Human Natural Killer Cell Differentiation Research Articles

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

Single-cell transcriptomics in bone marrow delineates CD56dimGranzymeK+ subset as intermediate stage in NK cell differentiation.

Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56brightCD16+, CD56dimCD16+ and CD69+CXCR6+ lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56dimGzmK+ subset was identified that shared features with CD56bright and CD56dimGzmK- NK cells based on transcriptome, phenotype (NKG2AhighCD16lowKLRG1highTIGIThigh) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56bright, CD56dimGzmK+ and CD56dimGzmK- cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34+ progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

Discrete Modes of Migration Define Human NK Cell Development from Hematopoietic Stem Cells

Human innate lymphoid cells play a critical role in the control of infection and the shaping of the immune response. Furthermore, natural killer (NK) cells are the first reconstituting lymphocyte population after hematopoietic stem cell transplant and play a key role in shaping engraftment and moderating graft vs. leukemic effects. Relative to their adaptive immune counterparts, the molecular interactions that drive innate immune cell differentiation development are poorly understood. Human NK cells arise from bone marrow CD34+ hematopoietic stem cells (HSC) but undergo maturation in distal microenvironments, the best characterized of which is secondary lymphoid tissue. Human NK cells can also be derived in vitro from CD34+ HSC through co-culture with developmentally supportive stromal cells. Despite the requirement for direct stromal cell contact in NK cell maturation, the nature of these contacts is not known. We have previously shown that freshly isolated human NK cell developmental intermediates from in vitro culture, secondary lymphoid tissue or peripheral blood undergo spontaneous migration on the EL08.1D2 stromal cell line. We have further defined the NK cell ‘developmental synapse’, a site of productive and directional signaling in developmental intermediates arrested on stromal cells. Despite these interesting insights, the regulation of, and requirement for, NK cell migration during differentiation has not been defined.To determine the modes of migration associated with human NK cell differentiation, we performed highly resolved continuous live cell imaging of NK cells developing in vitro from CD34+ HSC over 28 days of culture. These analyses were performed in parallel with flow cytometry to monitor the progression of developmental intermediates towards a functionally mature NK cell phenotype. We found that while CD34+ HSC had constrained motility, progressive NK cell differentiation was associated with more directed migratory phenotype. NK cell maturation was associated with increased track length and velocity, and decreased frequency of time of cells spent in arrest on stromal cells. We further defined the acquisition of complex, Levy-walk behavior as has previously been described for mature T cells imaged within tissue. The consistency of these behaviors between in vitro derived cells and fresh ex vivo NK cells isolated from healthy human donors confirms that in vitro differentiation effectively recapitulates migratory phenotypes in addition to functional and cell surface phenotypes associated with NK cell maturation.Together, these data define distinct modes of migration that are acquired by human NK cells. We propose that this reflects a previously unappreciated requirement for cell migration that enables the functional maturation of human NK cells. Better defining the bidirectional signaling between developing NK cells and their microenvironment will enable a complete understanding of the signals that help shape the acquisition of key innate immune cell functions. This understanding is critical for better predicting and therapeutically enhancing lymphocyte reconstitution after transplant. DisclosuresNo relevant conflicts of interest to declare.

Live Cell Tracking of Human NK Cell Precursors Identifies Complex Modes of Cell Migration Throughout Differentiation

Determining the complex interactions between immune cells and their microenvironment is key to understanding their functions in human health and disease. In particular, defining the nature and regulation of cell migration and its role in the development and functional maturation of immune cells will lead to improved outcomes of immune therapy and transplantation. Natural killer (NK) cells are innate immune effectors that play an important role in the control of viral infection and tumorigenesis. The development of human NK cells from hematopoietic precursors has not been well defined, in part because of a lack of satisfactory models of human hematopoiesis. We have developed a model for in vitro differentiation of human NK cells on developmentally supportive stromal cells that includes continuous phase-contrast imaging of NK cell developmental intermediates (NKDI) with high temporal and spatial resolution. Here, we combine this model with tracking and analyses to define the modes of migration that mark human NK cell development from CD34+ hematopoietic stem cells over the period of differentiation (28 days). We find that cells progress from a largely constrained mode of migration to increasingly random and directed modes. Overall heterogeneity of cell behavior increased throughout the time of imaging. This was in part due to an increase in the frequency of cell tracks that included multiple modes of migration (random, constrained, directed) within a single track. These complex modes were reminiscent of Levy-walk tracks previously demonstrated to improve efficiency of seeking behaviors. The consistency of these behaviors and those of freshly isolated NK cell subsets demonstrates that acquisition of complex modes of migration is a key component of human NK cell differentiation.

A long noncoding RNA positively regulates CD56 in human natural killer cells.

Natural killer (NK) cells are innate immune lymphocytes that play critical roles in host defense against viral infection and surveillance against malignant transformation. Long noncoding RNAs (lncRNAs) are important immune system regulators. Here, we analyzed human primary lymphocyte lncRNA expression profiles to identify NK-lncRNA signatures. We detected numerous novel NK-specific lncRNAs with potential roles in regulating human NK cell differentiation and function. Expression of lnc-CD56, an NK-specific lncRNA, was positively correlated with that of CD56, a classical human NK cell surface marker. We showed that lnc-CD56 may function as a positive regulator of CD56 in primary human NK cells and differentiated NK cells from human CD34+ hematopoietic progenitor cells. Our data provide an annotated human NK cell lncRNA expression catalog and demonstrate a key role for lncRNAs in NK cell biology.

Delineation of Natural Killer Cell Differentiation from Myeloid Progenitors in Human.

Understanding of natural killer (NK) cell development in human is incomplete partly because of limited access to appropriate human tissues. We have developed a cytokine-enhanced humanized mouse model with greatly improved reconstitution and function of human NK cells. Here we report the presence of a cell population in the bone marrow of the cytokine-treated humanized mice that express both NK cell marker CD56 and myeloid markers such as CD36 and CD33. The CD56+CD33+CD36+ cells are also found in human cord blood, fetal and adult bone marrow. Although the CD56+CD33+CD36+ cells do not express the common NK cell functional receptors and exhibit little cytotoxic and cytokine-producing activities, they readily differentiate into mature NK cells by acquiring expression of NK cell receptors and losing expression of the myeloid markers. Further studies show that CD33+CD36+ myeloid NK precursors are derived from granulo-myelomonocytic progenitors. These results delineate the pathway of human NK cell differentiation from myeloid progenitors in the bone marrow and suggest the utility of humanized mice for studying human hematopoiesis.

Integrated mRNA-microRNA profiling of human NK cell differentiation identifies MiR-583 as a negative regulator of IL2Rγ expression.

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

Proteome Analysis of Distinct Developmental Stages of Human Natural Killer (NK) Cells

The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.

Dendritic Cell Derived Cytokines in Human Natural Killer Cell Differentiation and Activation

Dendritic cells (DCs) and natural killer (NK) cells shape each other’s functions early during immune responses. DCs activate NK cells and NK cells can mature or kill DCs. In this review we will discuss which DC and NK cell subsets are mainly affected by this interaction, where these encounters might take place and which signals are exchanged. Finally, we will point out what the clinical benefit of understanding this interaction might be and how it changed our view on NK cells as innate lymphocytes.

CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells

The identification of developmental stages in natural killer (NK) cells, especially in human NK cells, has lagged for decades. We characterize four novel populations defined by CD11b and CD27, which can represent the distinct stages of human NK cells from different tissues. Nearly all NK cells from peripheral blood are CD11b(+) CD27(-) populations whereas NK cells from cord blood have CD11b(+) CD27(-) and CD11b(+) CD27(+) populations. Interestingly, we have found large CD11b(-) CD27(-) populations of NK cells from deciduas. We also demonstrate that each population could be characterized by unique functional and phenotypic attributes. CD11b(-) CD27(-) NK cells display an immature phenotype and potential for differentiation. CD11b(-) CD27(+) and CD11b(+) CD27(+) NK cells show the best ability to secrete cytokines. CD11b(+) CD27(-) NK cells exhibit high cytolytic function. We demonstrate that human NK cells at different developmental stages have special functions and describe a new model of human NK cell differentiation.

LFA-1 antigen identifies immature stages of human NK cell differentiation

Background . Human Natural killer (NK) cells are characterized by NK cell receptors (NKRs) with inhibitory and activatory function that finely control their functional activities. In particular, they express inhibitory receptors for MHC class I molecules, named killer cell immunoglobulin (Ig)-like receptors (KIRs) and C-type lectin CD94-CD159a, and many triggering molecules like NKp30, NKp44, NKp46, (called natural cytotoxicity receptors, NCRs), NKG2D, CD161, and CD244. The majority of peripheral blood human NK cells are characterized by a phenotype with a low density expression of CD56 (CD56 dim ) and a high expression of CD16 (CD16 bright ), whereas a minority (approximately 5–10%) shows a bright expression of CD56 (CD56 bright ). This latter NK subset presents relatively high expression of some cytokine receptors (CD117 and CD25) and the CD94-CD159a heterodimeric inhibitory receptor. CD56bright NK cells are widely expressed in lymphoid tissues and can be generated from CD34+ cells when cultured with combinations of flt-3 ligand (FL) or stem cell factor plus IL-15 or IL-2. During their development, NK cells sequentially acquire many different antigens but there is still limited knowledge on differentiation antigens able to identify immature human NK cells and the specific sequence through which developing NK cells acquire the expression of NKR. Methods . NK cells obtained from human CD34+ hematopoietic progenitor cells after 30-day culture with FL plus IL-15, or from peripheral and umbilical cord blood samples were characterized. Results . Virtually, all CD56 NK cells differentiated in vitro expressed CD117, CD25, NCRs, NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56 bright /NCRs+/NKG2D+/LFA-1 - /CD94 - subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34-/LFA-1 low /NKp46 dim /NKG2D dim /CD94 - subset that resembled an immature stage of NK cells present in lymph nodes. Conclusions . Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of human NK cell differentiation.

TOX regulates the differentiation of human natural killer cells from hematopoietic stem cells in vitro

Natural killer (NK) cells act important roles in innate immunity and adaptive immunity. However, the mechanisms governing NK cell development have not been clearly elucidated. Previous studies have shown that an HMG (high-mobility group) protein, TOX, is important for regulating the differentiation program of developing T cells in mice. In this study, we examined the role of TOX in differentiation of human NK cells. Knockdown of TOX in differentiating cells decreased the NK cell population identified by expression of NK surface markers and receptors. In addition, over-expression of TOX enhanced the differentiation of NK cells which give rise to a population showing effector functions of mature NK cells. Moreover, TOX influenced expression of T-bet (T-box expressed in T cells, also as known as Tbx21) during NK cell development. Overall, these results suggest that TOX is required for IL-15-mediated NK cell differentiation and affected expression of T-bet that plays critical roles in NK differentiation and maturation.

IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16+KIR+ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56hiCD16−KIR− to CD56loCD16+KIR−, and finally to CD56loCD16+KIR+. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.

Stromal Cells Support a Myeloid Pathway of Human NK Cell Differentiation.

Natural killer (NK) cells belong to the lymphocyte lineage; however a myeloid origin has been debated in the past based on nascent experimental evidence. We studied the in vitro development of human NK cells from UCB-derived CD34+ cells following culture with cytokines (IL15, IL7, SCF, FLT3L, IL3) on a murine fetal stromal cell line EL08.1D2 (Blood , 2006; 108: 3824–3833). We investigated the differential requirement of CD34+ subsets for stromal cell support. Limiting dilution experiments showed that CD34+ cells negative for phenotypic markers of NK commitment (CD7, CD161, integrin B7, CD122, CD45RA) absolutely require stromal cells and/or addition of hydrocortisone (HC) to differentiate into functional NK cells. Without stromal cells or HC those progenitors give rise to myeloid lineage cells, but not NK cells. Thus, we hypothesized that stromal cells could instruct myeloid precursors to convert to the NK lineage. Indeed, CD56+ cells generated in stroma supported cultures frequently co-express CD33 and CD13. To determine whether myeloid cells developing from CD34+ cells after 2–3 wk cultures could give rise to NK cells, we FACS sorted the CD56−CD33+CD13high and CD56−CD14+ populations. Such CD33+CD13high and CD14+ cells express macrosialin (CD68) and acquire lyzozyme (by FACS), confirming their myeloid characteristics. Sorted cells cultured further in cytokines alone (IL15, IL7, SCF, FLT3L) did not give rise to NK cells. However, in the presence of cytokines, stromal cells and HC, NK cells were generated. To exclude the possibility of NK cell contamination, CD33+CD13high and CD14+ cells were isolated from cultures of CD34+ cells in conditions not supportive of NK cell development (GM-CSF, IL3, FLT3L, SCF, without stroma, IL15 or IL7). Such cells gave the same results as above (i.e., NK cells developed only with stroma and HC). In additional studies, a fraction (∼16%) of CFU-GM colonies isolated from methylocellulose cultures could generate NK cells only in the presence of stromal cells, HC and cytokines, but not cytokines alone. As more of a definitive marker of the monocytic lineage, we used the surface expression of M-CSF receptor (CD115) on hematopoietic precursors. CD56−CD117+CD115+ and CD56−CD117+CD115− fractions were FACS sorted from 2–3 wk cultures of CD34+ cells. While both populations could differentiate into NK cells, only the CD115+ monocytic precursors required stromal cells. Quantitatively the CD117+CD115− cells were the main source of NK cells in this culture system. Notably the NK cells derived from CD115+ precursors were remarkably different, showing significantly higher expression of Killer Immunoglobulin-like Receptors (KIR: CD158a, CD158b and CD158e) than their CD115− derived counterparts (52% vs 15% KIR+, n=3, p=0.002). With respect to the repertoire of HLA-specific inhibitory receptors, NK cells derived from monocytic precursors resemble the dominant fraction of peripheral blood NK cells, including potentially alloreactive NK cells (KIR+CD94/NKG2A−). Collectively we present evidence that NK cells can be derived from developmental intermediates of the monocytic lineage and this differentiation pathway is dependent upon interaction with stroma. Our data indicate that the developmental trajectory shapes the pattern of inhibitory receptor expression on mature NK cells. Such findings have bearing on our understanding of NK cell biology, post transplant NK cell reconstitution and could explain the paucity of recognized immature NK cell leukemias coinciding with the occurrence of AML variants with NK specific antigen expression.

Evidence for discrete stages of human natural killer cell differentiation in vivo

Human natural killer (NK) cells originate from CD34(+) hematopoietic progenitor cells, but the discrete stages of NK cell differentiation in vivo have not been elucidated. We identify and functionally characterize, from human lymph nodes and tonsils, four NK cell developmental intermediates spanning the continuum of differentiation from a CD34(+) NK cell progenitor to a functionally mature NK cell. Analyses of each intermediate stage for CD34, CD117, and CD94 cell surface expression, lineage differentiation potentials, capacity for cytokine production and natural cytotoxicity, and ETS-1, GATA-3, and T-BET expression provide evidence for a new model of human NK cell differentiation in secondary lymphoid tissues.

Effects of IL-12 on human natural killer cell differentiation.

Effects of IL-12 on human natural killer cell differentiation.