Phenotypic Profile of Peripheral Blood NK Cells under Culturing with Trophoblast Cells and IL-15 and IL-18 Cytokines

Natural killer cells (NK cells) are innate immunity lymphocytes. NK cell differentiation is controlled by the cellular microenvironment and locally produced cytokines, including IL-2, IL-15 and IL-18. NK cells are present in various tissues, forming pools of tissue-resident NK cells, e.g., decidual NK cell pool. Peripheral blood NK cells (pNK cells) are considered a supposed source of cells for decidual NK cell differentiation. In the uterus, NK cells contact with trophoblast cells, which can affect their phenotype. Contribution of trophoblast cells and IL-2, IL-15 and IL-18 cytokines to the pNK cell phenotype regulation is scarcely studied. In this regard, the aim of our research was to evaluate the effect of trophoblast cells on the phenotype of pNK cells when cultured in medium with IL-2, IL-15, and IL-18. We used mononuclear cells obtained from peripheral blood of healthy non-pregnant women at their reproductive age, with regular menstrual cycle (n = 21). Mononuclear cells were cultured in presence of IL-2, and either of cytokines regulating NK cell differentiation (IL-15, or IL-18). JEG-3 cells were used as trophoblast cells. We evaluated expression of CD45, CD3, CD56, CD14, KIR3DL1, KIR2DL3, KIR2DL4, KIR2DS4, NKp44, CD215, CD122, CD127, NKG2D, KIR2DL1, NKG2C receptors by pNK cells. It was found that pNK cells cultured in presence of trophoblast cells (JEG-3 cell line) were characterized by lower intensity of CD56 receptor expression, compared to pNK cells cultured without trophoblast cells. These changes were detected upon culturing both in medium supplied by IL-15, and with IL-18. A reduced number of NKG2C+ pNK cells was detected in presence of JEG-3 trophoblast cells, compared to NK cells cultured without trophoblast cells in medium with IL-15. The detected changes in the CD56 and NKG2C expression by pNK cells in presence of trophoblast cells proved to be opposite to those previously detected for NK cells derived from NK-92 cell line. Along with trophoblast cells, the monocytes isolated among mononuclear cells and being affected by cytokines, can apparently influence the phenotype of pNK cells in the model system used. Since monocytes/macrophages are present in decidua, further research is required to study the effect of cytokines and cellular microenvironment, including monocytes, on pNK cells.

NK Cell Anti-Tumor Surveillance in a Myeloid Cell-Shaped Environment

NK cells are innate lymphoid cells endowed with cytotoxic capacity that play key roles in the immune surveillance of tumors. Increasing evidence indicates that NK cell anti-tumor response is shaped by bidirectional interactions with myeloid cell subsets such as dendritic cells (DCs) and macrophages. DC-NK cell crosstalk in the tumor microenvironment (TME) strongly impacts on the overall NK cell anti-tumor response as DCs can affect NK cell survival and optimal activation while, in turn, NK cells can stimulate DCs survival, maturation and tumor infiltration through the release of soluble factors. Similarly, macrophages can either shape NK cell differentiation and function by expressing activating receptor ligands and/or cytokines, or they can contribute to the establishment of an immune-suppressive microenvironment through the expression and secretion of molecules that ultimately lead to NK cell inhibition. Consequently, the exploitation of NK cell interaction with DCs or macrophages in the tumor context may result in an improvement of efficacy of immunotherapeutic approaches.

Ex Vivo Production of Large Numbers of Genetically Modified NK Cells from Cord Blood or Mobilized Peripheral Blood CD34 + Cells Using Notch Ligand Delta-like 4 Culture System

Natural killer (NK) cells are an essential component of human innate immune, with a remarkable ability to provide protection against cancer and viral infections. NK-based immune therapy has several advantages over T cell immunotherapy: NK cells do not cause graft-versus-host disease and do not induce T-cell driven inflammatory cytokine storm. Moreover, unlike T cell, NK cells do not need prior sensitization, specific antigen recognition and clonal expansion for their cytotoxic effector functions and they can rapidly trigger cytotoxicity against their targets. NK cells can be expanded from multiple sources including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), or mobilized peripheral blood (mPB) CD34 + cells. Yet the lack of efficient methods for in-vitro NK cell expansion, purification and genetic modification limits the clinical use of NK cell therapy. We aimed at developing a simple, scalable and clinically feasible technique for therapeutic NK cell production based on our recently published culture system that can produce large numbers of unmodified or genetically modified CD7 + T lymphoid progenitors from UCB and mPB CD34 + cells in 7 days on immobilized Notch-ligand delta-like 4 recombinant protein DLL4. Since T cells and NK cells share a common T/NK cell progenitor, we are interested to investigate the NK cell potential of DLL4 culture-generated T lymphoid progenitors. Firstly, we tested the NK cell potential of the transduced or non- transduced UCB or mPB CD34 +-derived progenitors from DLL4 culture by subjecting them to a feeder free NK cell differentiation for 3 weeks. HSPC-derived progenitors can be efficiently transduced with lentiviral vectors (average transduction efficacy: 50%). The transduced/ non-transduced progenitors were able to efficiently differentiate into NK (CD3 -CD56 +) cells beginning from one week of differentiation, reaching a frequencies of NK cells of >90% without any detectable T cell contamination at week 2. The phenotypic characterization of the NK cells demonstrates the presence of activation receptors such as NKG2D, DNAM-1, NKp30, NKp44 and NKp46 while lacking the inhibitory receptors like KLRG1, KIR2DL2/DL3 and KIR3DL1/DL2. Interestingly, these cells express the transcription factors known to be essential for NK cell differentiation and functions such as Eomes, T-bet and ID2. Additionally, the CB or mPB HSPC-derived NK cells (both transduced/ non-transduced) express perforin and granzyme B reflecting their ability to show cytotoxic potential. Further, the stimulation of UCB or mPB derived NK cells with myelogenous leukemia cell line K562 cells showed high level of degranulation, which is the key step for Interferon gamma (IFNg) induction. An analysis cytotoxic activity of the CB or mPB derived NK cells against K562 cells and monocytic leukemia cell line THP1 cells showed their ability to efficiently kill K562 cells compared to THP1 cells, however the UCB derived NK cells were highly cytotoxic compared to mPB derived NK cells. These data suggests that our DLL4 culture system, along with feeder-free NK cell differentiation is a unique combination that is able to give rise to high number of pure NK cell population with high cytotoxic potential. These results lay a foundation towards an easier approach to NK cell therapy for effective treatment of cancers and viral infections, which will be developed in collaboration with Smart Immune Inc. Disclosures Cavazzana: Smart Immune: Other: co-founder.

Single-Cell Sequencing Reveals the Novel Role of Ezh2 in NK Cell Maturation and Function

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and exhibit important functional properties in antimicrobial and antitumoral responses. Our previous work indicated that the enhancer of zeste homolog 2 (Ezh2) is a negative regulator of early NK cell differentiation and function through trimethylation of histone H3 lysine 27 (H3K27me3). Here, we deleted Ezh2 from immature NK cells and downstream progeny to explore its role in NK cell maturation by single-cell RNA sequencing (scRNA-seq). We identified six distinct NK stages based on the transcriptional signature during NK cell maturation. Conditional deletion of Ezh2 in NK cells resulted in a maturation trajectory toward NK cell arrest in CD11b SP stage 5, which was clustered with genes related to the activating function of NK cells. Mechanistically, we speculated that Ezh2 plays a critical role in NK development by activating AP-1 family gene expression independent of PRC2 function. Our results implied a novel role for the Ezh2-AP-1-Klrg1 axis in altering the NK cell maturation trajectory and NK cell-mediated cytotoxicity.

The Effect of Telomerase Inhibition on NK Cell Activity in Acute Myeloid Leukemia

Purpose: Acute myeloid leukemia (AML) is known to be an invasive and highly lethal hematological malignancy in adults and children. Resistance to the present treatments, including radiotherapy and chemotherapy with their side effects and telomere length shortening are the main cause of the mortality in AML patients. Telomeres sequence which are located at the end of eukaryotic chromosome play pivotal role in genomic stability. Recent studies have shown that apoptosis process is blocked in AML patient by the excessive telomerase activity in cancerous blasts. Therefore, the find of effective ways to prevent disease progression has been considered by the researchers. Natural killer (NK) cells as granular effector cells play a critical role in elimination of abnormal and tumor cells. Given that the cytotoxic function of NK cells is disrupted in the AML patients, we investigated the effect of telomerase inhibitors on NK cell differentiation. Methods: To evaluate telomerase inhibition on NK cell differentiation, the expression of CD105, CD56, CD57, and KIRs was evaluated in CD34+ derived NK cells after incubation of them with BIBR1532. Results: The results showed that the expression of CD105, CD56, CD57, and KIRs receptors reduces after telomerase inhibition. According to these findings, BIBR1532 affected the final differentiation of NK cells. Conclusion: The results revealed that telomerase inhibitor drugs suppress cancer cell progression in a NK cells-independent process.

Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.

The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.