of the pNK were T-bet+. Additionally, western blot analysis showed that ID2was exclusively expressed by dNK cells. Furthermore, we detected many known cell-surface markers related to NK-cell maturation. Consistent with published observations, dNK cells expressed many immature cell markers, including CD27and CD94, whereas pNK cells displayed a more mature phenotype with higher expression of CD57and CDllb. Overall, these results demonstrate that dNK cells exhibit immature characteristics relative to pNK cells.To investigate novel molecular signatures and transcriptional regulators of immature and mature human NK cells, we performed whole-genome microarray analysis on purified dNK, cord-blood NK (cNK), and pNK cells, and peripheral CD56+T cells and CD3+CD56-T cells were used as controls. Using high-resolution microarray analyses with verification of results by realtime-PCR and flow cytometry, we describe a comprehensive comparative analysis between idNK cells with a CD56brightCD16-T-bet-phenotype and mpNK cells with a CD56dimCD16+T-bet+phenotype. Many common characteristics and differences between immature and mature human NK cells were indicated here.Firstly, we describe a comparative analysis of the integral membrane proteins between idNK and mpNK cells and found that the differences in cell-surface molecule expression were consistent with the known functions of these cells. While idNK cells expressed more inhibitory receptors (including NKG2A, CD158b, and GITR) that may relate to their weak cytotoxic ability and maintenance of self-tolerance, mpNK cells expressed more activating receptors and co-stimulatory factors (including CD2, CD8, and CD226) that may be required for their strong cytotoxic ability to respond to pathogens and tumors.Secondly, we also identified many growth factor, cytokine, and chemokine transcripts that were distinct between idNK and mpNK cells. Numerous growth factors, cytokines, and chemokines are expressed by NK cells and may regulate NK cell development, or function in an autocrine/paracrine manner. These growth factors include bone morphogenetic protein2(BMP2), Jagged2, and osteopontin (OPN). In particular, idNK cells contained higher mRNA levels of cytokines linked to osteoclast and osteoblast genesis (e.g., Tnfsfll, Tnfrsf11b, Sppl, and Bmp2) compared with mpNK cells.Thirdly, we found that idNK cells had higher expression of chemokine genes, including Xcll, Cxcll, Cxcl10, and Cxcl14, which confer idNK cells with an increased ability to recruit other NK cells or lymphocytes, such as immature DCs and neutrophils. In contrast to our finding that idNK cells showed enriched expression of many chemokine genes, mpNK cells were instead enriched for many chemokine receptor genes (including Cxcr4, Cxcr2, Cxcrl and Ccr6). While several chemokine receptor genes were overexpressed in mpNK cells compared with idNK cells, indicating that mpNK cells may have more migration ability than idNK cells.Fourthly, we analysis the transcription factor expression profiles of the immature and mature NK cells. Our study is the first to describe the probable transcriptional regulators of the different human NK populations, including immature (dNK and CD56bright pNK cells) and mature (CD56dim pNK and CD34+cell-derived NK cells) NK cells. We found that idNK and mpNK cells are enriched for homeobox family TFs (such as Hoxa5, Hoxa9, Pbxl, and Hop) and zinc-finger proteins (such as Klf9, Znfl43, Znf483, and Znf831), respectively. We presume that the homeobox family TFs highly expressed by idNK cells may contribute to their immaturity, while zinc-finger proteins highly expressed by mpNK cells may regulate genes important for NK-cell cytotoxicity. Additionally, through comparative analysis with T cells, we identified many novel candidate NK cell regulators. Thus far, many transcriptional regulators in the mouse immune system have been identified, including Id2, Nfil3, Eomes, Mitf, and Klf4, and we confirm here that many lessons learned from mouse models can also be applied to the human system. In addition to confirming well-known TFs, we identified many novel TFs (including Etv5, Nfe2, Mycn, Nr2f2, and Hoxa10) that potentially regulate NK-cell development and function.Lastly, according to our data and the putative genes targeted by the selected TFs, we propose a self-regulatory network for NK cells. From this, we can infer that during HSCs development into NK cells, autocrine and/or paracrine cytokines and chemokines induce or activate TFs that regulate each other and conversely activate or promote the expression of molecules important for NK-cell development and maturation. This may be one feasible mechanism underlying the generation of programmed mature and functional NK cells. Additionally, we highlight that autocrine BMP2and TGF-β2, in coordination with their regulated cytokines (such as Osteoprotegerin/OPG, OPN, and Pleiotrophin/PTN) and the TFs enriched in idNK cells (such as homeobox TFs, CREB, and MYCN), may contribute to idNK-cell immaturity.The major highlights and significance from our study are as follows:(1) We identify a number of novel autocrine cytokines expressed by immature decidual NK cells that may play a role in early NK cell development and maintenance of the immaturity. Intriguingly, these cytokines include six factors (including Osteoprotegerin and Pleiotrophin) important for bone formation and development, indicating a new putative feature of NK cells and a virgin area for further research.(2) Notably, we identify and describe the transcriptional regulators of the immature and mature human NK cells. We find that immature decidual NK cells are enriched in homeobox-family transcription factors, which contribute to their immaturity; mature peripheral NK cells are enriched in zinc-finger proteins, which may contribute to their cytotoxic function. (3) Our data suggest that autocrine cytokines and transcription factors in NK cells regulate with each other, forming a complicated regulatory network. To the best of our knowledge, our study is the first to describe a comprehensive NK cell self-regulatory network.(4) Through a comprehensive comparative analysis with T cells, we identified many novel candidate regulators of NK cells, the collective actions of which may be essential for NK cell differentiation.Overall, we demonstrate our study from the perspective of cell development and differentiation. We analyzed not only the cell-surface receptors but also other NK-cell signatures, including growth factors, cytokines, chemokines, and transcriptional regulators, which may direct NK-cell fate. In conclusion, these results provide a detailed and comprehensive catalog of the signatures representing immature and mature human NK cell populations. The novel candidates for key transcription factors identified here and their downstream targets can be used to describe and further understand the transcriptional regulatory network underlying NK cell differentiation and function. Gaining a better understanding of the molecular signatures and transcriptional regulators that govern NK cell development can allow us to harness better NK cell functions in multiple clinical settings.