Liver Natural Killer Research Articles

Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.

Activated Natural Killer Cell Promotes Nonalcoholic Steatohepatitis Through Mediating JAK/STAT Pathway.

Background & AimsHepatic immune microenvironment plays a pivotal role in the development of nonalcoholic steatohepatitis (NASH). However, the role of natural killer (NK) cells, accounting for 10%–20% of liver lymphocytes, in NASH is still unclear. In this study, we aim to investigate the functional significance of NK cells in NASH evolution.MethodsNASH was induced in mice fed methionine- and choline-deficient diet (MCD), choline-deficient high-fat diet (CD-HFD), or high-fat diet with streptozotocin injection (STAM model). NK cell deficient mice (Nfil3-/-) and neutralization antibody (PK136) were used in this study.ResultsActivated liver NK cells were identified with increased expression of NKG2D, CD107a, and interferon-γ but decreased inhibitory NKG2A. With NK cell deficiency Nfil3-/- mice, the absence of NK cells ameliorated both MCD- and CDHF- induced NASH development with significantly decreased hepatic triglycerides, peroxides, alanine aminotransferase, and aspartate aminotransferase compared with Nfil3+/+ mice. Further molecular analysis unveiled suppressed pro-inflammatory cytokines and associated signaling. Mechanistically, NK cells isolated from NASH liver secreted higher levels of pro-inflammatory cytokines (interferon-γ, interleukin 1β, interleukin 12, CCL4, CCL5, and granulocyte-macrophage colony-stimulating factor), which could activate hepatic JAK-STAT1/3 and nuclear factor kappa B signaling and induce hepatocyte damage evidenced by elevated reactive oxygen species and apoptosis rate. Moreover, neutralization antibody PK136-dependent NK cell depletion can significantly alleviate MCD-induced steatohepatitis with suppressed cytokine levels and JAK-STAT1/3 activity.ConclusionsNK cells in NASH liver are activated with a more pro-inflammatory cytokine milieu and promote NASH development via cytokine-JAK-STAT1/3 axis. Modulation of NK cells provides a potential therapeutic strategy for NASH.

Pilot study to determine the safety and feasibility of deceased donor liver natural killer cell infusion to liver transplant recipients with hepatocellular carcinoma.

Liver transplantation (LT) is a viable treatment option for cirrhosis patients with hepatocellular carcinoma (HCC). However, recurrence is the rate-limiting factor of long-term survival. To prevent this, we conducted the phase I study of the adoptive transfer of deceased donor liver-derived natural killer (NK) cells. Liver NK cells were extracted from donor liver graft perfusate and were stimulated in vitro with IL-2. The patient received an intravenous infusion of NK cells 3-5days after LT. Eighteen LT recipients were treated. There were no severe cell infusion-related adverse events or acute rejection episodes. One patient withdrew from the study because the pathological observation revealed sarcoma instead of HCC. All patients who received this immunotherapy completed the follow-up for at least 2years without evidence of HCC recurrence (median follow-up, 96months [range, 17-121months]). Considering that 9 (52.9%) of the 17 patients pathologically exceeded the Milan criteria, liver NK cell infusion is likely to be useful for preventing HCC recurrence after LT. This is the first-in-human immunotherapy study using deceased donor liver-derived NK cells to prevent HCC recurrence after LT. This treatment was well tolerated and resulted in no HCC recurrence after LT.Clinical trial registration www.clinicaltrials.gov ; NCT01147380; registration date: June 17, 2010.

OX40-targeted immune agonist antibodies induce potent antitumor immune responses without inducing liver damage in mice.

Despite promising preclinical and clinical data demonstrating that immune agonist antibody immunotherapies (IAAs) such as αOX40 induce strong antitumor immune responses, clinical translation has been significantly hampered by the propensity of some IAAs to induce dose‐limiting and sometimes life‐threatening immunotoxicities such as cytokine release syndrome and hepatotoxicity. For example, in a recent study αOX40 was shown to induce significant liver damage in mice by inducing the pyroptosis of liver natural killer T cells (NKT) cells. Surprisingly; however, given these previous reports, αOX40 treatment in our hands did not induce NKT cell pyroptosis or liver damage. We investigated numerous potential confounding factors including age, sex, tumor burden, dosing strategy, and the gut microbiota, which could have explained this discrepancy with the previous study. In none of these experiments did we find that αOX40 induced any more than very mild inflammation in the liver. Our study therefore suggests that, preclinically, αOX40 is a safe and effective immunotherapy and further studies into the clinical benefit of αOX40 are warranted.

Diet-induced obesity aggravates NK cell-mediated contact hypersensitivity reaction in Rag1-/- mice.

Previous studies showed that natural killer (NK) cells mediate contact hypersensitivity (CHS) reaction. Many reports are showing that obesity promotes several inflammatory diseases. It was shown that diet-induced obesity (DIO) aggravates classical T cell-mediated CHS in mice. To determine whether the high-fat diet (HFD)-induced obesity modulates antigen-specific NK cell-mediated response. We evaluated the effect of DIO on NK cell-mediated CHS reaction using a model of dinitrofluorobenzene (DNFB)-induced CHS in Rag1-/- mice. Rag1-/- mice fed HFD for 8 but not for 4 weeks developed aggravated CHS reaction determined by ear swelling measurement when compared to animals kept on normal diet (ND) prior to DNFB sensitization. The obese Rag1-/- mice presented the adipose tissue inflammation. Furthermore, in vitro analysis showed that feeding with HFD significantly increases interferon γ (IFN-γ) and interleukin (IL)-12p70 and decreases adiponectin concentration in liver mononuclear cell (LMNC) culture supernatants. The flow cytometry analysis of LMNC revealed that HFD treatment prior to DNFB sensitization increases the percentage of NK1.1+ IFN-γ+ cell population and affects the development and maturation of NK1.1+ cells. In summary, current results suggest that the DIO significantly modulates the local and systemic inflammatory response, contributing to exacerbation of the CHS response mediated by liver NK cells.

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.

A combination of pirfenidone and TGF-β inhibition mitigates cystic echinococcosis-associated hepatic injury.

Cystic echinococcosis (CE) occurs in the intermediate host's liver, assuming a bladder-like structure surrounded by the host-derived collagen capsule mainly derived from activated hepatic stellate cells (HSCs). However, the effect of CE on liver natural killer (NK) cells and the potential of transforming growth factor-β (TGF-β) signalling inhibition on alleviating CE-related liver damage remain to be explored. Here, by using the CE-mouse model, we revealed that the inhibitory receptors on the surface of liver NK cells were up-regulated, whereas the activating receptors were down-regulated over time. TGF-β1 secretion was elevated in liver tissues and mainly derived from macrophages. A combination of TGF-β signalling inhibitors SB525334 and pirfenidone could reduce the expression of TGF-β1 signalling pathway-related proteins and collagen production. Based on the secretion of TGF-β1, only the pirfenidone group showed a depressing effect. Also, the combination of SB525334 and pirfenidone exhibited a higher potential in effectively alleviating the senescence of the hepatocytes and restoring liver function. Together, TGF-β1 may be a potential target for the treatment of CE-associated liver fibrosis.

A subset of liver resident natural killer cells is expanded in hepatitis C-infected patients with better liver function

Viral hepatitis leads to immune-mediated liver injury. The rate of disease progression varies between individuals. We aimed to phenotype immune cells associated with preservation of normal liver function during hepatitis C virus (HCV) infection. Clinical data and specimens were obtained from 19 HCV-infected patients undergoing liver transplantation. Liver and peripheral blood mononuclear cells were isolated and eight subsets of innate immune cells were delineated by multiparameter flow cytometry. Cytokine assays and microarrays were performed. Intrahepatic CD56Bright/CD16- natural killer (NK) cells comprised the only subset correlating with better liver function, i.e., lower bilirubin (p = 0.0002) and lower model for end stage of liver disease scores (p = 0.03). The signature of liver NK cells from HCV-infected patients included genes expressed by NK cells in normal liver and by decidual NK cells. Portal vein blood had a higher concentration of interleukin (IL)-10 than peripheral blood (p = 0.03). LMCs were less responsive to toll-like receptor (TLR) stimulation than PBMCs, with fewer pro-inflammatory gene-expression pathways up-regulated after in vitro exposure to lipopolysaccharide and a TLR-7/8 agonist. Hepatic CD56Bright/CD16- NK cells may be critical for maintaining liver homeostasis. Portal vein IL-10 may prime inhibitory pathways, attenuating TLR signaling and reducing responsiveness to pro-inflammatory stimuli.

Lipopolysaccharide preconditioning reduces liver metastasis of Colon26 cells by enhancing antitumor activity of natural killer cells and natural killer T cells in murine liver.

Lipopolysaccharide (LPS) preconditioning drastically augments bactericidal activity but reduces the host inflammatory response. Therefore, it may be beneficial to prevent postoperative infectious complications and mitigate host damage by surgical stress. Considering its clinical application, how LPS preconditioning influences the antitumor effect in the liver is an important issue. We then investigated the effect of LPS preconditioning on antitumor activity against Colon26 tumor cells in mice. Lipopolysaccharide preconditioning was induced in mice by the intraperitoneal injection of 5μg/kg LPS for three consecutive days. Intraportal inoculation of Colon26 cells, which express luminescent protein called Nano-lantern, was performed to evaluate the effect of LPS preconditioning on tumor liver metastasis. The antitumor activities of cytotoxic liver lymphocytes, especially natural killer (NK) cells and natural killer T (NKT) cells, against Colon26 cells were also examined in LPS preconditioned mice. Lipopolysaccharide preconditioning remarkably prevented liver metastasis of Colon26 cells, as observed by IVIS imaging system, and prolonged survival after tumor inoculation. LPS preconditioning increased the proportions and number of liver NK cells and NKT cells and augmented their intracellular perforin and granzyme B expression, while reducing their intracellular expression of IFN-γ. An in vitro antitumor cytotoxicity assay revealed that LPS preconditioning significantly augmented antitumor cytotoxicities of the liver NK cells and NKT cells, especially NKT cells, against Colon26 cells. Lipopolysaccharide preconditioning potently augmented antitumor cytotoxicity of liver NK cells and NKT cells, thereby improving mouse survival after intraportal inoculation of Colon26 tumor cells. It may be useful for perioperative care in oncological patients.

Protective Vaccination Reshapes Hepatic Response to Blood-Stage Malaria of Genes Preferentially Expressed by NK Cells.

The role of natural killer (NK) cells in the liver as first-line post infectionem (p.i.) effectors against blood-stage malaria and their responsiveness to protective vaccination is poorly understood. Here, we investigate the effect of vaccination on NK cell-associated genes induced in the liver by blood-stage malaria of Plasmodium chabaudi. Female Balb/c mice were vaccinated at weeks 3 and 1 before being infected with 106 P. chabaudi-parasitized erythrocytes. Genes preferentially expressed by NK cells were investigated in livers of vaccination-protected and non-protected mice on days 0, 1, 4, 8, and 11 p.i. using microarrays, qRT-PCR, and chromosome landscape analysis. Blood-stage malaria induces expression of specific genes in the liver at different phases of infection, i.e., Itga1 in expanding liver-resident NK (lrNK) cells, Itga2 in immigrating conventional NK (cNK) cells; Eomes and Tbx21 encoding transcription factors; Ncr1, Tnfsf10, Prf1, Gzma, Gzmb, Gzmc, Gzmm, and Gzmk encoding cytolytic effectors; natural killer gene complex (NKC)-localized genes encoding the NK cell receptors KLRG1, KLRK1, KLRAs1, 2, 5, 7, KLRD1, KLRC1, KLRC3, as well as the three receptors KLRB1A, KLRB1C, KLRB1F and their potential ligands CLEC2D and CLEC2I. Vaccination enhances this malaria-induced expression of genes, but impairs Gzmm expression, accelerates decline of Tnfsf10 and Clec2d expression, whereas it accelerates increased expression of Clec2i, taking a very similar time course as that of genes encoding plasma membrane proteins of erythroblasts, whose malaria-induced extramedullary generation in the liver is known to be accelerated by vaccination. Collectively, vaccination reshapes the response of the liver NK cell compartment to blood-stage malaria. Particularly, the malaria-induced expansion of lrNK cells peaking on day 4 p.i. is highly significantly (p < 0.0001) reduced by enhanced immigration of peripheral cNK cells, and KLRB1F:CLEC2I interactions between NK cells and erythroid cells facilitate extramedullary erythroblastosis in the liver, thus critically contributing to vaccination-induced survival of otherwise lethal blood-stage malaria of P. chabaudi.

Mesenchymal stem cells regulated DX5 – liver natural killer cells in rescuing acute-on-chronic liver failure murine

Mesenchymal stem cells regulated DX5 – liver natural killer cells in rescuing acute-on-chronic liver failure murine

β2-adrenergic signals downregulate the innate immune response and reduce host resistance to viral infection.

In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the β2-adrenergic receptor (β2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a β2-AR agonist were more susceptible to MCMV infection. By contrast, β2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the β2-AR signaling could be used to increase resistance to infectious diseases.

29-Color Flow Cytometry: Unraveling Human Liver NK Cell Repertoire Diversity.

Recent studies have demonstrated extraordinary diversity in peripheral blood human natural killer (NK) cells and have suggested environmental control of receptor expression patterns on distinct subsets of NK cells. However, tissue localization may influence NK cell differentiation to an even higher extent and less is known about the receptor repertoire of human tissue-resident NK cells. Advances in single-cell technologies have allowed higher resolution studies of these cells. Here, the power of high-dimensional flow cytometry was harnessed to unravel the complexity of NK cell repertoire diversity in liver since recent studies had indicated high heterogeneity within liver NK cells. A 29-color flow cytometry panel allowing simultaneous measurement of surface tissue-residency markers, activating and inhibitory receptors, differentiation markers, chemokine receptors, and transcription factors was established. This panel was applied to lymphocytes across three tissues (liver, peripheral blood, and tonsil) with different distribution of distinct NK cell subsets. Dimensionality reduction of this data ordered events according to their lineage, rather than tissue of origin. Notably, narrowing the scope of the analysis to the NK cell lineage in liver and peripheral blood separated subsets according to tissue, enabling phenotypic characterization of NK cell subpopulations in individual tissues. Such dimensionality reduction, coupled with a clustering algorithm, identified CD49e as the preferred marker for future studies of liver-resident NK cell subsets. We present a robust approach for diversity profiling of tissue-resident NK cells that can be applied in various homeostatic and pathological conditions such as reproduction, infection, and cancer.

Potently Cytotoxic Natural Killer Cell Potential Initially Emerges from Erythro-Myeloid Progenitors during Mammalian Development

Natural killer (NK) cells are innate immune cells that target and kill virally infected and malignant cells, making them an attractive target for adoptive immunotherapies. An alternative to donor-derived NK cells is the use of human pluripotent stem cell (hPSC)-derived NK cells, as a renewable "off the shelf" product. Previous studies have identified hPSC-derived NK cells as potently cytotoxic, compared to donor-derived NK cells. As the differentiation of hPSCs mimics early embryonic development, this raises the possibility that hPSC-derived NK cells are ontogenically distinct from adult NK cells. NK cells are present during embryonic hematopoiesis, but their ontogenic origins are poorly understood. NK cells are thought to arise from a common lymphoid progenitor (CLP), lying downstream of hematopoietic stem cells (HSCs), but evidence exists that NK cells may arise from HSC-independent progenitors as NK cells are found in the early murine fetal liver, and NK cell progenitors are found in the early human yolk sac (YS). In this study, we investigated the emergence of NK cells during murine and human embryonic hematopoietic development. During murine embryogenesis, overlapping HSC-independent waves of hematopoietic progenitors occur in the YS that give rise to hematopoietic cells prior to HSC emergence at E10.5. The "primitive" wave occurs at E7.5, followed by an "erythro-myeloid progenitor" (EMP) wave at E8.5. To study NK cell potential during murine YS hematopoiesis, we cultured total YS and sorted hematopoietic progenitors under NK cell promoting conditions. Strikingly, we found that the YS contains NK cell potential. Further, sorted E8.5 kit+CD41+CD16/32+ EMP progenitors, but not primitive hematopoietic progenitors, contain robust NK cell potential. EMP-derived NK (EMP-NK) cells were larger and more granular than adult CLP-derived NK cells. Additionally, NK cells from the E15.5 fetal liver were larger and more granular than NK cells from the adult spleen. Both EMP-NK cells and E15.5 fetal liver NK cells had a more robust degranulation response than their HSC-derived counterparts. Together, these data support the concept that EMP in the YS serve as an initial source of physiologically relevant, functional embryonic NK cells that are phenotypically and functionally distinct from adult NK cells. As hPSC-derived NK cells were described as potently cytotoxic, and we observed that murine HSC-independent NK cells robustly degranulate, we next asked whether NK cell development from hPSCs recapitulates that found in the murine embryo. We have demonstrated previously, using a stage-specific WNT signal manipulation approach that specifies ontogenically distinct hematopoietic progenitors, that hPSC-derived NK cell progenitors can be obtained from two distinct progenitors in vitro. In this study, we sought to better understand the development and function of these two NK cell populations. Stage-specific WNT inhibition (WNTi) during hPSC mesodermal patterning yielded extra-embryonic-like HOXA-/low CD34+ populations that possessed erythroid, myeloid and NK cell potential, but lacked T cell potential. The CD56+ NK cells in these cultures co-emerged with CD15+ granulocytes, indicating that these NK cells may arise from a committed myeloid progenitor. In contrast, HOXA+ CD34+ cells, obtained in a WNT-dependent (WNTd) manner, harbored erythro-myelo-lymphoid multi-lineage potential, including NK cell potential. Phenotypically, WNTi-NK cells were larger, more granular and more mature, compared to WNTd-NK and cord blood (CB)-derived NK cells, reminiscent of murine EMP-NK cells. Further, following multiple stimulation assays, WNTi-NK and WNTd-NK cells had different effector biases. WNTi-NK cells are biased for potent cytotoxic degranulation and exhibited superior cell killing in an ADCC assay. In contrast, WNTd-NK and CB-NK had an attenuated degranulation response, but robustly produced inflammatory cytokines. Finally, RNA-seq analysis demonstrated that WNTd-NK cells were most similar to CB-NK cells. Collectively, these studies identify for the first time that the murine EMP harbor NK cell potential, and these NK cells are functionally unique. These observations raise new questions regarding which ontogenic origin of NK cells should be used in future hPSC-derived adoptive immunotherapy strategies. Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Palis:Rubius Therapeutics: Consultancy.

Identification of a Porcine Liver EomeshighT-betlow NK Cell Subset That Resembles Human Liver Resident NK Cells.

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (EomeshighT-betlow in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3−CD8αhigh cells, the porcine liver contains an abundant additional CD3−CD8αdim NK cell subpopulation. In the current study, we show that this porcine CD3−CD8αdim liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an EomeshighT-betlow expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e− and CXCR6+. Furthermore, the porcine EomeshighT-betlow liver NK cell population is able to produce IFN-γ upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine EomeshighT-betlow NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease.

Mitochondrial fragmentation limits NK cell-based tumor immunosurveillance.

Natural killer (NK) cells have crucial roles in tumor surveillance. We found that tumor-infiltrating NK cells in human liver cancers had small, fragmented mitochondria in their cytoplasm, whereas liver NK cells outside tumors, as well as peripheral NK cells, had normal large, tubular mitochondria. This fragmentation was correlated with reduced cytotoxicity and NK cell loss, resulting in tumor evasion of NK cell-mediated surveillance, which predicted poor survival in patients with liver cancer. The hypoxic tumor microenvironment drove the sustained activation of mechanistic target of rapamycin-GTPase dynamin-related protein 1 (mTOR-Drp1) in NK cells, resulting in excessive mitochondrial fission into fragments. Inhibition of mitochondrial fragmentation improved mitochondrial metabolism, survival and the antitumor capacity of NK cells. These data reveal a mechanism of immune escape that might be targetable and could invigorate NK cell-based cancer treatments.

Defective FasL expression is associated with increased resistance to melanoma liver metastases and enhanced natural killer cell activity.

The objective was to determine if the absence of FasL signaling would affect melanoma liver metastases by influencing the antimelanoma properties of liver natural killer (NK) cells. Melanoma liver metastases were induced in wild-type C57BL/6 mice and the gld/gld mutant C57BL/6 mouse strain that expresses a defective form of FasL (CD95L) that fails to engage and signal via the Fas receptor (CD95). Liver metastases were produced by intrasplenic injection of B16LS9 melanoma cells. Liver NK cell activity directed against murine B16LS9 melanoma cells was determined in a 24 h in-vitro cytotoxicity assay. Liver NK cells, NK T cells, and the NK cell surface activation marker, NKG2D, were measured by flow cytometry. Mice expressing defective FasL displayed reduced, rather than enhanced, melanoma liver metastases that coincided with increased liver NK cell-mediated tumor cell cytotoxicity. Enhanced cytotoxicity was not mediated by perforin, tumor necrosis factor-α, or tumor necrosis-associated apoptosis-inducing ligand but was closely associated with elevated interferon-γ in the tumor-bearing liver. FasL-defective gld/gld mice also displayed reduced numbers of liver NK T cells, which have been previously implicated in suppression on liver NK cell activity. The absence of functional FasL in the liver correlates with a heightened, not diminished, resistance to melanoma liver metastases. The resistance to liver metastases coincides with a significant, albeit transient, increase in liver NK cytotoxicity and elevated levels of interferon-γ in the liver.

Retained NK Cell Phenotype and Functionality in Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), and the progressive stage non-alcoholic steatohepatitis (NASH), is the predominant cause of chronic liver disease globally. As part of the complex pathogenesis, natural killer (NK) cells have been implicated in the development of liver inflammation in experimental murine models of NASH. However, there is a lack of knowledge on how NK cells are affected in humans with this disease. Here, we explored the presence of disease-specific changes within circulating and tissue-resident NK cell populations, as well as within other major immune cell subsets, in patients with liver biopsy-confirmed NAFLD. Using 18-color-flow cytometry, substantial changes were observed in certain myeloid populations in patients as compared to controls. NK cell numbers, on the other hand, were not altered. Furthermore, only minor differences in expression of activating and inhibitory NK cell receptors were noted, with the exception of an increased expression of NKG2D on NK cells from patients with NASH. NK cell differentiation remained constant, and NK cells from these patients retain their ability to respond adequately upon stimulation. Instead, considerable alterations were observed between liver, adipose tissue, and peripheral blood NK cells, independently of disease status. Taken together, these results increase our understanding of the importance of the local microenvironment in shaping the NK cell compartment and stress the need for further studies exploring how NASH affects intrahepatic NK cells in humans.

Cotransplantation of preactivated mesenchymal stem cells improves intraportal engraftment of islets by inhibiting liver natural killer cells in mice.

The activation of natural killer (NK) cells in the liver inhibits engraftment of intraportally transplanted islets. We attempted to modulate the activity of NK cells by cotransplanting mesenchymal stem cells (MSCs) with islets in mice. We first investigated the ability of MSCs to secrete prostaglandin E2 , a predominant inhibitor of NK cell function, in various combinations of inflammatory cytokines. Notably, we found that prostaglandin E2 production was partially delayed in MSCs activated by inflammatory cytokines in vitro, whereas liver NK cells were activated early after islet transplant in vivo. Accordingly, preactivated MSCs, but not naive MSCs, substantially suppressed the expression of activation markers in liver NK cells after cotransplant with islets. Similarly, cotransplant with preactivated MSCs, but not naive MSCs, markedly improved the survival of islet grafts. These results highlight MSC cotransplant as an effective and clinically feasible method for enhancing engraftment efficiency.

Sirtuin2 enhances the tumoricidal function of liver natural killer cells in a mouse hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is the third most lethal cancer in the world. Natural killer (NK) cell-mediated immunity is crucial for tumor surveillance and therapy. Characterization of the regulatory mechanisms of NK cell function is important for developing novel immunotherapies against HCC. In this study, we used a chemical-induced mouse HCC model to identify the upregulation of Sirtuin2 (SIRT2) in liver NK cells. In particular, SIRT2 was predominantly expressed in liver CD94+ NK cells. The HCC liver microenvironment induced SIRT2 expression in NK cells. In addition, overexpression of exogenous SIRT2 significantly upregulated the production of cytokines and cytotoxic mediators in activated NK cells. Consistently, SIRT2-overexpressing NK cells showed a stronger tumoricidal effect on hepatoma cells. Moreover, SIRT2 remarkably promoted the phosphorylation of Extracellular-signal-regulated kinase 1/2 (Erk1/2) and p38 Mitogen-activated protein kinases (MAPK) in activated NK cells. SIRT2 knockdown in liver CD94+ NK cells impaired their cytotoxic effect on hepatoma cells. Our study indicates that SIRT2 enhances the tumoricidal activity of liver NK cells in HCC.