Extracellular Vesicle-Mediated Secretion of HLA-E by Trophoblasts Maintains Pregnancy by Regulating the Metabolism of Decidual NK Cells

Extracellular vesicles derived from trophoblasts (T-EVs) play an important role in pregnancy, but the mechanism is not entirely clear. In this study, we found that HLA-E, which is mostly confined to the cytoplasm of trophoblast cells, was secreted by T-EVs. The level of HLA-E in T-EVs from unexplained recurrent spontaneous abortion (URSA) patients was lower than that in normal pregnancy (NP) and RSA patients who had an abnormal embryo karyotype (AK-RSA). T-EVs promoted secretion of IFN-γ and VEGFα by decidual NK (dNK) cells from URSA patients via HLA-E, VEGFα was necessary for angiogenesis and trophoblast growth, and IFN-γ inhibited Th17 induction. Glycolysis and oxidative phosphorylation (OxPhos) were involved in this process. Glycolysis but not OxPhos of dNK cells facilitated by T-EVs was dependent on mTORC1 activation. Inhibition of T-EV production in vivo increased the susceptibility of mice to embryo absorption, which was reversed by transferring exogenous T-EVs. T-EVs promoted secretion of IFN-γ and VEGFα by dNK cells to maintain pregnancy via Qa-1 in abortion-prone mouse models. This study reveals a new mechanism of pregnancy maintenance mediated by HLA-E via T-EVs.     

Tim-3 Negatively Mediates Natural Killer Cell Function in LPS-Induced Endotoxic Shock

Sepsis is an exaggerated inflammatory condition response to different microorganisms with high mortality rates and extremely poor prognosis. Natural killer (NK) cells have been reported to be the major producers of IFN-γ and key players in promoting systematic inflammation in lipopolysaccharide (LPS)-induced endotoxic shock. T-cell immunoglobulin and mucin domain (Tim)-3 pathway has been demonstrated to play an important role in the process of sepsis, however, the effect of Tim-3 on NK cell function remains largely unknown. In this study, we observed a dynamic inverse correlation between Tim-3 expression and IFN-γ production in NK cells from LPS-induced septic mice. Blockade of the Tim-3 pathway could increase IFN-γ production and decrease apoptosis of NK cells in vitro, but had no effect on the expression of CD107a. Furthermore, NK cell cytotoxicity against K562 target cells was enhanced after blocking Tim-3 pathway. In conclusion, our results suggest that Tim-3 pathway plays an inhibitory role in NK cell function, which might be a potential target in modulating the excessive inflammatory response of LPS-induced endotoxic shock.     

Decidual NK cells regulate key developmental processes at the human fetal-maternal interface

Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.     

Development of Functional Human NK Cells in an Immunodeficient Mouse Model with the Ability to Provide Protection against Tumor Challenge

Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2^−/−/γ_c ^−/− and NOD/Scid mice, newborn BALB/c Rag2^−/−/γ_c ^−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56⁺ cells in BALB/c Rag2^−/−/γ_c ^−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2^−/−/γ_c ^−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2^−/−/γ_c ^−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2^−/−/γ_c ^−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2^−/−/γ_c ^−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment.     

PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

CD8⁺ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3⁺PD-1⁺CD8⁺ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8⁺ T cells upregulated PD-1⁺ and/or Tim-3⁺ immune cells. Furthermore, the population of CD8⁺ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8⁺ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8⁺ T-cell dysfunction. Importantly, the number and function of Tim-3⁺PD-1⁺CD8⁺ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8⁺ T-cell function and maintaining normal pregnancy.Successful pregnancy requires the maternal immune system to tolerate the semi-allogeneic fetus. A failure in immune tolerance may result in abnormal pregnancies, such as recurrent spontaneous abortion. For many years, the model of immune regulation during pregnancy has been based on a shift in the maternal immune response towards a Th2 bias. The shift from producing inflammatory Th1-type cytokines toward Th2-type cytokines promotes maternal–fetal tolerance.^{1, 2} In addition, maternal administration of the Th2-type cytokine interleukin (IL)-10 or blockade of the Th1-type cytokine tumor necrosis factor (TNF)-α is known to prevent pregnancy loss induced by lipopolysaccharide.^{3, 4}Compared with CD4⁺ T cells, our understanding of the role of CD8⁺ T cells during pregnancy remains poorly understood. CD8⁺ T cells, which directly recognize allogeneic major histocompatibility complex (MHC) class I molecules, have important roles in defense against viral infections. Studies on several murine models have demonstrated the existence of CD8⁺ T cells at the maternal–fetal interface.⁵ During normal pregnancy, the major antigen present is the embryo-derived paternal antigen expressed on extravillous trophoblast (EVT) cells. These cells do not express MHC class I human leukocyte antigens (HLA)-A and HLA-B,⁶ which are the main causes of CD8⁺ T cell-mediated rejection. However, HLA-C and HLA-G, highly expressed on EVT cells,⁶ can elicit a direct cytotoxic response by CD8⁺ T cells during hematopoietic stem cell and allogeneic organ transplantation.^{7, 8} Therefore, whether suppressor or regulatory CD8⁺ T cells are present at the maternal–fetal interface, and how they function to maintain normal pregnancy, remain to be explored.Inhibitory co-stimulatory signals have crucial roles in regulating CD8⁺ T-cell activation or tolerance. It has been shown that exhausted T cells express up to seven different inhibitory molecules,⁹ including PD-1 and Tim-3. PD-1 has been identified as a marker for dysfunctional T cells, and blockade of PD-1 signals has been shown to revert the dysfunctional state of exhausted CD8⁺ T cells in most cases.^{10, 11} Tim-3 has been similarly associated with CD8⁺ T-cell exhaustion as Tim-3 blockade restores proliferation and cytokine production.^{12, 13} Tim-3 and PD-1 co-expression on T cells characterizes the most severely exhausted CD8⁺ T-cell subset, and combined blockade of Tim-3 and PD-1 restores the function of exhausted CD8⁺ T cells.^{14, 15, 16} However, much less is known about the functional regulation of Tim-3 and PD-1 on CD8⁺ T cells during pregnancy.In this study, we investigated Tim-3 and PD-1 expression on CD8⁺ T cells from decidua and peripheral blood in normal pregnant women and those who underwent miscarriage. In particular, we used surface and intracellular phenotype analysis, as well as multifunctional assays, to study the role of Tim-3 and PD-1 signaling pathways in regulating decidual CD8⁺ (dCD8⁺) T-cell function and maintenance of pregnancy. Our data indicate that Tim-3 and PD-1 co-expression on CD8⁺ T cells might be important in maintaining maternal–fetal immune tolerance and successful pregnancy. These results could provide a strategy for developing novel therapies that enhance Tim-3 and PD-1 signals to promote maternal–fetal tolerance and prevent pregnancy loss.     

Transforming Growth Factor-β1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein

Human trophoblast cells express transforming growth factor-β (TGF-β) and TGF-β receptors. It has been shown that TGF-β1 treatment decreases the invasiveness of trophoblast cells. However, the molecular mechanisms underlying TGF-β1-decreased trophoblast invasion are still not fully understood. In the current study, we demonstrated that treatment of HTR-8/SVneo human trophoblast cells with TGF-β1 decreased cell invasion and down-regulated the expression of vascular endothelial cadherin (VE-cadherin). In addition, the inhibitory effect of TGF-β1 on VE-cadherin was confirmed in primary cultures of human trophoblast cells. Moreover, knockdown of VE-cadherin using siRNA decreased the invasiveness of HTR-8/SVneo cells and primary cultures of trophoblast cells. Treatment with TGF-β1 induced the activation of Smad-dependent signaling pathways and the expression of Snail and Slug. Knockdown of Smads attenuated TGF-β1-induced up-regulation of Snail and Slug and down-regulation of VE-cadherin. Interestingly, depletion of Snail, but not Slug, attenuated TGF-β1-induced down-regulation of VE-cadherin. Furthermore, overexpression of Snail suppressed VE-cadherin expression. Chromatin immunoprecipitation analyses showed the direct binding of Snail to the VE-cadherin promoter. These results provide evidence that Snail mediates TGF-β1-induced down-regulation of VE-cadherin, which subsequently contributed to TGF-β1-decreased trophoblast cell invasion.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

Tumor-released Galectin-3, a Soluble Inhibitory Ligand of Human NKp30, Plays an Important Role in Tumor Escape from NK Cell Attack

Human Galectin-3 (Gal-3), a β-galactoside-binding protein expressed by tumor cells, has been reported to act as an immune regulator in antitumor T cells. However, its effect on natural killer (NK) cells is elusive. Using a recombinant human NK cell-activating receptor, NKp30 fusion protein (NKp30-Fc), we found that soluble NKp30-Fc could immunoprecipitate Galectin-3. The direct interaction between NKp30 and Galectin-3 was further confirmed using surface plasmon resonance experiments. Because Galectin-3 was mainly released from tumor cells in a soluble form in our study, the binding assay was performed to show that soluble Galectin-3 specifically bound to NK cells and NKp30 on the surface of the NK cells. Functionally, when soluble Galectin-3 was added to the NK-tumor cell coculture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by preincubation of soluble Galectin-3 with NKp30-Fc fusion protein or the addition of anti-Gal-3 antibody alone. Moreover, genetic down-regulation of Galectin-3 (shGal-3) resulted in tumor cells being more sensitive to NK cell lysis, and, reversely, Galectin-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft non-obese diabetic/severe combined immunodeficiency mice after NK cell adoptive immunotherapy, indicating that Galectin-3 strongly antagonizes human NK cell attack against tumors in vivo. These findings indicate that Galectin-3 may function as an immune regulator to inhibit NK cell function against tumors, therefore providing a new therapeutic target for tumor treatment.     

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.     

Galectin-9 Ameliorates Clinical Severity of MRL/lpr Lupus-Prone Mice by Inducing Plasma Cell Apoptosis Independently of Tim-3

Galectin-9 ameliorates various murine autoimmune disease models by regulating T cells and macrophages, although it is not known what role it may have in B cells. The present experiment shows that galectin-9 ameliorates a variety of clinical symptoms, such as proteinuria, arthritis, and hematocrit in MRL/lpr lupus-prone mice. As previously reported, galectin-9 reduces the frequency of Th1, Th17, and activated CD8⁺ T cells. Although anti-dsDNA antibody was increased in MRL/lpr lupus-prone mice, galectin-9 suppressed anti-dsDNA antibody production, at least partly, by decreasing the number of plasma cells. Galectin-9 seemed to decrease the number of plasma cells by inducing plasma cell apoptosis, and not by suppressing BAFF production. Although about 20% of CD19^−/low CD138⁺ plasma cells expressed Tim-3 in MRL/lpr lupus-prone mice, Tim-3 may not be directly involved in the galectin-9-induced apoptosis, because anti-Tim-3 blocking antibody did not block galectin-9-induced apoptosis. This is the first report of plasma cell apoptosis being induced by galectin-9. Collectively, it is likely that galectin-9 attenuates the clinical severity of MRL lupus-prone mice by regulating T cell function and inducing plasma cell apoptosis.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

T-cell Immunoglobulin and ITIM Domain (TIGIT) Receptor/Poliovirus Receptor (PVR) Ligand Engagement Suppresses Interferon-γ Production of Natural Killer Cells via β-Arrestin 2-mediated Negative Signaling

Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8⁺ T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor β-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.     

Loss of Cytotoxicity and Gain of Cytokine Production in Murine Tumor-Activated NK Cells

NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25⁺ NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFNγ, TNFα, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines.     

Galectin-1, -3 and -9 Expression and Clinical Significance in Squamous Cervical Cancer

Galectins are proteins that bind β-galactoside sugars and provide a new type of potential biomarkers and therapeutic targets in cancer. Galectin-1, -3 and -9 have become the focus of different research groups, but their expression and function in cervical cancer is still unclear. The aim of this study was to determine the phenotype of galectin-1, -3 and -9 expressing cells and the association with clinico-pathological parameters in cervical cancer. Galectin expression was scored in tumor cells, tumor epithelium infiltrating immune cells and stromal cells in squamous cervical cancer (n = 160). Correlations with clinico-pathological parameters and survival were studied according to the REMARK recommendations. We additionally investigated whether the galectins were expressed by tumor cells, fibroblasts, macrophages and T cells. Galectin-1 and -9 were both expressed by tumor cells in 11% of samples, while 84% expressed galectin-3. Strong galectin-1 expression by tumor cells was an independent predictor for poor survival (hazard ratio: 8.02, p = 0.001) and correlated with increased tumor invasion (p = 0.032) and receiving post-operative radiotherapy (p = 0.020). Weak and positive tumor cell galectin-3 expression were correlated with increased and decreased tumor invasion, respectively (p = 0.012). Tumor cell expression of galectin-9 showed a trend toward improved survival (p = 0.087). The predominant immune cell type expressing galectin-1, -3 and -9 were CD163⁺ macrophages. Galectin-1 and -3 were expressed by a minor population of T cells. Galectin-1 was mainly expressed by fibroblasts in the tumor stroma. To conclude, while tumor cell expression of galectin-9 seemed to represent a beneficial response, galectin-1 expression might be used as a marker for a more aggressive anti-cancer treatment.     

Essential Role of NK Cells in IgG Therapy for Experimental Autoimmune Encephalomyelitis

Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fcγ receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fcγ receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon γ and interleukin 17 production. The percentage of CD4⁺Foxp3⁺ regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4⁺ T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4⁺Foxp3⁺ T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.     

Reduced Expression of Galectin-9 Contributes to a Poor Outcome in Colon Cancer by Inhibiting NK Cell Chemotaxis Partially through the Rho/ROCK1 Signaling Pathway

Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearman''s rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearman''s rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56⁺ NK cell infiltration (R² = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.