Mouse Natural Killer Cells Research Articles

TL1A Aggravates Cytokine-Induced Acute Gut Inflammation and Potentiates Infiltration of Intraepithelial Natural Killer Cells in Mice

The tumor necrosis factor alpha (TNFα)-homologous cytokine TL1A is emerging as a major player in intestinal inflammation. From in vitro experiments on human lymphocytes, TNF-like molecule 1A (TL1A) is known to activate a highly inflammatory lymphoid response in synergy with interleukin (IL)-12 and IL-18. Carriers of specific genetic polymorphisms associated with IL-12, IL-18, or TL1A signaling have increased Crohn's disease risk, and all 3 cytokines are upregulated during active disease. The study aim was to investigate whether the type 1-polarizing cytokines IL-12 and IL-18 could directly initiate intestinal pathology in mice and how TL1A would influence the resulting inflammatory response. Conventional barrier-bred and germ-free mice were randomly allocated to different groups and injected twice with different combinations of IL-12, IL-18, and TL1A, and killed 3 days after the first injection. All treatment groups were co-housed and fed a piroxicam-supplemented chow diet. Intestinal pathology was evident in IL-12- and IL-18-treated mice and highly exacerbated by TL1A in both the colon and ileum. The cytokine-induced intestinal inflammation was characterized by epithelial damage, increased colonic levels of TNFα, IL-1β, IFN-γ, and IL-6, and various chemokines along with gut microbiota alterations exhibiting high abundance of Enterobacteriaceae. Furthermore, the inflamed ileum and colon exhibited a TL1A-specific increased infiltration of intraepithelial natural killer cells co-expressing NKG2D and IL-18Ra and a higher frequency of unconventional T cells in the colonic epithelium. Upon cytokine injection, germ-free mice exhibited similar intraepithelial lymphoid infiltration and increased colonic levels of IFNγ and TNFα. This study demonstrates that TL1A aggravates IL-12- and IL-18-induced intestinal inflammation in the presence and absence of microbiota.

The C-type lectin-like receptor Nkrp1b: Structural proteomics reveals features affecting protein conformation and interactions

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo. SignificanceDespite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation; therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets

ABSTRACTNKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function.SignificanceInnate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

Monomethylarsonous acid: Induction of DNA damage and oxidative stress in mouse natural killer cells at environmentally-relevant concentrations

The carcinogenicity of arsenic has been confirmed in many studies, but its mechanism of action is still unclear. A lymphocyte component of the innate immune system, natural killer (NK) cells are responsible for killing cancer cells. Although inorganic arsenical species are the prevalent forms of arsenic in the environment, monomethylarsonous acid (MMA+3) is the major arsenical species found in immune-system cells, in this 30 d drinking-water-exposure study in mice. Therefore, the effect of MMA+3 on NK cells should be studied as a possible contributor to arsenic-induced toxicity at environmental exposure levels. In the mouse drinking-water-exposure model, As+3 induces dose-dependent DNA damage in NK cells. In in vitro studies, MMA+3 inhibited cell growth and induced DNA damage and oxidative stress at low concentration (20 and 50 nM) in isolated mouse NK cells. Strong correlations were found between DNA damage and oxidative stress in MMA+3-treated mouse NK cells. Even at low concentrations relevant to environmental arsenic exposures, MMA+3 is genotoxic to primary mouse NK cells.

Real-Time Tracking of Ex Vivo-Expanded Natural Killer Cells Toward Human Triple-Negative Breast Cancers.

Ex vivo-expanded natural killer (NK) cells are a potential candidate for cancer immunotherapy based on high cytotoxicity against malignant tumor cells. However, a limited understanding of the migration of activated NK cells toward solid tumors is a critical dilemma in the development of effective and adoptive NK cell-based immunotherapy. Ex vivo-expanded NK cells from healthy donors were stained with near-infrared fluorophores at different concentrations. NK cell proliferation and cytotoxicity were assessed using a WST-8 assay, while the expression levels of surface molecules were analyzed by flow cytometry. To investigate the biodistribution of NK cells in both normal and tumor-bearing NSG mice, NK cells labeled with ESNF13 were subjected to NIR fluorescence imaging using the Mini-FLARE imaging system. Finally, mice were sacrificed and histopathological tests were performed in resected organs. The signal intensity of ESNF-stained NK cells was long-lasting at 72 h using concentrations as low as 0.04 µM. At a low dose range, ESNF13 did not affect NK cell purity, expression levels of surface receptors, or cytotoxic functions against MDA-MB-231 cancer cells. Ex vivo-expanded NK cells labeled with ESNF13 had a 4-h biodistribution in non-tumor-bearing NSG mice that mainly localized to the lungs immediately after injection and then fully migrated to the kidney after 4 h. In an MDA-MB-231 tumor-bearing NSG mice with extensive metastasis in both lungs, the fluorescence signal was dominant in both lungs and steady at 1, 2, and 4 h post-injection. In a early phase of tumor progression, administered NK cell migrated to the lungs and tumor sites within 30 min post-injection, the signal dominated the tumor site after 1 h, and remained steady at 4 h. Optical imaging with NIR fluorophore ESNF13 is a highly sensitive, applicable, and inexpensive method for the real-time tracking of ex vivo-expanded NK cells both in vitro and in vivo. Administered NK cells had different patterns of NK cell distribution and accumulation to the tumor site according to tumor progression in triple-negative breast cancer xenograft models.

MRI-guided interventional natural killer cell delivery for liver tumor treatment.

While natural killer (NK) cell‐based adoptive transfer immunotherapy (ATI) provides only modest clinical success in cancer patients. This study was hypothesized that MRI‐guided transcatheter intra‐hepatic arterial (IHA) infusion permits local delivery to liver tumors to improve outcomes during NK‐based ATI in a rat model of hepatocellular carcinoma (HCC). Mouse NK cells were labeled with clinically applicable iron nanocomplexes. Twenty rat HCC models were assigned to three groups: transcatheter IHA saline infusion as the control group, transcatheter IHA NK infusion group, and intravenous (IV) NK infusion group. MRI studies were performed at baseline and at 24 h, 48 h, and 8 days postinfusion. There was a significant difference in tumor R2* values between baseline and 24 h following the selective transcatheter IHA NK delivery to the tumors (P = 0.039) when compared to IV NK infusion (P = 0.803). At 8 days postinfusion, there were significant differences in tumor volumes between the control, IV, and IHA NK infusion groups (control vs. IV, P = 0.196; control vs. IHA, P < 0.001; and IV vs. IHA, P = 0.001). Moreover, there was a strong correlation between tumor R2* value change (∆R2*) at 24 h postinfusion and tumor volume change (∆volume) at 8 days in IHA group (R 2 = 0.704, P < 0.001). Clinically applicable labeled NK cells with 12‐h labeling time can be tracked by MRI. Transcatheter IHA infusion improves NK cell homing efficacy and immunotherapeutic efficiency. The change in tumor R2* value 24 h postinfusion is an important early biomarker for prediction of longitudinal response.

Interleukin (IL)-23 Stimulates IFN-γ Secretion by CD56bright Natural Killer Cells and Enhances IL-18-Driven Dendritic Cells Activation.

Interleukin (IL)-23 is a member of the IL-12 family of cytokines that, as the other members of this family, is secreted by monocytes, macrophages, and dendritic cells (DC) upon recognition of bacterial, viral, and fungal components. IL-23 is critical during immunity against acute infections, and it is also involved in the development of autoimmune diseases. Although immunoregulatory effects of IL-23 on mouse natural killer (NK) cells have been described, the effect of IL-23 on human NK cells remains ill-defined. In this study, we observed that monocytes stimulated with LPS secreted IL-23 and that blockade of this cytokine during monocyte and NK cell coculture led to a diminished production of IFN-γ by NK cells. Accordingly, rIL-23-induced NK cell activation and stimulated IFN-γ production by CD56bright NK cells. This effect involved MEK1/MEK2, JNK, PI3K, mammalian target of rapamycin, and NF-κB, but not STAT-1, STAT-3, nor p38 MAPK pathways. Moreover, while NK cell-mediated cytotoxicity remained unaltered, antibody-dependent cellular cytotoxicity (ADCC) was enhanced after IL-23 stimulation. In addition, IL-23 displayed a synergistic effect with IL-18 for IFN-γ production by both CD56bright and CD56dim NK cells, and this effect was due to a priming effect of IL-23 for IL-18 responsiveness. Furthermore, NK cells pre-stimulated with IL-18 promoted an increase in CD86 expression and IL-12 secretion by DC treated with LPS, and IL-23 potentiated these effects. Moreover, IL-23-driven enhancement of NK cell “helper” function was dependent on NK cell-derived IFN-γ. Therefore, our results suggest that IL-23 may trigger NK cell-mediated “helper” effects on adaptive immunity, shaping T cell responses during different pathological situations through the regulation of DC maturation.

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

Sex Hormone Contributes to Sexually Dimorphic Susceptibility in CVB3-Induced Viral Myocarditis via Modulating IFN-γ+ NK Cell Production

BackgroundViral myocarditis is a widespread cardiac disease associated with inflammation and myocardial injury and is predominantly caused by coxsackievirus B3 (CVB3) infection in humans as well as in mice. CVB3-induced myocarditis shows sexually dimorphic sensitivity and is more prevalent in male mice. Our previous studies showed that natural killer (NK) cells played an indispensable role in CVB3-induced myocarditis, and female mice exhibited less pathological cardiac interferon gamma (IFN-γ)+ NK cell infiltration than did male mice. However, the precise mechanisms were not well elucidated. MethodsWe investigated the influence of estrogen on cardiac IFN-γ+ NK cell enrichment in CVB3-induced myocarditis and explored the underlying molecular mechanism. ResultsIn this study, we found that CVB3 stimulation could clearly induce IFN-γ expression by NK cells; however, this trend could be blunted by estrogen treatment. Consistently, ovariectomized female mice with decreased estrogen levels exhibited substantially increased enrichment of cardiac IFN-γ+ NK cells and displayed significantly aggravated myocarditis. Similarly, estrogen-treated male mice showed less cardiac IFN-γ+ NK cell infiltration, accompanied by significantly alleviated viral myocarditis. In sharp contrast, sexually immature female and male mice (with similar estrogen levels) showed comparable levels of cardiac IFN-γ+ NK cell infiltration and similar levels of myocarditis severity. Upon further exploration of the underlying mechanisms, we found that estrogen could downregulate expression of Th1-specific T box transcription factor (T-bet), the key transcription factor associated with IFN-γ production, in CVB3-stimulated NK cells. ConclusionsOverall, this study might help us understand the mechanism of increased cardiac infiltration by IFN-γ+ NK cells in CVB3-infected male mice compared with that in female mice and might provide new clues for the sex bias in CVB3-induced myocarditis.

NKp46 Receptor-Mediated Interferon-γ Production by Natural Killer Cells Increases Fibronectin 1 to Alter Tumor Architecture and Control Metastasis

Natural killer (NK) cells are innate lymphoid cells, and their presence within human tumors correlates with better prognosis. However, the mechanisms by which NK cells control tumors invivo are unclear. Here, we used reflectance confocal microscopy (RCM) imaging in humans and in mice to visualize tumor architecture invivo. We demonstrated that signaling via the NK cell receptor NKp46 (human) and Ncr1 (mouse) induced interferon-γ (IFN-γ) secretion from intratumoral NKcells. NKp46- and Ncr1-mediated IFN-γ production led to the increased expression of the extracellular matrix protein fibronectin 1 (FN1) in the tumors, which altered primary tumor architecture and resulted in decreased metastases formation. Injection of IFN-γ into tumor-bearing mice or transgenic overexpression of Ncr1 in NK cells in mice resulted in decreased metastasis formation. Thus, we have defined a mechanism of NK cell-mediated control of metastases invivo thatmay help develop NK cell-dependent cancer therapies.

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

Humanized mouse model supports development, function, and tissue residency of human natural killer cells

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18.

The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor.

Persistent infection due to a small-colony variant of Burkholderia pseudomallei leads to PD-1 upregulation on circulating immune cells and mononuclear infiltration in viscera of experimental BALB/c mice.

BackgroundMelioidosis is a neglected tropical disease endemic across South East Asia and Northern Australia. The etiological agent, Burkholderia pseudomallei (B.pseudomallei), is a Gram-negative, rod-shaped, motile bacterium residing in the soil and muddy water across endemic regions of the tropical world. The bacterium is known to cause persistent infections by remaining latent within host cells for prolonged duration. Reactivation of the recrudescent disease often occurs in elders whose immunity wanes. Moreover, recurrence rates in melioidosis patients can be up to ~13% despite appropriate antibiotic therapy, suggestive of bacterial persistence and inefficacy of antibiotic regimens. The mechanisms behind bacterial persistence in the host remain unclear, and hence understanding host immunity during persistent B. pseudomallei infections may help designing potential immunotherapy.Methodology/Principal findingsA persistent infection was generated using a small-colony variant (SCV) and a wild-type (WT) B. pseudomallei in BALB/c mice via intranasal administration. Infected mice that survived for >60 days were sacrificed. Lungs, livers, spleens, and peripheral blood mononuclear cells were harvested for experimental investigations. Histopathological changes of organs were observed in the infected mice, suggestive of successful establishment of persistent infections. Moreover, natural killer (NK) cell frequency was increased in SCV- and WT-infected mice. We observed programmed death-1 (PD-1) upregulation on B cells of SCV- and WT-infected mice. Interestingly, PD-1 upregulation was only observed on NK cells and monocytes of SCV-infected mice. In contrast, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) downregulation was seen on NK cells of WT-infected mice, and on monocytes of SCV- and WT-infected mice.Conclusions/SignificanceThe SCV and the WT of B. pseudomallei distinctly upregulated PD-1 expression on B cells, NK cells, and monocytes to dampen host immunity, which likely facilitates bacterial persistence. PD-1/PD-L1 pathway appears to play an important role in the persistence of B. pseudomallei in the host.

IL-6/Stat3-Dependent Induction of a Distinct, Obesity-Associated NK Cell Subpopulation Deteriorates Energy and Glucose Homeostasis

Natural killer (NK) cells contribute to the development of obesity-associated insulin resistance. We demonstrate that in mice obesity promotes expansion of a distinct, interleukin-6 receptor (IL6R)a-expressing NK subpopulation, which also expresses a number of other myeloid lineage genes such asthe colony-stimulating factor 1 receptor (Csf1r). Selective ablation of this Csf1r-expressing NK cell population prevents obesity and insulin resistance. Moreover, conditional inactivation of IL6Ra or Stat3 in NK cells limits obesity-associated formation of these myeloid signature NK cells, protecting from obesity, insulin resistance, and obesity-associated inflammation. Also in humans IL6Ra+ NK cells increase in obesity and correlate with markers of systemic low-grade inflammation, and their gene expression profile overlaps with characteristic gene sets of NK cells in obese mice. Collectively, we demonstrate that obesity-associated inflammation and metabolic disturbances depend on interleukin-6/Stat3-dependent formation of a distinct NK population, which may provide a target for the treatment of obesity, metaflammation-associated pathologies, and diabetes.

Transcriptome analysis reveals similarities between human blood CD3\u2212 CD56bright cells and mouse CD127+ innate lymphoid cells

For many years, human peripheral blood natural killer (NK) cells have been divided into functionally distinct CD3− CD56bright CD16− and CD3− CD56dim CD16+ subsets. Recently, several groups of innate lymphoid cells (ILC), distinct from NK cells in development and function, have been defined in mouse. A signature of genes present in mouse ILC except NK cells, defined by Immunological Genome Project studies, is significantly over-represented in human CD56bright cells, by gene set enrichment analysis. Conversely, the signature genes of mouse NK cells are enriched in human CD56dim cells. Correlations are based upon large differences in expression of a few key genes. CD56bright cells show preferential expression of ILC-associated IL7R (CD127), TNFSF10 (TRAIL), KIT (CD117), IL2RA (CD25), CD27, CXCR3, DPP4 (CD26), GPR183, and MHC class II transcripts and proteins. This could indicate an ontological relationship between human CD56bright cells and mouse CD127+ ILC, or conserved networks of transcriptional regulation. In line with the latter hypothesis, among transcription factors known to impact ILC or NK cell development, GATA3, TCF7 (TCF-1), AHR, SOX4, RUNX2, and ZEB1 transcript levels are higher in CD56bright cells, while IKZF3 (AIOLOS), TBX21 (T-bet), NFIL3 (E4BP4), ZEB2, PRDM1 (BLIMP1), and RORA mRNA levels are higher in CD56dim cells.

NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

FHL2 Regulates Natural Killer Cell Development and Activation during Streptococcus pneumoniae Infection.

Recent in silico studies suggested that the transcription cofactor LIM-only protein FHL2 is a major transcriptional regulator of mouse natural killer (NK) cells. However, the expression and role of FHL2 in NK cell biology are unknown. Here, we confirm that FHL2 is expressed in both mouse and human NK cells. Using FHL2−/− mice, we found that FHL2 controls NK cell development in the bone marrow and maturation in peripheral organs. To evaluate the importance of FHL2 in NK cell activation, FHL2−/− mice were infected with Streptococcus pneumoniae. FHL2−/− mice are highly susceptible to this infection. The activation of lung NK cells is altered in FHL2−/− mice, leading to decreased IFNγ production and a loss of control of bacterial burden. Collectively, our data reveal that FHL2 is a new transcription cofactor implicated in NK cell development and activation during pulmonary bacterial infection.

EBI3 regulates the NK cell response to mouse cytomegalovirus infection

Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection. The induction of EBI3 protein expression in mouse NK cells is a late activation event. Thus, early activation events of NK cells, such as IFNγ production and CD69 expression, were not affected in EBI3-deficient (Ebi3-/- ) C57BL/6 (B6) mice during MCMV infection. Furthermore, comparable levels of early viral replication in spleen and liver were observed in MCMV-infected Ebi3-/- and wild-type (WT) B6 mice. Interestingly, the viral load in salivary glands and oral lavage was strongly decreased in the MCMV-infected Ebi3-/- B6 mice, suggesting that EBI3 plays a role in the establishment of MCMV latency. We detected a decrease in the sustained IL-10 production by NK cells and lower serum levels of IL-10 in the MCMV-infected Ebi3-/- B6 mice. Furthermore, we observed an increase in dendritic cell maturation markers and an increase in activated CD8+ T cells. Thus, EBI3 dampens the immune response against MCMV infection, resulting in prolonged viral persistence.

MICA/IL-12: A novel bifunctional protein for killer cell activation.

Natural killer (NK) cells have the potential to be effective killers of tumor cells. They are governed by inhibitory and activating receptors such as NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have ways to reduce the expression of these ligands, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, such as IL-12. A previous study in our laboratory showed that a fusion protein of the extracellular domain of mouse UL-16 binding protein-like transcript 1 (MULT1E) and mouse interleukin 12 (IL-12) can effectively activate mouse NK cells by invitro assays and invivo in animal tumor models. The aim of the present study was to expand the concept of developing a novel bifunctional fusion protein for enhanced NK cell activation to human killer cells. The proposed protein combines the extracellular domain of a human NKG2D ligand, MHC class I polypeptide-related sequence A (MICA) and IL-12. It is hypothesized that when expressed by tumor cells, the protein will activate human NK and other killer cells using the NKG2D receptor, and deliver IL-12 to the NK cells where it can interact with the IL-12R and enhance cytotoxicity. The fusion protein, when expressed by engineered tumor cells, indeed activated NK92 cells as measured by an increase in interferon-γ (IFN-γ) production and an increase in cytotoxicity of tumor cells. The fusion protein was also able to increase the proliferation of human peripheral blood mononuclear cells (PBMCs) and augment their production of IFN-γ. This study along with the data from the previous mouse studies suggest that the MICA/IL-12 bifunctional fusion protein represents an effective activator of killer cells for cancer treatment.