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Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces severe pneumonia and is the cause of a worldwide pandemic. Coronaviruses, including SARS-CoV-2, have RNA proofreading enzymes in their genomes, resulting in fewer gene mutations than other RNA viruses. Nevertheless, variants of SARS-CoV-2 exist and may induce different symptoms; however, the factors and the impacts of these mutations are not well understood. We found that there is a bias to the mutations occurring in SARS-CoV-2 variants, with disproportionate mutation to uracil (U). These point mutations to U are mainly derived from cytosine (C), which is consistent with the substrate specificity of host RNA editing enzymes, APOBECs. We also found the point mutations which are consistent with other RNA editing enzymes, ADARs. For the C-to-U mutations, the context of the upstream uracil and downstream guanine from mutated position was found to be most prevalent. Further, the degree of increase of U in SARS-CoV-2 variants correlates with enhanced production of cytokines, such as TNF-a and IL-6, in cell lines when compared with stimulation by the ssRNA sequence of the isolated virus in Wuhan. Therefore, RNA editing is a factor for mutation bias in SARS-CoV-2 variants, which affects host inflammatory cytokines production.

Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution., T cell mediated anti-tumour immune responses result in the emergence of an immune-resistant population in a process called immunoediting. Here, the authors show that immunoediting is associated with an increase in genomic rearrangements of tumour cells that requires both cytotoxic T cells and IFNγ exposure.

Chromium (Cr) is commonly added into various metal alloys to improve some mechanical properties such as corrosion resistance, strength, and workability. However, Cr is also known to be a metal allergen for some individuals. Metal allergy is a T cell-mediated disease with symptoms of inflammation and swelling that involve inflammatory cytokines and prostaglandins. Hence, suppressing these inflammation paths by using COX-2 inhibitor might be useful in treating Cr allergy. In this study, mice were used with Cr-induced allergy challenge model. The mice were injected with celecoxib once per day for 7 days one hour after the challenge. Footpad samples were stained with haematoxylin and eosin (H&E), and lymphocytes were isolated from popliteal lymph nodes for the flow cytometric analysis. The results show that both prostaglandin E2 (PGE2), a known mediator of inflammation, and cyclooxygenases (COX)-2 have important roles in the development of Cr allergy. Further, COX-2 inhibitor, celecoxib, was effective in relieving swelling and inflammation in Cr-allergic mice concordant with suppression of IFN-γ production by CD8+ T cells and T cell accumulation in the lymph nodes. Therefore, the inhibition of COX-2 may be a therapeutic target for Cr allergy, and additional molecules in the PGE2 signalling pathway may also be an effective therapeutic target for the treatment of metal allergy.

Nickel (Ni) eluted from metallic biomaterials is widely accepted as a major cause of allergies and inflammation. To improve the safety of cobalt-chromium-molybdenum (Co-Cr-Mo) alloy implants, new ultralow-Ni Co-Cr-Mo alloys with and without zirconium (Zr) have been developed, with Ni contents of less than 0.01%. In the present study, we investigated the biocompatibility of these new alloys in vivo by subcutaneously implanting pure Ni, conventional Co-Cr-Mo, ultralow-Ni Co-Cr-Mo, and ultralow-Ni Co-Cr-Mo with Zr wires into the dorsal sides of mice. After 3 and 7 days, tissues around the wire were excised, and inflammation; the expression of IL-1β, IL-6, and TNF-α; and Ni, Co, Cr, and Mo ion release were analyzed using histological analyses, qRT-PCR, and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Significantly larger amounts of Ni eluted from pure Ni wires than from the other wires, and the degree of inflammation depended on the amount of eluted Ni. Although no significant differences in inflammatory reactions were identified among new alloys and conventional Co-Cr-Mo alloys in histological and qRT-PCR analyses, ICP-MS analysis revealed that Ni ion elution from ultralow-Ni Co-Cr-Mo alloys with and without Zr was significantly lower than from conventional Co-Cr-Mo alloys. Our study, suggests that the present ultralow-Ni Co-Cr-Mo alloys with and without Zr have greater safety and utility than conventional Co-Cr-Mo alloys. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1505-1513, 2016.

The transcriptional regulator IκB-ζ is important for the control of apoptosis in keratinocytes. Thus, IκB-ζ-deficient mice develop autoimmune diseases, such as Sjögren’s syndrome. However, T cells also play a pivotal role in Sjögren’s syndrome. To study the role of IκB-ζ in T cells, we generated T cell-specific, IκB-ζ-deficient mice. We observed increased numbers of peripheral effector/memory CD4+ cells and IFN-γ-producing CD4+ cells in 3-week-old mice. We found that IκB-ζ can be up-regulated by TGF-β1 in naϊve CD4+ T cells and that it negatively regulates IFN-γ expression. In addition, we generated Treg-specific, IκB-ζ deficient mice and found that IκB-ζ is dispensable for the plasticity and stability of Tregs. However, Tregs from T cell-specific, IκB-ζ-deficient mice have reduced immunoregulatory function. Thus, our data reveal a previously unappreciated role for IκB-ζ in IFN-γ production in T cells and the immunoregulatory function of Tregs.

Nickel ions (Ni2+) are eluted from various metallic materials, such as medical devices implanted in human tissues. Previous studies have shown that Ni2+ enters inflammatory cells inducing inflammation. However, the regulation of Ni2+ uptake in cells has not yet been reported in detail. In the present study, we investigated the effects of various divalent cations on Ni2+ uptake and Ni2+-induced interleukin (IL)-8 production in the human monocytic cell line, THP-1. We demonstrated that ZnCl2, MnCl2, and CoCl2 inhibited the Ni2+ uptake, while CuCl2, FeCl2, MgCl2, and divalent metal transporter (DMT)-1 inhibitor, Chlorazol Black, did not. Furthermore, ZnCl2 inhibited Ni2+-induced IL-8 production, correlating with the inhibition of Ni2+ uptake. These results suggested that Ni2+ uptake occurred through Zn2+, Mn2+, and Co2+-sensitive transporters and that the inhibition of Ni2+ uptake resulted in the inhibition of IL-8 production. Furthermore, using an Ni wire-implanted mouse model, we found that Ni wire-induced expression of mouse macrophage inflammatory protein-2 (MIP-2) and cyclooxygenase-2 (COX-2) mRNA in the skin tissue surrounding the wire were enhanced by low Zn conditions. These results suggested that the physiological concentration of Zn2+ modulates Ni2+ uptake by inflammatory cells, and a Zn deficient state might increase sensitivity to Ni.

While metallic biomaterials have led to an improvement in the quality of life, metal allergies, especially to palladium (Pd), has caused a recent increase in allergic patients. Metal allergy is known to be a T cell-mediated delayed-type hypersensitivity (DTH); however, the pathogenic T cell subsets and the specific T cell receptor (TCR) have not been identified. Therefore, we attempted to identify the pathogenic T cells responsible for Pd allergy. We found that activating CD8+ T cells significantly increased and that the TRAV (TCRα variable) 7-2*02 chain skewed in Pd allergic mice. Furthermore, adoptive transfer experiments revealed that in vitro-cultured Pd-stimulated antigen presenting cells (APCs) function as memory APCs with recipient mice developing Pd allergy and that the frequency of TRAV7-2*02 increases the same as conventional Pd allergic mice. In contrast, neither proliferation of CD8+ T cells nor increasing of TRAV7-2*02 was observed in major histocompatibility complex I (MHC I)-deficient Pd-APCs transferred to mice. Taken together, we revealed that TRAV7-2*02-expressing CD8+ T cells are the pathogenic T cells for the development of Pd allergy. We also identified the CDR3 consensus motif of pathogenic TCRs as CAAXSGSWQLIF in TRAV7-2*02/TRAJ (TCRα junction)22*01 positive cells. These results suggest that the specific TCRs represent novel targets for the development of diagnostics and treatments for metal allergy.

The natural killer group 2 membrane D (NKG2D) activating receptor plays crucial roles not only in host defense against tumors and viral infections, but also in autoimmune diseases. After NKG2D-mediated activation, Natural killer (NK) cells must be regulated to avoid potentially harmful reactivity. However, the negative regulation of these activated NK cells is poorly understood. Here, we reveal that the engagement of NKG2D by its ligand elicits not only target cell lysis, but also NK cell fratricide. Conventional mouse NK cells underwent cell death when cocultured with RMA cells expressing the NKG2D ligand retinoic acid early-inducible protein 1 (Rae-1), but not with RMA cells lacking MHC class I. NK cells from mice deficient for DAP10 and DAP12 or perforin did not undergo death, highlighting the importance of the NKG2D pathway for NK cell death. However, NKG2D does not transmit direct death signals in NK cells. Rather, the interaction between NKG2D and Rae-1 allowed NK cells to acquire tumor-derived Rae-1 by a membrane transfer process known as ”trogocytosis,” which was associated with clathrin-dependent NKG2D endocytosis. NK cells dressed with Rae-1 were lysed by neighboring NK cells through the NKG2D-induced perforin pathway in vitro and in vivo. These results provide the unique NKG2D function in negative regulation of activated NK cells.

Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. However, cellular activation during disease development induces Opn expression in various immune cells. In this study, using Opn-EGFP knock-in (KI) mice we found that CD8α⁺ T cells in the intestinal tissues, including Peyer’s patch, lamina propria and epithelium, express Opn under steady state conditions. Therefore, we examined the role of Opn-expressing CD8α⁺ T cells in intestinal homeostasis. Interestingly, Opn knockout (KO) mice had altered fecal microflora concordant with a reduction of TCRγδ⁺ intraepithelial lymphocytes (IELs). Consistent with this result, both treatment with anti-Opn blocking antibody and deficiency of Opn resulted in decreased survival of TCRγδ⁺ and TCRαβ⁺ IELs. This data suggests that a possibility that Opn may function as a survival factor for IELs in the intestinal tissue. Collectively, these data suggest the possibility that Opn might regulate the homeostasis of intestinal microflora through maintenance of TCRγδ⁺ IELs, possibly by support of IEL survival.

In this chapter, bioceramic coatings on Ti and its alloys are examined. Surface modification processes of metallic biomaterials are reviewed, and the formation and evaluation of amorphous calcium phosphate (ACP) and anatase-rich TiO2 coatings are described. Dry processes such as radio-frequency (RF) magnetron sputtering and thermal oxidation are employed for coating. Ag-containing ACP coating films exhibited antibacterial activity through the continuous release of Ag ions, caused by the resorbability of ACP. Anatase-rich TiO2 layers fabricated by a two-step thermal oxidation process showed photodegradation of organic compounds under both UV- and visible-light irradiation. The introduction of Au into TiO2 layers from Ti-Au alloy substrates by thermal oxidation contributed to the expression of visible-light response.

Bronchiolitis obliterans after lung transplantation is a major cause of postoperative mortality in which T cell-mediated immunity is known to play an important role. However, the exact contribution of natural killer (NK) cells, which have functions similar to CD8+ T cells, has not been defined. Here, we assessed the role of NK cells in murine bronchiolitis obliterans through heterotopic tracheal transplantations and found a greater percentage of NK cells in allografts than in isografts. Depletion of NK cells using an anti-NK1.1 antibody attenuated bronchiolitis obliterans in transplant recipients compared with controls. In terms of NK cell effector functions, an improvement in bronchiolitis obliterans was observed in perforin-KO recipient mice compared to wild type (WT). Furthermore, we found upregulation of NKG2D-ligand in allografts and demonstrated the significance of this using grafts expressing Rae-1, a murine NKG2D-ligand, which induced severe bronchiolitis obliterans in WT and Rag-1 KO recipients. This effect was ameliorated by injection of anti-NKG2D blocking antibody. Together, these results suggest that cytotoxicity resulting from activation of NK cells through NKG2D leads to the development of murine bronchiolitis obliterans.

The innate immune system has developed to acquire a wide variety of pattern-recognition receptors (PRRs) to identify potential pathogens, whereas pathogens have also developed to escape host innate immune responses. ITIM-bearing receptors are attractive targets for pathogens to attenuate immune responses against them; however, the in vivo role of the inhibitory PRRs in host–bacteria interactions remains unknown. We demonstrate in this article that Staphylococcus aureus, a major Gram-positive bacteria, exploits inhibitory PRR paired Ig-like receptor (PIR)-B on macrophages to suppress ERK1/2 and inflammasome activation, and subsequent IL-6 and IL-1β secretion. Consequently, Pirb−/− mice infected with S. aureus showed enhanced inflammation and more effective bacterial clearance, resulting in resistance to the sepsis. Screening of S. aureus mutants identified lipoteichoic acid (LTA) as an essential bacterial cell wall component required for binding to PIR-B and modulating inflammatory responses. In vivo, however, an LTA-deficient S. aureus mutant was highly virulent and poorly recognized by macrophages in both wild-type and Pirb−/− mice, demonstrating that LTA recognition by PRRs other than PIR-B mediates effective bacterial elimination. These results provide direct evidence that bacteria exploit the inhibitory receptor for virulence, and host immune system counterbalances the infection.

CD30+ lymphoproliferative disorders represent a spectrum of diseases from lymphomatoid papulosis (LyP) to primary cutaneous anaplastic large-cell lymphoma (PCALCL) (1, 2). LyP is characterized by recurrent papulonodular lesions, which undergo spontaneous regression (1, 2). Survival is unaffected by LyP, but patients are at risk of a second cutaneous or lymphoid malignancy (1). PCALCL has a favourable prognosis with a 5-year survival rate between 76% and 96%. In this report, we used immunohistochemical (IHC) staining of LyP type A and PCALCL specimens to examine for macrophage markers as well as dendritic cell (DC)-specific markers and their functional markers.

Natural killer (NK) cells contribute to not only innate but also to adaptive immunity by interacting with dendritic cells (DCs) and T cells. All activated human NK cells express HLA-DR and can initiate MHCII-dependent CD4 + T-cell proliferation; however, the expression of MHCII by mouse NK cells and its functional significance are controversial. In this study, we show that NK–DC interactions result in the emergence of MHCII-positive NK cells. Upon in vitro or in vivo activation, mouse conventional NK cells did not induce MHCII transcripts, but rapidly acquired MHCII protein from DCs. MHCII H2-Ab1 –deficient NK cells turned I-A b -positive when adoptively transferred into wild-type mice or when cultured with WT splenic DCs. NK acquisition of MHCII was mediated by intercellular membrane transfer called “trogocytosis,” but not upon DAP10/12- and MHCI-binding NK cell receptor signaling. MHCII-dressed NK cells concurrently acquired costimulatory molecules such as CD80 and CD86 from DCs; however, their expression did not reach functional levels. Therefore, MHCII-dressed NK cells inhibited DC-induced CD4 + T-cell responses rather than activated CD4 + T cells by competitive antigen presentation. In a mouse model for delayed-type hypersensitivity, adoptive transfer of MHCII-dressed NK cells attenuated footpad swelling. These results suggest that MHCII-dressed NK cells generated through NK–DC interactions regulate T cell-mediated immune responses.

Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1-124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.

Cellular labile iron, which contains chelatable redox-active Fe(2+), has been implicated in iron-mediated cellular toxicity leading to multiple organ dysfunction. Iron homeostasis is controlled by monocytes/macrophages through their iron recycling and storage capacities. Furthermore, iron sequestration by monocytes/macrophages is regulated by pro-inflammatory cytokines including interleukin-1, highlighting the importance of these cells in the crosstalk between inflammation and iron homeostasis. However, a role for cellular labile iron in monocyte/macrophage-mediated inflammatory responses has not been defined. Here we describe how cellular labile iron activates the NLRP3 inflammasome in human monocytes. Stimulation of lipopolysaccharide-primed peripheral blood mononuclear cells with ferric ammonium citrate increases the level of cellular Fe(2+) levels in monocytes and induces production of interleukin-1β in a dose-dependent manner. This ferric ammonium citrate-induced interleukin-1β production is dependent on caspase-1 and is significantly inhibited by an Fe(2+)-specific chelator. Ferric ammonium citrate consistently induced interleukin-1β secretion in THP1 cells, but not in NLRP3-deficient THP1 cells, indicating a requirement for the NLRP3 inflammasome. Additionally, activation of the inflammasome is mediated by potassium efflux, reactive oxygen species-mediated mitochondrial dysfunction, and lysosomal membrane permeabilization. Thus, these results suggest that monocytes/macrophages not only sequestrate iron during inflammation, but also mediate inflammation in response to cellular labile iron, which provides novel insights into the role of iron in chronic inflammation.

Irradiation-resistant natural killer (NK) cells in an F(1) recipient can reject parental bone marrow, and host NK cells can also prevent engraftment of allogeneic bone marrow. We show here that repopulating bone marrow cells in certain mouse strains expressed retinoic acid early inducible 1 proteins, which are ligands for the activating NKG2D NK cell receptor. Treatment with a neutralizing antibody to NKG2D prevented rejection of parental BALB/c bone marrow in (C57BL/6 x BALB/c) F(1) recipients and allowed engraftment of allogeneic BALB.B bone marrow in C57BL/6 recipients. Additionally, bone marrow from C57BL/6 mice transgenic for retinoic acid early inducible 1epsilon was rejected by syngeneic mice but was accepted after treatment with antibody to NKG2D. If other stem cells or tissues upregulate expression of NKG2D ligands after transplantation, NKG2D may contribute to graft rejection in immunocompetent hosts.

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a key effector molecule expressed by natural killer (NK) cells and has been shown to prevent tumor initiation, growth, and metastasis. Here we demonstrate that TRAIL is the dominant cytotoxic effector molecule expressed by NK cells in fetal mice. On birth and with age, NK cells develop full functional capacity, including the ability to secrete interferon γ (IFN-γ) and interleukin 13 (IL-13) and mediate perforin- and Fas ligand-mediated cytotoxicity. However, interestingly, a phenotypically immature TRAIL+ NK cell subpopulation is retained in the liver of adult mice, and its retention is dependent on IFN-γ but not dependent on host IL-12, IL-18, or endogenous host pathogens. Adoptive transfer of either adult liver or neonatal TRAIL+ NK cells resulted in the appearance of TRAIL- NK cells with a mature phenotype, suggesting that these TRAIL+ NK cells were indeed a precursor. Although inducers of IFN-γ stimulated TRAIL expression on mature NK cells, our data indicated that constitutive TRAIL expression was a hallmark of immature cytotoxic NK cells. This study is the first to describe the concomitant maturation of NK cell effector function with surface phenotype in vivo and implies an important defense role for NK cell TRAIL in the developing immune system.

Macrophages recognize the presence of infection by using the Toll-like receptor (TLR) family of proteins that detect ligands on bacterial, viral, and fungal pathogens. We show that murine macrophages stimulated with pathogen products known to signal through TLRs express ligands for the NKG2D receptor, found on NK cells, activated CD8+ T cells and activated macrophages. TLR signaling, through the MyD88 adaptor, up-regulates transcription of the retinoic acid early inducible-1 (RAE-1) family of NKG2D ligands, but not H-60 or murine UL16-binding protein-like transcript-1. RAE-1 proteins are found on the surface of activated, but not resting, macrophages and can be detected by NKG2D on NK cells resulting in down-regulation of this receptor both in vitro and in vivo. RAE-1-NKG2D interactions provide a mechanism by which NK cells and infected macrophages communicate directly during an innate immune response to infection.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral, modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1alpha, RAE-1beta, RAE-1delta, RAE-1epsilon, and RAE-1gamma), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon gamma production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Deltam152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c nice with a neutralizing anti-NKG2D antibody before infection increased titers of Deltam152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1-NKG2D interaction.

The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16+ CD56−, CD16− CD56+ and CD16− CD56− cells. NKG2D expression on marmoset CD16+ CD56− and CD16− CD56+ splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16+ CD56− and CD16− CD56+. In addition, CD16+ CD56+ cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

Natural killer (NK) cells play an important role in early defense against viral infection. The cytotoxic activity of NK cells is increased by interferon-alpha/beta (IFN-alpha/beta), produced en masse in virally infected cells. However, the mechanism(s) by which IFN-alpha/beta contribute to the NK-cell-mediated antiviral response is not well understood. Here we provide evidence that the cytotoxicity of NK cells is enhanced by IFN-alpha/beta through induction of TNF-related apoptosis-inducing ligand (TRAIL). Isolation and analysis of the murine TRAIL promoter revealed the presence of an IFN-stimulated response element (ISRE), which binds to the transcription factor ISGF3 (interferon stimulated gene factor-3). This promoter is indeed activated by IFN-beta in ISGF3-dependent manner. We also show that virally infected cells, but not uninfected cells, are susceptible to TRAIL-mediated cytotoxicity in vitro, and that the TRAIL expressed in NK cells is indeed crucial in limiting virus replication in vivo. Thus, our study reveals a new molecular link between IFN-alpha/beta signaling and activation of NK cells in antiviral response of the host.

Bone resorption is regulated by the immune system, where T-cell expression of RANKL (receptor activator of nuclear factor (NF)-kappaB ligand), a member of the tumour-necrosis factor family that is essential for osteoclastogenesis, may contribute to pathological conditions, such as autoimmune arthritis. However, whether activated T cells maintain bone homeostasis by counterbalancing the action of RANKL remains unknown. Here we show that T-cell production of interferon (IFN)-gamma strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signalling pathway. IFN-gamma induces rapid degradation of the RANK adapter protein, TRAF6 (tumour necrosis factor receptor-associated factor 6), which results in strong inhibition of the RANKL-induced activation of the transcription factor NF-kappaB and JNK. This inhibition of osteoclastogenesis is rescued by overexpressing TRAF6 in precursor cells, which indicates that TRAF6 is the target critical for the IFN-gamma action. Furthermore, we provide evidence that the accelerated degradation of TRAF6 requires both its ubiquitination, which is initiated by RANKL, and IFN-gamma-induced activation of the ubiquitin-proteasome system. Our study shows that there is cross-talk between the tumour necrosis factor and IFN families of cytokines, through which IFN-gamma provides a negative link between T-cell activation and bone resorption. Our results may offer a therapeutic approach to treat the inflammation-induced tissue breakdown.

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1(-/-) mice) have been shown to exhibit a severe NK-cell deficiency. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1(-/-) bone-marrow cells can generate functional NK cells when cultured with the cytokine interleukin-15 and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

Depletion of both natural killer 1.1+ (NK1+) intermediate alpha beta T-cell receptor (int T) cells and NK cells by in vivo treatment with anti-NK1 antibody greatly increased hepatic metastases of intravenously injected EL4 cells as well as pulmonary metastases of 3LL cells in C57BL/6 mice. However, depletion of NK cells alone by anti-asialo GM1 (AGM1) antibody treatment did not increase the metastases in either organ. Interleukin-12 (IL-12) administration into mice induced strong cytotoxicities of NK cell-depleted liver and lung mononuclear cells (MNC) comparable to those without NK-cell depletion and inhibited metastases in either organ. In contrast, in both NK cell- and NK1+ int T-cell-depleted mice, IL-12 could not induce cytotoxic activity of liver and lung MNC and metastases in both organs increased with or without IL-12 treatment. These results confirmed the fact that NK+ int T cells are more potent antitumour effectors than NK cells against experimental haematogenous tumour metastases.

Eradication of a given pathogen is dependent on the selective differentiation of T helper (Th) cells into Th1 or Th2 types. We show here that T cells from mice lacking the transcription factor IRF-1 fail to mount Th1 responses and instead exclusively undergo Th2 differentiation in vitro. Compromised Th1 differentiation is found to be associated with defects in multiple cell types, namely impaired production of interleukin-12 by macrophages, hyporesponsiveness of CD4 + T cells to interleukin-12, and defective development of natural killer cells. These results indicate the involve- ment of IRF-1 in multiple stages of the Th1 limb of the immune response.

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.

A gene encoding a novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) was cloned, and its expression system was constructed in Escherichia coli. The 1.4-kilobase pair DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with KpnI and SmaI was found to express a protein binding FMN as a prosthetic group under control of the lac promoter in E. coli. This DNA fragment contained several putative open reading frames. The partial amino acid sequence of the polypeptide portion of the purified FMN-binding protein and its tryptic peptides were completely consistent with those deduced from the nucleotide sequence of the third open reading frame in the cloned SmaI-SmaI fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of FMN-binding protein indicated that the protein is composed of 122 amino acids including an initiator Met residue and lacks a signal peptide for secretion. The main redox potential of the FMN-binding protein was measured as -325 mV using direct current cyclic and differential pulse voltammetric techniques and an electroreflectance method, suggesting that this FMN-binding protein functions as a redox protein like other FMN-binding proteins. Immunoblot analysis of the whole proteins from D. vulgaris (Miyazaki F) clearly indicated that this protein is expressed in this bacteria. However, the protein was found to have a primary structure distinct from those of other FMN-binding proteins and to be the smallest FMN-binding protein yet reported.

NK cells are effector lymphocytes playing a critical role in the natural resistance against tumors. However, the precise mechanisms underlying NK cell-mediated natural resistance against tumor metastasis are still unrevealed. B16 cells, mouse melanoma cells, were resistant to freshly isolated NK cell-mediated killing; nevertheless, NK cells were critical for natural resistance against experimental lung metastasis of B16 cells. We found that lung metastasis was increased significantly in IFN-γ–/– mice but not pfp–/–, IFN-αR–/–, or IL-12/IL-18–/– mice. Interestingly, freshly isolated lung NK cells, but not spleen or liver NK cells, displayed augmented IFN-γ production after B16 inoculation. Adoptive transfer of pfp–/– NK cells, but not IFN-γ–/– NK cells, significantly decreased B16 lung metastasis in IFN-γ–/– and pfp/IFN-γ–/–mice. Lung metastases of IFN-γRDN B16 was also increased in NK cell-depleted or IFN-γ–/– mice, suggesting that the IFN-γ response of host cells was required in the NK cell and IFN-γ-mediated antimetastatic effect. Our results demonstrate that IFN-γ production from lung resident NK cells is a key response in the natural resistance to the experimental lung metastasis of NK cell-resistant tumor cells.

Human tumors frequently express membrane-bound or soluble NK group 2, member D (NKG2D) ligands. This results in chronic engagement of NKG2D on the surfaces of NK and CD8+ T cells and rapid internalization of the receptor. Although it is well appreciated that this phenomenon impairs NKG2D-dependent function, careful analysis of NKG2D-independent functions in cells chronically stimulated through NKG2D is lacking. Using a mouse model of chronic NKG2D ligand expression, we show that constant exposure to NKG2D ligands does not functionally impair NK cells and CD8+ T cells in the context of viral infection.