An engineered NKp46 antibody for construction of multi-specific NK cell engagers.

Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.

Reciprocal transcription factor networks govern tissue-resident ILC3 subset function and identity.

Innate lymphoid cells (ILCs) are guardians of mucosal immunity, yet the transcriptional networks that support their function remain poorly understood. We used inducible combinatorial deletion of key transcription factors (TFs) required for ILC development (RORγt, RORα and T-bet) to determine their necessity in maintaining ILC3 identity and function. Both RORγt and RORα were required to preserve optimum effector functions; however, RORα was sufficient to support robust interleukin-22 production among the lymphoid tissue inducer (LTi)-like ILC3 subset, but not natural cytotoxicity receptor (NCR)+ ILC3s. Lymphoid tissue inducer-like ILC3s persisted with only selective loss of phenotype and effector functions even after the loss of both TFs. In contrast, continued RORγt expression was essential to restrain transcriptional networks associated with type 1 immunity within NCR+ ILC3s, which coexpress T-bet. Full differentiation to an ILC1-like population required the additional loss of RORα. Together, these data demonstrate how TF networks integrate within mature ILCs after development to sustain effector functions, imprint phenotype and restrict alternative differentiation programs.

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin-NK1.1+NKp46+ cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1+NKp46+ cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1+NKp46+ cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Host genetic control of natural killer cell diversity revealed in the Collaborative Cross.

Natural killer (NK) cells are innate effectors armed with cytotoxic and cytokine-secreting capacities whose spontaneous antitumor activity is key to numerous immunotherapeutic strategies. However, current mouse models fail to mirror the extensive immune system variation that exists in the human population which may impact on NK cell-based therapies. We performed a comprehensive profiling of NK cells in the Collaborative Cross (CC), a collection of novel recombinant inbred mouse strains whose genetic diversity matches that of humans, thereby providing a unique and highly diverse small animal model for the study of immune variation. We demonstrate that NK cells from CC strains displayed a breadth of phenotypic and functional variation reminiscent of that reported for humans with regards to cell numbers, key marker expression, and functional capacities. We took advantage of the vast genetic diversity of the CC and identified nine genomic loci through quantitative trait locus mapping driving these phenotypic variations. SNP haplotype patterns and variant effect analyses identified candidate genes associated with lung NK cell numbers, frequencies of CD94+ NK cells, and expression levels of NKp46. Thus, we demonstrate that the CC represents an outstanding resource to study NK cell diversity and its regulation by host genetics.

Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30.

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1+  cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ T cells (TRM  cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM  cells by NK1.1+  cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM  cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

Selective reconstitution of IFN­Î³ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection.

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

Quantitative analysis of human NK cell reactivity using latex beads coated with defined amounts of antibodies.

Natural Killer (NK) cell responses are regulated by a variety of different surface receptors. While we can determine the overall positive or negative effect of a given receptor on NK cell functions, investigating NK cell regulation in a quantitative way is challenging. To quantitatively investigate individual receptors for their effect on NK cell activation, we chose to functionalize latex beads that have approximately the same size as lymphocytes with defined amounts of specific antibodies directed against distinct activating receptors. This enabled us to investigate NK cell reactivity in a defined, clean, and controllable system. Only CD16 and NKp30 could activate the degranulation of resting human NK cells. CD16, NKG2D, NKp30, NKp44, and NKp46 were able to activate cultured NK cells. NK cell activation resulted in the induction of polyfunctional cells that degranulated and produced IFN-γ and MIP-1ß. Interestingly, polyfunctional NK cells were only induced by triggering ITAM-coupled receptors. NKp44 showed a very sensitive response pattern, where a small increase in receptor stimulation caused maximal NK cell activity. In contrast, stimulation of 2B4 induced very little NK cell degranulation, while providing sufficient signal for NK cell adhesion. Our data demonstrate that activating receptors differ in their effectiveness to stimulate NK cells.

Low-dose IL-2 induces CD56bright NK regulation of T cells via NKp44 and NKp46.

Low-dose interleukin (IL)-2 has shown clinical benefits in patients with autoimmune and inflammatory diseases. Both regulatory T cells (Tregs ) and natural killer (NK) cells are increased in response to low-dose IL-2 immunotherapy. The role of regulatory T cells in autoimmune diseases has been extensively studied; however, NK cells have not been as thoroughly explored. It has not been well reported whether the increase in NK cells is purely an epiphenomenon or carries actual benefits for patients with autoimmune diseases. We demonstrate that low-dose IL-2 expands the primary human CD56bright NK cells resulting in a contact-dependent cell cycle arrest of effector T cells (Teffs ) via retention of the cycle inhibitor p21. We further show that NK cells respond via IL-2R-ß, which has been shown to be significant for immunity by regulating T cell expansion. Moreover, we demonstrate that blocking NK receptors NKp44 and NKp46 but not NKp30 could abrogate the regulation of proliferation associated with low-dose IL-2. The increase in NK cells was also accompanied by an increase in Treg cells, which is dependent on the presence of CD56bright NK cells. These results not only heighten the importance of NK cells in low-dose IL-2 therapy but also identify key human NK targets, which may provide further insights into the therapeutic mechanisms of low-dose IL-2 in autoimmunity.

Expressions of natural cytotoxicity receptor, NKG2D and NKG2D ligands in endometriosis.

Pathogenesis of endometriosis is still unknown, and the relationship between NK cell activating receptors and endometriosis remains to be explored. We investigated the expression of NCRs and NKG2D in NK cells in peripheral blood (PB) and peritoneal fluid (PF) as well as expression of NKG2D ligands in endometrial cells, and illuminated their relationship with ovarian endometriosis. 20 patients with ovarian endometriosis and 13 subjects for control group were recruited. Flow cytometry was used for examining expressions of NCRs and NKG2D on NK cells. In PF with endometriosis, the expressions of NKp30 (P = 0. 006) and NKG2D (P = 0. 010) on CD56+NK cells were decreased, whereas the expression of NKp46 (P = 0. 040) on CD16+NK cells was higher than that of control. Real time PCR and Western blotting were used for detecting expression of NKG2D ligands. mRNA level of NKG2D ligands on endometrial cells showed no noticeable difference. As for protein expression, the ULBP-2 expression on eutopic endometrial cells with pelvic endometriosis was lower than that on ectopic endometrial cells and eutopic endometrial cells without endometriosis (P < 0.05), and the ULBP-3 expression on ectopic endometrial cells was lower than that on eutopic endometrial cells with or without endometriosis (P < 0.05). These findings indicate that change of NKp30, NKp46 and NKG2D on NK cells in PF and ULBP-2, 3 on endometrial cells may relate to the pathogenesis of pelvic endometriosis. Especially, change of NK cell activating receptors in PF implies that pelvic endometriosis is probably due to local immune changes.

Activation Receptor-Dependent IFN-γ Production by NK Cells Is Controlled by Transcription, Translation, and the Proteasome.

NK cells can recognize target cells such as virus-infected and tumor cells through integration of activation and inhibitory receptors. Recognition by NK cells can lead to direct lysis of the target cell and production of the signature cytokine IFN-γ. However, it is unclear whether stimulation through activation receptors alone is sufficient for IFN-γ production. In this study, we show that NK activation receptor engagement requires additional signals for optimal IFN-γ production, which could be provided by IFN-ß or IL-12. Stimulation of murine NK cells with soluble Abs directed against NK1.1, Ly49H, Ly49D, or NKp46 required additional stimulation with cytokines, indicating that a range of activation receptors with distinct adaptor molecules require additional stimulation for IFN-γ production. The requirement for multiple signals extends to stimulation with primary m157-transgenic target cells, which triggers the activation receptor Ly49H, suggesting that NK cells do require multiple signals for IFN-γ production in the context of target cell recognition. Using quantitative PCR and RNA flow cytometry, we found that cytokines, not activating ligands, act on NK cells to express Ifng transcripts. Ly49H engagement is required for IFN-γ translational initiation. Results using inhibitors suggest that the proteasome-ubiquitin-IKK-TPL2-MNK1 axis was required during activation receptor engagement. Thus, this study indicates that activation receptor-dependent IFN-γ production is regulated on the transcriptional and translational levels.

Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity.

Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer. VIDEO ABSTRACT.

NK cell-intrinsic FcεRIγ limits CD8+ T-cell expansion and thereby turns an acute into a chronic viral infection.

During viral infection, tight regulation of CD8+ T-cell functions determines the outcome of the disease. Recently, others and we determined that the natural killer (NK) cells kill hyperproliferative CD8+ T cells in the context of viral infection, but molecules that are involved in shaping the regulatory capability of NK cells remain virtually unknown. Here we used mice lacking the Fc-receptor common gamma chain (FcRγ, FcεRIγ, Fcer1g-/- mice) to determine the role of Fc-receptor and NK-receptor signaling in the process of CD8+ T-cell regulation. We found that the lack of FcRγ on NK cells limits their ability to restrain virus-specific CD8+ T cells and that the lack of FcRγ in Fcer1g-/- mice leads to enhanced CD8+ T-cell responses and rapid control of the chronic docile strain of the lymphocytic choriomeningitis virus (LCMV). Mechanistically, FcRγ stabilized the expression of NKp46 but not that of other killer cell-activating receptors on NK cells. Although FcRγ did not influence the development or activation of NK cell during LCMV infection, it specifically limited their ability to modulate CD8+ T-cell functions. In conclusion, we determined that FcRγ plays an important role in regulating CD8+ T-cell functions during chronic LCMV infection.

TGF-ß in Mice Ameliorates Experimental Autoimmune Encephalomyelitis in Regulating NK Cell Activity.

Multiple sclerosis is a disease characterized by inflammation and demyelination located in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for multiple sclerosis (MS). Although the roles of T cells in MS/EAE have been well investigated, little is known about the functions of other immune cells in the neuroinflammation model. Here we found that an essential cytokine transforming growth factor ß (TGF-ß) which could mediate the differentiation of Th17/regulatory T cells was implicated in the natural killer (NK) cells' activity in EAE. In EAE mice, TGF-ß expression was first increased at the onset and then decreased at the peak, but the expressions of TGF-ß receptors and downstream molecules were not affected in EAE. When we immunized the mice with MOG antigen, it was revealed that TGF-ß treatment reduced susceptibility to EAE with a lower clinical score than the control mice without TGF-ß. Consistently, inflammatory cytokine production was reduced in the TGF-ß treated group, especially with downregulated pathogenic interleukin-17 in the central nervous system tissue. Furthermore, TGF-ß could increase the transcription level of NK cell marker NCR1 both in the spleen and in the CNS without changing other T cell markers. Meanwhile TGF-ß promoted the proliferation of NK cell proliferation. Taken together, our data demonstrated that TGF-ß could confer protection against EAE model in mice through NK cells, which would be useful for the clinical therapy of MS.

The Natural Cytotoxicity Receptors in Health and Disease.

The Natural Cytotoxicity Receptors (NCRs), NKp46, NKp44, and NKp30, were some of the first human activating Natural Killer (NK) cell receptors involved in the non-MHC-restricted recognition of tumor cells to be cloned over 20 years ago. Since this time many host- and pathogen-encoded ligands have been proposed to bind the NCRs and regulate the cytotoxic and cytokine-secreting functions of tissue NK cells. This diverse set of NCR ligands can manifest on the surface of tumor or virus-infected cells or can be secreted extracellularly, suggesting a remarkable NCR polyfunctionality that regulates the activity of NK cells in different tissue compartments during steady state or inflammation. Moreover, the NCRs can also be expressed by other innate and adaptive immune cell subsets under certain tissue conditions potentially conferring NK recognition programs to these cells. Here we review NCR biology in health and disease with particular reference to how this important class of receptors regulates the functions of tissue NK cells as well as confer NK cell recognition patterns to other innate and adaptive lymphocyte subsets. Finally, we highlight how NCR biology is being harnessed for novel therapeutic interventions particularly for enhanced tumor surveillance.

Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile.

OBJECTIVE: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. METHODS: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. RESULTS: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. CONCLUSION: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

ILC3 function as a double-edged sword in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), composed mainly of Crohn's disease (CD) and ulcerative colitis (UC), are strongly implicated in the development of intestinal inflammation lesions. Its exact etiology and pathogenesis are still undetermined. Recently accumulating evidence supports that group 3 innate lymphoid cells (ILC3) are responsible for gastrointestinal mucosal homeostasis through moderate generation of IL-22, IL-17, and GM-CSF in the physiological state. ILC3 contribute to the progression and aggravation of IBD while both IL-22 and IL-17, along with IFN-γ, are overexpressed by the dysregulation of NCR- ILC3 or NCR+ ILC3 function and the bias of NCR+ ILC3 towards ILC1 as well as regulatory ILC dysfunction in the pathological state. Herein, we feature the group 3 innate lymphoid cells' development, biological function, maintenance of gut homeostasis, mediation of IBD occurrence, and potential application to IBD therapy.

Aberrant natural killer (NK) cell activation and dysfunction among ART-treated HIV-infected adults in an African cohort.

BACKGROUND: We examined NK cell phenotypes and functions after seven years of ART and undetectable viral loads (<50 copies/ml) with restored CD4 T-cell counts (≥500 cells/µl) and age-matched healthy-HIV-uninfected individuals from the same community. METHODS: Using flow-cytometry, NK cell phenotypes were described using lineage markers (CD56+/-CD16+/-). NK cell activation was determined by expression of activation receptors (NKG2D, NKp44 and NKp46) and activation marker CD69. NK cell function was determined by CD107a, granzyme-b, and IFN-gamma production. RESULTS: CD56 dim and CD56 bright NK cells were lower among ART-treated-HIV-infected than among age-matched-HIV-negative individuals; p = 0.0016 and p = 0.05 respectively. Production of CD107a (P = 0.004) and Granzyme-B (P = 0.005) was lower among ART-treated-HIV-infected relative to the healthy-HIV-uninfected individuals. NKG2D and NKp46 were lower, while CD69 expression was higher among ART-treated-HIV-infected than healthy-HIV-uninfected individuals. CONCLUSION: NK cell activation and dysfunction persisted despite seven years of suppressive ART with "normalization" of peripheral CD4 counts.

NK cells suppress CD8+ T cell immunity via NKG2D in severe aplastic anemia.

The roles of natural killer (NK) cells in shaping the immune system had raised wide interests. Here we intended to explore the regulatory functions of NK cells on CD8+ T cells in severe aplastic anemia (SAA) using human participants and lymphocyte infusion-induced bone marrow failure (BMF) mouse model. In SAA patients, NK cells had over-expressions of NKG2D and NKp46, under-expression of NKG2A and enhanced cytotoxicity. NK cells limited autologous CD8+ T cell immunity in an effector/target ratio manner. The suppression was dependent on the existence of NKG2D. We also observed upregulated MICA expression on activated CD8+ T cells, which were susceptible to NK cell mediated lysis in SAA. Animal model concurred with the data from patients. Infusion of NK cells suppressed the proliferation of CD8+ T cells and decreased IFN-γ production. In conclusion, NK cells served NKG2D-dependent immunoregulatory roles by attenuating autologous CD8+ T cell response in SAA.

Conditional Genetic Ablation Mouse Models as a Tool to Study Cancer Immunosurveillance In Vivo.

Over the last decades, it has been established that the immune system is crucial for the impediment of cancer development by recognizing and destroying transformed cells. This process has been termed cancer immunosurveillance. Small animal models have significantly facilitated our understanding of it. Dissecting the contribution of any specific immune cell type participating in this process requires the ability to specifically target it while leaving the other immune components as well as the cancer model system unperturbed in vivo. Here, we provide a simple and rapid protocol for the generation of transgenic mice expressing Cre recombinase in a cell type-specific manner-in our example we chose cells expressing Ncr1, which encodes for the surface protein NKp46-and the use of those mice to ablate NKp46+ cells in order to study their role in a model of cancer immunosurveillance against experimental pulmonary metastases. This protocol can easily be adapted to target other cell types and other cancer models.