Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

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Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing

Blood Advances ◽

10.1182/bloodadvances.2019000699 ◽

2020 ◽

Vol 4 (7) ◽

pp. 1388-1406 ◽

Cited By ~ 9

Author(s):

Samantha L. Smith ◽

Philippa R. Kennedy ◽

Kevin B. Stacey ◽

Jonathan D. Worboys ◽

Annie Yarwood ◽

...

Keyword(s):

Nk Cells ◽

Single Cell ◽

Rna Sequencing ◽

Peripheral Blood ◽

Nk Cell ◽

Killer Cell ◽

Early Response ◽

Receptor Expression ◽

Type I ◽

Single Cell Rna Sequencing

Abstract Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2–activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16−, CD56dimCD16+CD57−, and CD56dimCD16+CD57+. CD56dimCD16+CD57− cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon–responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

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Insights Into Human Intrahepatic NK Cell Function From Single Cell RNA Sequencing Datasets

Frontiers in Immunology ◽

10.3389/fimmu.2021.649311 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gráinne Jameson ◽

Mark W. Robinson

Keyword(s):

Nk Cells ◽

Single Cell ◽

Rna Sequencing ◽

Human Liver ◽

Immune Cell ◽

Nk Cell ◽

Single Cells ◽

Cell Populations ◽

Cellular Interactions ◽

Single Cell Rna Sequencing

Diverse populations of natural killer (NK) cells have been identified in circulating peripheral blood and a wide variety of different tissues and organs. These tissue-resident NK cell populations are phenotypically distinct from circulating NK cells, however, functional descriptions of their roles within tissues are lacking. Recent advances in single cell RNA sequencing (scRNA-seq) have enabled detailed transcriptional profiling of tissues at the level of single cells and provide the opportunity to explore NK cell diversity within tissues. This review explores potential novel functions of human liver-resident (lr)NK cells identified in human liver scRNA-seq studies. By comparing these datasets we identified up-regulated and down-regulated genes associated with lrNK cells clusters. These genes encode a number of activating and inhibiting receptors, as well as signal transduction molecules, which highlight potential unique pathways that lrNK cells utilize to respond to stimuli within the human liver. This unique receptor repertoire of lrNK cells may confer the ability to regulate a number of immune cell populations, such as circulating monocytes and T cells, while avoiding activation by liver hepatocytes and Kupffer cells. Validating the expression of these receptors on lrNK cells and the proposed cellular interactions within the human liver will expand our understanding of the liver-specific homeostatic roles of this tissue-resident immune cell population.

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Varicella-Zoster Virus and Herpes Simplex Virus 1 Differentially Modulate NKG2D Ligand Expression during Productive Infection

Journal of Virology ◽

10.1128/jvi.00292-15 ◽

2015 ◽

Vol 89 (15) ◽

pp. 7932-7943 ◽

Cited By ~ 23

Author(s):

Tessa M. Campbell ◽

Brian P. McSharry ◽

Megan Steain ◽

Barry Slobedman ◽

Allison Abendroth

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Activity ◽

Nkg2d Ligand ◽

Nk Cell Activity ◽

Herpes Simplex Virus 1 ◽

Varicella Zoster ◽

Ligand Expression ◽

Simplex Virus ◽

Hsv 1

ABSTRACTNatural killer (NK) cell-deficient patients are particularly susceptible to severe infection with herpesviruses, especially varicella-zoster virus (VZV) and herpes simplex virus 1 (HSV-1). The critical role that NK cells play in controlling these infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. Our study addressed this absence of knowledge and found that infection with VZV was not associated with enhanced NK cell activation, suggesting that the virus uses specific mechanisms to limit NK cell activity. Analysis of viral regulation of ligands for NKG2D, a potent activating receptor ubiquitously expressed on NK cells, revealed that VZV differentially modulates expression of the NKG2D ligands MICA, ULBP2, and ULBP3 by upregulating MICA expression while reducing ULBP2 and ULBP3 expression on the surface of infected cells. Despite being closely related to VZV, infection with HSV-1 produced a remarkably different effect on NKG2D ligand expression. A significant decrease in MICA, ULBP2, and ULBP3 was observed with HSV-1 infection at a total cellular protein level, as well as on the cell surface. We also demonstrate that HSV-1 differentially regulates expression of an additional NKG2D ligand, ULBP1, by reducing cell surface expression while total protein levels are unchanged. Our findings illustrate both a striking point of difference between two closely related alphaherpesviruses, as well as suggest a powerful capacity for VZV and HSV-1 to evade antiviral NK cell activity through novel modulation of NKG2D ligand expression.IMPORTANCEPatients with deficiencies in NK cell function experience an extreme susceptibility to infection with herpesviruses, in particular, VZV and HSV-1. Despite this striking correlation, research into understanding how these two alphaherpesviruses interact with NK cells is surprisingly limited. Through examination of viral regulation of ligands to the activating NK cell receptor NKG2D, we reveal patterns of modulation by VZV, which were unexpectedly varied in response to regulation by HSV-1 infection. Our study begins to unravel the undoubtedly complex interactions that occur between NK cells and alphaherpesvirus infection by providing novel insights into how VZV and HSV-1 manipulate NKG2D ligand expression to modulate NK cell activity, while also illuminating a distinct variation between two closely related alphaherpesviruses.

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Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging

Biomedicines ◽

10.3390/biomedicines9050557 ◽

2021 ◽

Vol 9 (5) ◽

pp. 557

Author(s):

Xuewen Deng ◽

Hiroshi Terunuma ◽

Mie Nieda

Keyword(s):

Nk Cells ◽

Viral Infections ◽

Nk Cell ◽

Healthy Lifestyle ◽

Cell Activity ◽

Nk Cell Activity ◽

Effector Cells ◽

Cell Dysfunction ◽

Forest Bathing ◽

Infected Cells

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

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miR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling

Journal of Neuroinflammation ◽

10.1186/s12974-021-02181-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Yan Feng ◽

Yan Li ◽

Ying Zhang ◽

Bo-Hao Zhang ◽

Hui Zhao ◽

...

Keyword(s):

Ischemic Stroke ◽

Nk Cells ◽

Natural Killer ◽

Brain Ischemia ◽

Cell Activation ◽

Nk Cell ◽

Cell Activity ◽

Passive Transfer ◽

Immune Defense ◽

Nk Cell Activity

Abstract Background Brain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke. Methods Using a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia. Results We observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice. Conclusions These findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

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Role of Cytokines in the Development of Natural Killer (NK) Cells: Bone Marrow Colonies with NK Cell Activity

Natural Killer Cells: Biology and Clinical Application ◽

10.1159/000418923 ◽

2015 ◽

pp. 258-260

Author(s):

Carlo Riccardi ◽

Lorenza Cannarile ◽

Graziella Migliorati

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Cell Activity ◽

Nk Cell Activity

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Thermal stresses reduce natural killer cell cytotoxicity

Journal of Applied Physiology ◽

10.1152/jappl.1995.79.3.732 ◽

1995 ◽

Vol 79 (3) ◽

pp. 732-737 ◽

Cited By ~ 29

Author(s):

S. J. Won ◽

M. T. Lin

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Target Cell ◽

Nk Cell ◽

Cell Activity ◽

Cortisol Concentration ◽

Target Cells ◽

Nk Cell Activity ◽

Colonic Temperature ◽

Effector Target

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7–10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.

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Hitting More Birds with a Stone: Impact of TGF-β on ILC Activity in Cancer

Journal of Clinical Medicine ◽

10.3390/jcm9010143 ◽

2020 ◽

Vol 9 (1) ◽

pp. 143 ◽

Cited By ~ 6

Author(s):

Cinzia Fionda ◽

Helena Stabile ◽

Cristina Cerboni ◽

Alessandra Soriani ◽

Angela Gismondi ◽

...

Keyword(s):

Tumor Microenvironment ◽

Nk Cells ◽

Cancer Cells ◽

Nk Cell ◽

Critical Role ◽

Cell Activity ◽

Lymphoid Cells ◽

Receptor Expression ◽

Nk Cell Activity ◽

Group I

Transforming growth factor (TGF)-β is a central immunosuppressive cytokine within tumor microenvironment inhibiting the expansion and function of major cellular components of adaptive and innate immune system. Among them, compelling evidence has demonstrated that TGF-β is a key regulator of natural killer (NK) cells, innate lymphoid cells (ILCs) with a critical role in immunosurveillance against different kinds of cancer cells. A TGF-β rich tumor microenvironment blocks NK cell activity at multiple levels. This immunosuppressive factor exerts direct regulatory effects on NK cells including inhibition of cytokine production, alteration of activating/inhibitory receptor expression, and promotion of the conversion into non cytotoxic group I ILC (ILC1). Concomitantly, TGF-β can render tumor cells less susceptible to NK cell-mediated recognition and lysis. Indeed, accumulating evidence suggest that changes in levels of NKG2D ligands, mainly MICA, as well as an increase of immune checkpoint inhibitors (e.g., PD-L1) and other inhibitory ligands on cancer cells significantly contribute to TGF-β-mediated suppression of NK cell activity. Here, we will take into consideration two major mechanisms underlying the negative regulation of ILC function by TGF-β in cancer. First, we will address how TGF-β impacts the balance of signals governing NK cell activity. Second, we will review recent advances on the role of this cytokine in driving ILC plasticity in cancer. Finally, we will discuss how the development of therapeutic approaches blocking TGF-β may reverse the suppression of host immune surveillance and improve anti-tumor NK cell response in the clinic.

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IPH2101, a Novel Anti-Inhibitory KIR Monoclonal Antibody, and Lenalidomide Combine to Enhance the Natural Killer (NK) Cell Versus Multiple Myeloma (MM) Effect.

Blood ◽

10.1182/blood.v114.22.3870.3870 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3870-3870 ◽

Cited By ~ 1

Author(s):

Don Benson ◽

Courtney E Bakan ◽

Shuhong Zhang ◽

Lana Alghothani ◽

Jing Liang ◽

...

Keyword(s):

Clinical Trial ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Phase 1 ◽

Granzyme B ◽

Cell Activity ◽

Target Cells ◽

Nk Cell Activity ◽

Immune Complex Formation

Abstract Abstract 3870 Poster Board III-806 Background NK cell activity against tumor cells is regulated by a balance of inhibitory and activating signals mediated by receptors on NK cells that recognize inhibitory and activating ligands expressed by cancer cells. IPH2101 (1-7F9) is a novel monoclonal anti-inhibitor KIR blocking antibody that has been shown to augment NK cell function against MM targets. Moreover, lenalidomide has been shown to expand and activate NK cells in vivo and in vitro. We have previously reported that the combination of IPH2101 and lenalidomide enhances NK cell mediated cytotoxicity against MM cells compared to each agent alone (Zhang et al., AACR 2009). We expand our studies to investigate potential mechanisms for the enhancement of NK cell activity by the combination of IPH2101 and lenalidomide. Methods The effects of IPH2101 and lenalidomide alone and in combination were studied using primary human NK cells from healthy donors as well as from MM patients. The MM cell lines U266 and RPMI 8226 as well as primary tumor cells from marrow aspirates of MM patients served as target cells. The effect of lenalidomide on MM activating and inhibitory ligand expression was studied by flow cytometry. NK cell trafficking was investigated with standard transwell plate migration assay. Immune complex formation between NK cell effectors and MM tumor targets was characterized by flow cytometry in control conditions and with NK cells pre-treated with IPH2101 and lenalidomide. The effects of IPH2101 and lenalidomide were studied regarding interferon-gamma and granzyme B production by ELISPOT and target-specific cytotoxicity studies were conducted to complement effector-based assays. Results IPH2101 (30 ug/ml) significantly enhanced cytotoxicity against U266 cells and primary MM tumor cells by both purified NK cells at effector:target (E:T) ratios of 10:1 or less, and also of freshly isolated peripheral blood mononuclear cells (PBMC) at E:T ratios of 60:1 or less, from more than 10 random donors. In addition, treatment of PBMC with 5-10 μmol/L lenalidomide for 72h without interleukin (IL)-2 increased NK cell lysis of U266. Treatment of PBMC from normal donors did not enhance the expression of the NK receptors KIR, NKG2D, NCR, TRAIL, and DNAM-1. Incubation of U266 cells with lenalidomide (5 uM) for 3-5 days resulted in significant enhancement of cytotoxicity by normal donor NK cells. This was associated with upregulation of the activating ligands, MICA, ULBP-2, DR4, and CD112. Using blocking antibodies to NKG2D, TRAIL, and DNAM-1, lenalidomide enhancement of MM cell killing was abrogated indicating the importance of the modulation of the ligands to the latter receptors by lenalidomide. Although IPH2101 and lenalidomide did not significantly increase NK cell migration into normal media, migration was enhanced 2.98-fold (+/− 0.36, p < 0.05) towards U266 cell targets (n= 3, p < 0.05) and MM patient serum 3.2-fold (+/− 0.4, n=3, p < 0.05). IPH2101 and lenalidomide also led to a 2.3-fold (+/− 0.43, p < 0.05) increase in immune complex formation between NK cells and MM tumor cells. IPH2101 and lenalidomide also augmented NK cell interferon gamma production against MM (control mean 303 spots/well +/− 13 versus 525 +/− 83, n=3, p < 0.05) and granzyme B production (control mean 115 +/− 98 versus 449 +/−72, n=3, p < 0.05). Importantly, in all experiments described herein, the effects of IPH2101 and lenalidomide together were greater than either agent alone. Conclusions Taken together, our data suggest that IPH2101 and lenalidomide may exert complementary mechanisms on both effector and target cells to enhance NK cell mediated killing of MM cells. Moreover, these agents have no predicted clinical cross-toxicities. A single-agent phase 1 clinical trial of IPH2101 has shown the mAb to be safe and well tolerated in MM patients. These findings support a phase 1/2 clinical trial of IPH2101 with lenalidomide as a first dual-innate immunotherapy for patients with MM. Disclosures: Andre: Innate Pharma: Employment. Squiban:Innate pharma: Employment. Romagne:Innate Pharma: Employment.

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