MicroRNA management of NK-cell developmental and functional programs.

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Helper innate lymphoid cells (ILCs) combat pathogen infection by rapid secretion of cytokines whereas killer ILCs (NK cells) eliminate virus-infected cells or tumor cells during early defense. However, the early development of NK cells and lineage relationships between NK cells and helper ILCs remain elusive. In PLZF lineage tracing model, normal NK cells are traced at a much lower ratio than helper ILCs, which indicates most NK cells develop from progenitors that do not express PLZF. However, it is also possible that most NK cells develop from PLZF-expressing ILC progenitors (ILCPs), but PLZF-labelled NK cells may proliferate less than unlabeled NK cells. To address these possibilities, we transferred ILCPs and its upstream progenitors early innate lymphoid progenitors (EILPs) and all lymphoid progenitors (ALPs), and then assessed PLZF labelling in NK cells derived from these progenitors. We found that ILCP-derived NK cells were traced at a ratio as high as in helper ILCs whereas EILP and ALP-derived NK cells were labelled at a much lower ratio, which is similar to the ratio in normal NK cells. These results indicate that NK cells develop from both ILCP-dependent and ILCP-independent pathways. Our findings establish novel lineage relationships between NK cells and helper ILCs. We are currently assessing whether different developmental pathways of NK cells lead to functionally distinct subsets of NK cells.

NK Cell Subsets Isolated From Human Thymus Differ From Peripheral Blood NK Cells in Their Cytotoxic and Cytokine Secreting Capability

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.

To investigate the relationship of T lymphocyte subsets, B lymphocytes and NK cells with the genesis, progression and prognosis of B cell lymphoma. The levels of T lymphocyte subsets, B lymphocytes and NK cells in peripheral blood of healthy control group and B cell lymphoma group were detected by flow cytometry (FCM). The clinical data were collected, and the relationship of these immune indexes with the general conditions, laboratory indexes, curative effect and prognosis were analyzed. Forty-four patients entolled in this study including 24 male and 20 females with the median age of 57 years old (17-82 years), all the patients were the first visit to our hospital and diagnosed. The total counts of lymphocytes, T, B and NK cells in the peripheral blood of patients with the first treatment of B-cell lymphoma were significantly lower than those in healthy controls, and the ratio of CD3+HLA-DR+ activated T cells was significantly higher than that of healthy controls (<0.05). In the newly diagnosed patients, the levels of total lymphocytes, total T cells, total B cells, CD4+ cells and NK cells in peripheral blood of patients with complete remission (CR) were higher than those in patients without complete remission ［partial remission (PR) and disease progression（PD）］ (<0.01, <0.01, <0.05, <0.01, <0.05, respectively). The logistic regression analysis showed that NK cells, FAC-1（FAC-1 is a component factor for linear regression of total lymphocytes, total T cells, and total B cells）, the ECOG score and β2-microglobulin level were significantly different between CR group and PR+SD group (<0.05, <0.01, <0.05, <0.01, respectively), among which ECOG score and β2-microglobulin were independent risk factor respectively for the prognosis of patients. The NK cells and FAC-1 were the protective factors for the prognosis of patients. The survival analysis performed according to the NK cell level of patients at initial diagnosis, the Kaplan-Meier survival curve found that the overall survival (OS) of the 2 groups was significantly different (<0.01), the survial rate in patients with NK cell absolute count> 61.5 /μl was higher than that in patients with low level of NK cells. The level of total lymphocytes, total T cells, total B cells, NK cells and advanced activated T cells in the patients with B cell lymphoma were significantly different from those in normal subjects. Total count of lymphocytes, T cells, B cells, CD4+ cells and NK cells in peripheral blood are important prognostic indicators for BCL. The ECOG score and β2-microglobulin level are independent risk factors for prognosis. The NK cell level and FAC-1 are independent protective factors for the prognosis of B cell lymphoma.

Preliminary Results of a Phase 1/2 Clinical Trial of Cndo-109-Activated Allogeneic Natural Killer Cells in High Risk Acute Myelogenous Leukemia Patients in First Complete Remission

This 29-color flow cytometry panel was developed and optimized for in-depth characterization of human peripheral blood NK cells for preclinical development and monitoring of NK cell therapies. The panel includes markers associated with NK cell differentiation, cytotoxicity, tissue residency, as well as NK cell dysfunction. Panel optimization was performed on freshly isolated and exvivo activated NK cells enriched from human peripheral blood mononuclear cells (PBMCs). Overall, this panel functions as a tool to extensively characterize human NK cells, paving the way for rapid and standardized approaches to evaluate the biological activity of therapeutic NK cell products.

Differential activation of cord blood and peripheral blood natural killer cells by cytokines

CD16 (Fc gammaRIIIA), the low affinity receptor for IgG, is expressed on the majority of human peripheral blood NK cells. Ligation of CD16 with mAb or immune complexes activates NK cell cytotoxicity and cytokine secretion, and stimulates death of activated NK cells by apoptosis. This study uses NK cells labeled with the stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester to provide quantitative data on the effect of CD16 ligation on NK cell division and NK cell survival. When NK cells are cultured with rIL-2 and CD16 is ligated, NK cell division is stimulated, but there also is a substantial loss of NK progenitor cells. When NK cell proliferation is stimulated by coculture with gamma-irradiated MM-170 malignant melanoma cells and rIL-2, CD16 ligation enhances entry of NK cells into division. In some cases, CD16 ligation is essential for NK cell proliferation stimulated by MM-170 cells. In these cultures, there is no loss of NK progenitor cells. This study demonstrates that CD16 is an activation receptor for NK cell proliferation, and suggests that cellular costimulation alters the balance between NK cell death and NK cell proliferation stimulated by CD16 ligation.

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

Human NK cell deficiency (NKD) is a primary immunodeficiency in which the main clinically relevant immunological defect involves missing or dysfunctional NK cells. Here, we describe a familial NKD case in which 2 siblings had a substantive NKD and neutropenia in the absence of other immune system abnormalities. Exome sequencing identified compound heterozygous variants in Go-Ichi-Ni-San (GINS) complex subunit 4 (GINS4, also known as SLD5), an essential component of the human replicative helicase, which we demonstrate to have a damaging impact upon the expression and assembly of the GINS complex. Cells derived from affected individuals and a GINS4-knockdown cell line demonstrate delayed cell cycle progression, without signs of improper DNA synthesis or increased replication stress. By modeling partial GINS4 depletion in differentiating NK cells in vitro, we demonstrate the causal relationship between the genotype and the NK cell phenotype, as well as a cell-intrinsic defect in NK cell development. Thus, biallelic partial loss-of-function mutations in GINS4 define a potentially novel disease-causing gene underlying NKD with neutropenia. Together with the previously described mutations in other helicase genes causing NKD, and with the mild defects observed in other human cells, these variants underscore the importance of this pathway in NK cell biology.

NK cells are innate lymphoid cells that play important roles in tumor eradication and viral clearance. We previously found that deletion or inhibition of the histone methyltransferase Ezh2 (enhancer of zeste homolog 2) in hematopoietic stem and progenitor cells (HSPCs) from both mice and humans enhanced the commitment and cytotoxicity of NK cells to tumor cells. This study tested the hypothesis that inhibiting Ezh2, especially in NK lineage cells, could also affect NK cell development and function. We crossed Ezh2fl/fl mice with Ncr1iCre mice to delete the Ezh2 gene in immature NK cells and downstream progeny. Ezh2 deficiency increased the total number of NK cells and promoted NK cell terminal differentiation, as the percentages of the most mature CD27- CD11b+ subsets were increased. The NK cell cytotoxicity against tumor cells in vitro was enhanced, with increased degranulation and IFN-γ production. In addition, during the process of human NK cells differentiating from HSPCs , inhibiting EZH2 catalytic activity at day 14 (when NK lineage commitment began) also resulted in increased proportions of mature NK cells and cytotoxicity. Furthermore, RNA-seq and CUT&RUN-qPCR assays showed that the effects of Ezh2 may be based on its direct modulation of the expression of the transcription factor Pbx1 (pre-B-cell leukemia transcription factor 1), which has been reported to promote NK cell development. In summary, we demonstrate that Ezh2 is a negative regulator of NK cell terminal maturation and function.

Recent studies have attempted to uncover the role of Group 1 Innate lymphoid cells (ILCs) in multiple physiological contexts, including cancer. However, the definition and precise contribution of Group 1 ILCs (constituting ILC1 and NK subsets) to metastasis is unclear due to the lack of well-defined cell markers. Here, we first identified ILC1 and NK cells in NSCLC patient blood and differentiated them based on the expression of transcription factors, T-bet and Eomes. Interestingly, Eomes downregulation in the peripheral blood NK cells of NSCLC patients positively correlated with disease progression. Additionally, we noted higher Eomes expression in NK cells (T-bet+Eomeshi) compared to ILC1s (T-bet+Eomeslo). We asked whether the decrease in Eomes was associated with the conversion of NK cells into ILC1 using Eomes as a reliable marker to differentiate ILC1s from NK cells. Utilizing a murine model of experimental metastasis, we observed an association between increase in metastasis and Eomes downregulation in NKp46+NK1.1+ Group 1 ILCs, which was consistent to that of human NSCLC samples. Further confirmation of this trend was achieved by flow cytometry, which identified tissue-specific Eomeslo ILC1-like and Eomeshi NK-like subsets in the murine metastatic lung based on cell surface markers and adoptive transfer experiments. Next, functional characterization of these cell subsets showed reduced cytotoxicity and IFNγ production in Eomeslo ILC1s compared to Eomeshi cells, suggesting that lower Eomes levels are associated with poor cancer immunosurveillance by Group 1 ILCs. These findings provide novel insights into the regulation of Group 1 ILC subsets during metastasis, through the use of Eomes as a reliable marker to differentiate between NK and ILC1s.

Human CD8+ T lymphocytes and NK cells can be successfully engrafted in highly immuno-deficient mouse strains such as NOD/shi-SCID/γgcnull (NOG), NOD/SCID/IL2Rγnull (NSG), NOD/Rag1KO/γcnull (NRG), and BALB/c-Rag2KO/γcnull (BRG) mice following reconstitution with human CD34+ hematopoietic stem cells (HSCs) or, alternatively, upon adoptive transfer of peripheral blood mononuclear cells (PBMC). These humanized immune system (HIS) mice have evolved as a promising tool to study human CD8+ T cell and NK cell-mediated immune responses to cancer and infectious diseases and to explore new approaches in adoptive immunotherapy and vaccination. However, long-term generation of CD8+ T lymphocytes and NK cells is limited in the absence of human leukocyte antigen (HLA) class I expression, appropriate chemokines and trafficking signals to support proper homing into lymphoid tissues and reasonable concentrations of human cytokines to promote CD8+ T and NK cell development, maintenance and function. In this chapter we review the current status and the existing limitations of CD8+ T cell and NK cell biology in HIS mice and outline recent progress made using new substrains expressing human HLA molecules and exogenous supplementation of human cytokines to improve humanization.

NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain-containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1 -/-) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1 -/- mice is also changed. Lower frequencies of Sostdc1 -/- splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27-CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1 -/- mice would correlate with NK killing ability and observed that Sostdc1-/- NK cells are hyporesponsive against MHC class I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1's known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1 -/- NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1-/- immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.