Natural killer cells and engagers: Powerful weapons against cancer.

Natural killer (NK) cells are innate immune effectors whose functions rely on receptors binding cytokines, recognizing self-molecules, or detecting danger signals expressed by virus-infected or tumor cells. The potent cytotoxic potential makes NK cells promising candidates for cancer immunotherapy. To enhance their activity strategies include cytokine administration, blocking of immune checkpoints, and designing of antibody-based NK cell engagers (NKCEs). NKCEs represent a cutting-edge approach to cancer therapy: they strengthen the NK-to-target cell interactions and optimize tumor killing, possibly overcoming the immunosuppressive tumor microenvironment. NK cells belong to the innate lymphoid cells (ILCs) and are categorized into different subsets also including cells with a memory-like phenotype: this complexity needs to be explored in the context of cancer immunotherapy, particularly when designing NKCEs. Two strategies to enhance NK cell activity in cancer patients can be adopted: activating patients' own NK cells versus the adoptive transfer of ex vivo activated NK cells. Furthermore, the capability of NKCEs to activate γδ T cells could have a significant synergistic effect in immunotherapy.

NKG2D engagement on human NK cells leads to DNAM-1 hypo-responsiveness through different converging mechanisms.

Natural killer (NK) cell activation is regulated by activating and inhibitory receptors that facilitate diseased cell recognition. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. During tumor progression, however, these receptors are frequently downmodulated and rendered functionally inactive. Of note, NKG2D internalization has been associated with the acquisition of a dysfunctional phenotype characterized by the cross-tolerization of unrelated activating receptors. However, our knowledge of the consequences of NKG2D engagement is still incomplete. Here, by cytotoxicity assays combined with confocal microscopy, we demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance.

Human NK cells, their receptors and function.

NK cells are cytotoxic components of innate lymphoid cells (ILC) that provide a first line of defense against viral infections and contribute to control tumor growth and metastasis. Their function is finely regulated by an array of HLA-specific and non-HLA-specific inhibitory and activating receptors which allow to discriminate between healthy and altered cells. Human NK cells gained a major attention in recent years because of the important progresses in understanding their biology and of some promising data in tumor therapy. In this review, we will outline well-established issues of human NK cells and discuss some of the open questions, debates, and recent advances regarding their origin, differentiation, and tissue distribution. Newly defined NK cell specializations, including the impact of inhibitory checkpoints on their function, their crosstalk with other cell types, and the remarkable adaptive features acquired in response to certain virus infections will also be discussed.

Comprehensive Phenotyping of Human PB NK Cells by Flow Cytometry.

The NK cell compartment provides powerful innate defenses against virus-infected and tumor cells. Specific NK cell receptors control this process and maintain the immune system homeostasis and prevent autoimmunity. A wide variety of NK cell subsets with different functional capabilities exist and this reflects not only the different maturation stages of NK cells but also different microenvironments in which they can operate. In this review, we will give an overview on the various NK cell subsets present in peripheral blood of healthy donors in order to clearly and univocally identify them on the basis of their phenotypic traits using flow cytometry. © 2020 International Society for Advancement of Cytometry.

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

HLA-DR+ NK cells are mostly characterized by less mature phenotype and high functional activity.

NK cells change their phenotype and functional characteristics during activation. In this work, we searched for a relationship of HLA-DR expression with differentiation stages and functional activity of NK cells ex vivo and stimulated in vitro with IL-2 challenged with gene modified feeder K562 cells expressing membrane-bound IL-21 (K562-mbIL21). This stimulation technique has been described for NK cell expansion in clinical use. We have observed that HLA-DR expression in freshly isolated circulating NK cells was mostly associated with less differentiated CD56bright CD57- cells, although in some individuals it could also be found in terminally differentiated CD57+ cells. Ex vivo HLA-DR+ NK cells possessed better capacity to produce IFN-γ in response to cytokine stimulation compared to their HLA-DR- counterparts. In vitro activation with IL-2 and K562-mbIL21 induces an increase in HLA-DR-positive NK cell proportion, again mostly among CD56bright CD57- NK cells. This happened in particular due to appearance of HLA-DR+ expression de novo in HLA-DR-negative cells. Acquired in vitro HLA-DR expression was associated with NK cell proliferation activity, more intense cytokine-induced IFN-γ production, increased degranulation toward feeder cells, and higher expression of CD86 and NKG2D. Thus, stimulation with IL-2/K562-mbIL21 causes a significant phenotype and functional shift during NK cell activation and expansion.

Expansion of CD16 positive and negative human NK cells in response to tumor stimulation.

NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.

TLR9 agonism differentially impacts human NK cell-mediated direct killing and antibody-dependent cell-mediated cytotoxicity.

There are two known mechanisms by which natural killer (NK) cells recognize and kill diseased targets: (i) direct killing and (ii) antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated an indirect NK cell activation strategy for the enhancement of human NK cell killing function. We did this by leveraging the fact that toll-like receptor 9 (TLR9) agonism within pools of human peripheral blood mononuclear cells (PBMCs) results in a robust interferon signaling cascade that leads to NK cell activation. After TLR9 agonist stimulation, NK cells were enriched and incorporated into assays to assess their ability to kill tumor cell line targets. Notably, differential impacts of TLR9 agonism were observed-direct killing was enhanced while ADCC was not increased. To ensure that the observed differential effects were not attributable to differences between human donors, we recapitulated the observation using our Natural Killer-Simultaneous ADCC and Direct Killing Assay (NK-SADKA) that controls for human-to-human differences. Next, we observed a treatment-induced decrease in NK cell surface CD16-known to be shed by NK cells post-activation. Given the essential role of CD16 in ADCC, such shedding could account for the observed differential impact of TLR9 agonism on NK cell-mediated killing capacity.

SMAD4 enhances the cytotoxic efficacy of human NK cells against colorectal cancer cells via the m6A reader YTHDF2.

Background: Colorectal cancer (CRC) ranks as the third most prevalent malignant neoplasm in terms of both morbidity and mortality. Within the tumor microenvironment (TME) of CRC, the diminished presence and diminished cytotoxic function of natural killer (NK) cells serve as important factors driving the advancement of CRC; however, the precise regulatory mechanisms governing this phenomenon remain incompletely understood. Consequently, the identification of novel, potential anti-CRC targets associated with NK cells emerges as a pressing and paramount concern warranting immediate attention. Methods: We examined the regulatory mechanism of SMAD4-mediated NK cell cytotoxicity on CRC by utilizing various experimental techniques, such as qRT-PCR, flow cytometry. Results: Our findings revealed that the expression of SMAD4 is decreased in NK cells within the TME of human CRC. Furthermore, we observed that enforced upregulation of SMAD4 resulted in enhanced cytotoxicity of NK cells towards CRC cells. Furthermore, our research has revealed that YTHDF2 functions as a downstream effector of SMAD4, playing a crucial role in the control of transcription and translation of m6A-modified RNA. Moreover, our investigation demonstrated that increased expression of SMAD4 promoted the activating receptor NKG2D by elevating levels of YTHDF2. Ultimately, the SMAD4-YTHDF2 regulatory axis significantly enhanced the cytotoxicity of NK cells against human CRC cells. Conclusion: Our study unveils a novel mechanism through which SMAD4 modulates the cytotoxicity of NK cells towards CRC cells, suggesting that SMAD4 may hold promise as a potential therapeutic target for NK cell therapy in CRC.

CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison.

BACKGROUND: CAR NK cells as vehicles for engineered "off-the-shelf" cellular cancer immunotherapy have attracted significant interest. Nonetheless, a comprehensive comparative assessment of the anticancer activity of CAR T cells and CAR NK cells carrying approved benchmark anti-CD19 CAR constructs is missing. Here, we report a direct head-to-head comparison of CD19-directed human T and NK cells. METHODS: We generated CAR T and CAR NK cells derived from healthy donor PBMC by retroviral transduction with the same benchmark second-generation anti-CD19 CAR construct, FMC63.28z. We investigated IFN-γ secretion and direct cytotoxicity in vitro against various CD19+ cancer cell lines as well as in autologous versus allogeneic settings. Furthermore, we have assessed anticancer activity of CAR T and CAR NK cells in vivo using a xenograft lymphoma model in an autologous versus allogeneic setting and a leukemia model. RESULTS: Our main findings are a drastically reduced capacity for CAR-mediated IFN-γ production and lower CAR-mediated cytotoxicity of CAR NK cells relative to CAR T cells in vitro. Consistent with these in vitro findings, we report superior anticancer activity of autologous CAR T cells compared with allogeneic CAR NK cells in vivo. CONCLUSIONS: CAR T cells had significantly higher CAR-mediated effector functions than CAR NK cells in vitro against several cancer cell lines and autologous CAR T cells outperformed allogeneic CAR NK cells both in vitro and in vivo. CAR NK cells will likely benefit from further engineering to enhance anticancer activity to ultimately fulfill the promise of an effective off-the-shelf product.

Methods to Analyze the Developmental Trajectory of Human Primary NK Cells Using Monocle and SCENIC Analyses.

Development of novel cellular therapies based on primary human NK cells is under active investigation. Human NK cells are comprised of distinct subsets with high transcriptomic heterogeneity. Unique methodologies are being developed to determine the transcriptomic profiles of human NK cells. NK cells account for 10-20% of total lymphocytes in the human peripheral blood, which mediates anti-tumor and anti-viral effector functions. Therapeutic success in the clinic depends on a better understanding of the single-cell transcriptome of human NK cell subsets. Moreover, a better understanding of the transcriptional network that regulates NK cell development, subset specification, and terminal maturation is obligatory for their in vitro generation and expansion toward clinical utilization. Here, we describe the procedure for single-cell RNA-sequencing of human NK cells and strategies for bioinformatic analyses. This protocol provides a data analysis roadmap for investigators who work on the basic biology and therapeutic applications of human NK cells.

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.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

T-BET and EOMES Accelerate and Enhance Functional Differentiation of Human Natural Killer Cells.

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.

Role of the Main Non HLA-Specific Activating NK Receptors in Pancreatic, Colorectal and Gastric Tumors Surveillance.

Human NK cells can control tumor growth and metastatic spread thanks to their powerful cytolytic activity which relies on the expression of an array of activating receptors. Natural cytotoxicity receptors (NCRs) NKG2D and DNAM-1 are those non-HLA-specific activating NK receptors that are mainly involved in sensing tumor transformation by the recognition of different ligands, often stress-induced molecules, on the surface of cancer cells. Tumors display several mechanisms aimed at dampening/evading NK-mediated responses, a relevant fraction of which is based on the downregulation of the expression of activating receptors and/or their ligands. In this review, we summarize the role of the main non-HLA-specific activating NK receptors, NCRs, NKG2D and DNAM-1, in controlling tumor growth and metastatic spread in solid malignancies affecting the gastrointestinal tract with high incidence in the world population, i.e., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and gastric cancer (GC), also describing the phenotypic and functional alterations induced on NK cells by their tumor microenvironment.

miRNAs in NK Cell-Based Immune Responses and Cancer Immunotherapy.

The incidence of certain forms of tumors has increased progressively in recent years and is expected to continue growing as life expectancy continues to increase. Tumor-infiltrating NK cells may contribute to develop an anti-tumor response. Optimized combinations of different cancer therapies, including NK cell-based approaches for targeting tumor cells, have the potential to open new avenues in cancer immunotherapy. Functional inhibitory receptors on NK cells are needed to prevent their attack on healthy cells. Nevertheless, disruption of inhibitory receptors function on NK cells increases the cytotoxic capacity of NK cells against cancer cells. MicroRNAs (miRNAs) are small non-coding RNA molecules that target mRNA and thus regulate the expression of genes involved in the development, maturation, and effector functions of NK cells. Therapeutic strategies that target the regulatory effects of miRNAs have the potential to improve the efficiency of cancer immunotherapy. Interestingly, emerging evidence points out that some miRNAs can, directly and indirectly, control the surface expression of immune checkpoints on NK cells or that of their ligands on tumor cells. This suggests a possible use of miRNAs in the context of anti-tumor therapy. This review provides the current overview of the connections between miRNAs and regulation of NK cell functions and discusses the potential of these miRNAs as innovative biomarkers/targets for cancer immunotherapy.

Human NK cells: surface receptors, inhibitory checkpoints, and translational applications.

NK cells play important roles in innate defenses against viruses and in the control of tumor growth and metastasis. The regulation/induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral infection, or tumor transformation. The HLA-class I-specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class-Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 ligand (PD-L1) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk leukemias.

Type I Interferon Receptor on NK Cells Negatively Regulates Interferon-γ Production.

NK cells are a key antiviral component of the innate immune response to HSV-2, particularly through their production of IFN-γ. It is still commonly thought that type I IFN activates NK cell function; however, rather than requiring the type I IFN receptor themselves, we have previously found that type I IFN activates NK cells through an indirect mechanism involving inflammatory monocytes and IL-18. Here, we further show that direct action of type I IFN on NK cells, rather than inducing IFN-γ, negatively regulates its production during HSV-2 infection and cytokine stimulation. During infection, IFN-γ is rapidly induced from NK cells at day 2 post-infection and then immediately downregulated at day 3 post-infection. We found that this downregulation of IFN-γ release was not due to a loss of NK cells at day 3 post-infection, but negatively regulated through IFN signaling on NK cells. Absence of IFNAR on NK cells led to a significantly increased level of IFN-γ compared to WT NK cells after HSV-2 infection in vitro. Further, priming of NK cells with type I IFN was able to suppress cytokine-induced IFN-γ production from both human and mouse NK cells. We found that this immunosuppression was not mediated by IL-10. Rather, we found that type I IFN induced a significant increase in Axl expression on human NK cells. Overall, our data suggests that type I IFN negatively regulates NK cell IFN-γ production through a direct mechanism in vitro and during HSV-2 infection.

Small-Molecule Immunosuppressive Drugs and Therapeutic Immunoglobulins Differentially Inhibit NK Cell Effector Functions in vitro.

Small-molecule immunosuppressive drugs (ISD) prevent graft rejection mainly by inhibiting T lymphocytes. Therapeutic immunoglobulins (IVIg) are used for substitution, antibody-mediated rejection (AbMR) and HLA-sensitized recipients by targeting distinct cell types. Since the effect of ISD and IVIg on natural killer (NK) cells remains somewhat controversial in the current literature, the aim of this comparative study was to investigate healthy donor's human NK cell functions after exposure to ISD and IVIg, and to comprehensively review the current literature. NK cells were incubated overnight with IL2/IL12 and different doses and combinations of ISD and IVIg. Proliferation was evaluated by 3[H]-thymidine incorporation; phenotype, degranulation and interferon gamma (IFNγ) production by flow cytometry and ELISA; direct NK cytotoxicity by standard 51[Cr]-release and non-radioactive DELFIA assays using K562 as stimulator and target cells; porcine endothelial cells coated with human anti-pig antibodies were used as targets in antibody-dependent cellular cytotoxicity (ADCC) assays. We found that CD69, CD25, CD54, and NKG2D were downregulated by ISD. Proliferation was inhibited by methylprednisolone (MePRD), mycophenolic acid (MPA), and everolimus (EVE). MePRD and MPA reduced degranulation, MPA only of CD56bright NK cells. MePRD and IVIg inhibited direct cytotoxicity and ADCC. Combinations of ISD demonstrated cumulative inhibitory effects. IFNγ production was inhibited by MePRD and ISD combinations, but not by IVIg. In conclusion, IVIg, ISD and combinations thereof differentially inhibit NK cell functions. The most potent drug with an effect on all NK functions was MePRD. The fact that MePRD and IVIg significantly block NK cytotoxicity, especially ADCC, has major implications for AbMR as well as therapeutic strategies targeting cancer and immune cells with monoclonal antibodies.