Activation Of NKT Cells Research Articles

Hypoxia-Inducible Factor-2α Limits Natural Killer T Cell Cytotoxicity in Renal Ischemia/Reperfusion Injury.

Natural killer T (NKT) cells are the major early-acting immune cell type and fundamental immune modulators in ischemia-reperfusion injury (IRI). Because lymphocytes are exposed to various oxygen tensions under pathophysiologic conditions, we hypothesize that hypoxia-inducible factors (HIFs) have roles in NKT cell activation, and thus determine the final outcome of renal IRI. In this study, we used Lck-Cre transgenic mice to specifically disrupt HIF-2α in T/NKT cells and found that HIF-2α knockout led to upregulated Fas ligand expression on peripheral NKT cells, but not on conventional T cells. HIF-2α knockout promoted infiltration of NKT cells into ischemic kidneys and exacerbated IRI, which could be mitigated by in vivo NK1.1(+) cell depletion or Fas ligand blockade. Compared with wild-type NKT cells, HIF-2α(-/-) NKT cells adoptively transferred to Rag1-knockout mice elicited more severe renal injury, and these mice were not protected by CGS21680, an adenosine A2A receptor agonist. Mechanistically, hypoxia-induced expression of adenosine A2A receptor in NKT cells and CGS21680-induced cAMP production in thymocytes were HIF-2α-dependent. Hydrogen peroxide-induced Fas ligand expression on thymic wild-type NKT cells was significantly attenuated by CGS21680 treatment, but this effect was lost in HIF-2α(-/-) NKT cells. Finally, CGS21680 and LPS, an inducer of HIF-2α in endothelium, synergistically reduced renal IRI substantially, but this effect was absent in Mx1-Cre-induced global HIF-2α-knockout mice. Taken together, our results reveal a hypoxia/HIF-2α/adenosine A2A receptor axis that restricts NKT cell activation when confronted with oxidative stress and thus protects against renal IRI.

Border patrol gone awry: lung NKT cell activation by F. tularensis exacerbates tularemia-like disease (MUC1P.908)

Abstract The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning for border patrol, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia—because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain results in rapid recruitment of NKT cells to the lung interstitium. Interstitial NKT cell activation resulted in protective interferon-γ production, and affected both local and systemic proinflammatory responses. Hence, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet survived the infection whilst their wild type counterparts did not. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F tularensis infection hampers iBALT formation, which in conjunction with NKT cell-dependent proinflammatory response causes severe pulmonary tularemia-like disease in mice.

NKT cell-mediated enhancement of humoral immunity against Clostridium difficile toxin B (IRC10P.406)

Abstract To develop more effective antibody-stimulating vaccines, we must improve our understanding of the mechanisms by which vaccines induce and maintain long-lasting humoral immunity. This is important for the development of new vaccines against emerging pathogens such as C. difficile. It is well-established that Natural Killer T (NKT) cells, activated with the glycolipid, α-galactosylceramide (α-GC) enhance humoral immunity, but the mechanisms by which they do so are poorly understood. Here we show that activated NKT cells enhance the antigen-specific IgG1 response and survival against C. difficile Toxin B (TcdB). We and others have shown that NKT cells may enhance priming of T helper (Th) cells. We therefore hypothesized that T helper and NKT subsets coordinate to enhance the humoral immune response against C. difficile. Flow cytometry analysis demonstrated that NKT cell activation with a-GC did not alter numbers of Th or follicular T helper (Tfh) cells, but increased the number of NKTfh cells. ELISPOT assays indicated that the α-GC enhanced secretion of IL-4 and IL-21 is mainly Th-derived. Adoptive transfer experiments show that α-GC did not alter the ability of Th cells to boost antibody production. This suggests that NKT cells may exert direct and indirect effects (via Th cells) on humoral immunity. Understanding the mechanisms by which NKT cells enhance humoral immunity will help in designing strategies to appropriately activate NKT cells and boost vaccine efficacy.

512. Tumor-Associated Macrophages Via Up-Regulation of PD1 Ligands Protect Neuroblastoma from Immunotherapy With NKT Cells Expressing GD2-Specific Chimeric Antigen Receptor

Immunotherapy with Natural Killer T cells engineered to express a GD2-specific chimeric antigen receptor (CAR.GD2 NKTs) demonstrated potent anti-tumor activity in a metastatic model of neuroblastoma in humanized (hu)NSG mice. However, tumors eventually progressed without the loss of GD2 expression suggesting that tumor escape is due to inhibition of effector function of CAR. GD2 NKTs. Since NKTs co-localize and specifically interact with M2-like tumor-associated macrophages (TAMs), we examined whether TAMs inhibit effector functions of NKT and CAR.GD2 NKT cells. In vitro experiments revealed a reciprocal inhibition of NKTs and M2-polarized macrophages. We found that NKTs selectively kill M2-polarized macrophages or induce their differentiation toward M1-like cells. However, both M2- and M1-macrophages suppressed NKT-cell proliferation and IFNγ production in response to TCR or CAR stimulation. This was associated with rapid up-regulation of PD-L1 and PD-L2 on both M2 and M1 macrophages upon addition of supernatant from activated NKT cells. Moreover, M1 macrophages constitutively expressed PD-L1 and were resistant to NKT-cell cytotoxicity. The analysis of tumor tissues in hu-NSG mice revealed strong up-regulation of PD-L1 expression exclusively on TAMs and not on neuroblastoma cells after treatment with CAR. GD2 NKTs. Neither neuroblastoma cells nor TAMs expressed PD1 ligands in untreated mice or in primary NB tissues from patients with stage 4 disease. Importantly, a combination of CAR.GD2 NKT cell immunotherapy with anti-PD1 blocking mAb produced durable tumor regression. The results provide a rationale for a combined use of CAR-redirected NKT cells with PD1-blocking therapeutics for immunotherapy of neuroblastoma and possibly other solid tumors.

Mantle cell lymphoma associated sphingosine-1 phosphate inhibits natural killer T cell mediated antitumor responses (TUM2P.1042)

Abstract The incidence of lymphoma in the US is rising. While chemoimmunotherapy causes remissions in mantle cell lymphoma (MCL), responses are not durable and most patients relapse. New strategies that focus on eliminating tumor-associated immunosuppressive factors are needed to improve patient outcomes. One of the earliest pathways to be activated during an immune response is CD1d-mediated presentation of lipid antigens to natural killer T (NKT) cells. We have found that MCL patients have a significant reduction in circulating NKT cell number and function. We hypothesized that MCL-associated sphingosine-1 phosphate (S1P) can down regulate CD1d-dependent NKT cell responses to MCL. We found that inhibition of S1P signaling in MCL cell lines led to increased CD1d-mediated NKT cell activation, as assessed by cytokine production and cytotoxicity. Furthermore, knockdown of sphingosine kinase 1 in MCL cell lines resulted in the presentation of an activating endogenous antigen. These results demonstrate that S1P regulates CD1d-mediated antigen processing and presentation to NKT cells by altering the endogenous repertoire of lipid antigens and suggest that targeting this pathway could be an important immunotherapeutic strategy.

The inhibition of CD1d-mediated antigen presentation upon an HSV-1 infection is VP22-dependent (VIR1P.1134)

Abstract CD1d-restricted T (NKT) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to down-regulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell’s cytoskeletal network and viral spreading. We have shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared to wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. Of importance, the effects of VP22 were shown to be exerted on the T322/S323 dyad, critical residues for the virus to reduced antigen presentation. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not ΔUL49 virus. Rather than inhibiting antigen presentation by CD1d, ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Therefore, VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

TLR stimulation of type II NKT cells requires cell to cell contact with DCs with involvement of CD40-CD40L and IL-12p40 (INC6P.318)

Abstract Natural killer T (NKT) cells are CD1d restricted innate like lymphocytes recognizing lipid antigens and mediating immune regulatory functions. NKT cells are divided into two subsets based on the type of T cell receptor (TCR) expressed. Type 1 or invariant NKT cells uses an invariant TCRα-chain (Vα14-Jα18 in mice/Vα24-Jα18 in humans) and type II or diverse NKT cells are all non Vα14 expressing NKT cells. Most of the knowledge about NKT cells have been derived from type I NKT cells, while type II NKT cells are less well explored. Here, we investigated the signals driving Toll-like receptor (TLR) ligand activation of type II NKT cells. We use the 24αβ mouse model expressing a transgenic Vα3.2 and Vβ9 type II NKT cell TCR on the C57/BL6 background. Using in vitro co-culture experiments with 24αβ type II NKT cells and FLT3L derived bone marrow dendritic cells (BMDCs) and TLR ligands, we have analyzed the factors required for type II NKT cell activation, applying BMDCs from different knock out mice. TLR stimulated DCs potently activated type II NKT cells to secrete IFN-γ in a CD1d and type I IFN independent manner. In contrast, an absolute requirement of IL12p40 and partial dependence on CD40, IL15 and IL18 for optimal IFN-γ induction was observed. Thus, our data suggest that TLR ligand - BMDC mediated activation of type II NKT cells requires cell - cell contact that includes CD40 - CD40L ligation, IL15, IL18 and IL-12, but not CD1d - TCR interaction.

Activated NKT cells imprint NK-cell differentiation, functionality and education.

NKcells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NKcells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross-talk between NKT cells and NKcells needs to be better understood. Our results provide strong evidence for NKT cell-induced effects on key biological features of NKcells. NKT-cell activation results in the generation of highly active CD27(high) NKcells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT-cell stimulation induced the generation of highly functional educated and uneducated NKcells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT-NKcell axis that provide important hints for the manipulation of NKcells in clinical settings.

Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

IL32γ activates natural killer receptor-expressing innate immune cells to produce IFNγ via dendritic cell-derived IL12

The inflammatory cytokine IL32γ acts on dendritic cells (DCs) to produce IL12 and IL6, which are involved in the differentiation of Th1 and Th17 cells. Natural killer (NK) and NKT cells play important roles in IL12-mediated adaptive immune responses, such as antitumor immunity. Herein we demonstrate the effect of IL32γ on the activation of NK and NKT cells. Upon IL32γ stimulation, splenic NK and NKT cells could be activated, and this activation was dependent on both IL12 and DCs, which was confirmed by using IL12p35 knockout and CD11c-diphtheria toxin receptor transgenic mouse models. Furthermore, IL32γ could induce the production of proinflammatory cytokines by NKDCs, a subset of DCs expressing NK cell markers, known to enhance NKT cell function. Unlike conventional DCs, NKDCs produced IFNγ and TNFα rather than IL12 upon stimulation with IL32γ. Taken together, IL32γ will be useful as an adjuvant to boost the cytotoxicities of NK and NKT cells that play critical roles in antitumor immunity.

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

Natural killer T cell activation overcomes immunosuppression to enhance clearance of postsurgical breast cancer metastasis in mice

Metastatic lesions are responsible for over 90% of breast cancer associated deaths. Therefore, strategies that target metastasis are of particular interest. This study examined the efficacy of natural killer T (NKT) cell activation as a post-surgical immunotherapy in a mouse model of metastatic breast cancer. Following surgical resection of orthotopic 4T1 mammary carcinoma tumors, BALB/c mice were treated with NKT cell activating glycolipid antigens (α-GalCer, α-C-GalCer or OCH) or α-GalCer-loaded dendritic cells (DCs). Low doses of glycolipids transiently reduced metastasis but did not increase survival. A high dose of α-GalCer enhanced overall survival, but was associated with increased toxicity and mortality at early time points. Treatment with α-GalCer-loaded DCs limited tumor metastasis, prolonged survival, and provided curative outcomes in ∼45% of mice. However, survival was not increased further by additional DC treatments or co-transfer of expanded NKT cells. NKT cell activation via glycolipid-loaded DCs decreased the frequency and immunosuppressive activity of myeloid derived suppressor cells (MDSCs) in tumor-resected mice. In vitro, NKT cells were resistant to the immunosuppressive effects of MDSCs and were able to reverse the inhibitory effects of MDSCs on T cell proliferation. NKT cell activation enhanced antitumor immunity in tumor-resected mice, increasing 4T1-specific cytotoxic responses and IFNγ production from natural killer (NK) cells and CD8+ T cells. Consistent with increased tumor immunity, mice surviving to day 150 were resistant to a second tumor challenge. This work provides a clear rationale for manipulating NKT cells to target metastatic disease.

NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

NKT cells are a subgroup of T cells, which express a restricted TCR repertoire and are critical for the innate immune responses to viral infections. Activation of NKT cells depends on the major histocompatibility complex-related molecule CD1d, which presents bioactive lipids to NKT cells. The marine sponge derived lipid αGalCer has recently been demonstrated as a specific agonist for activation of human and murine NKT cells. In the present study we investigated the applicability of αGalCer pre-treatment for immune protection against intra-vaginal HSV-2 infection. We found that C57BL/6 WT mice that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells in vaginal tissue and elevated levels of IFN-γ in the vaginal tissue and in vaginal fluids 24h after αGalCer pre-treatment. Collectively our data demonstrate a protective effect of αGalCer induced activation of NKT cells in the innate immune protection against viral infection.

Maintenance therapy with the TLR-9 agonist MGN1703 in the phase II IMPACT study of metastatic colorectal cancer patients: A subgroup with improved overall survival.

680 Background: Patients with mCRC and disease control after induction with first-line chemotherapy +/- bevacizumab were included in the double-blind placebo-controlled phase II IMPACT trial, aiming to assess the clinical efficacy, safety, and immunological effects of the immunomodulator MGN1703, a potent TLR9 agonist, given at the dose of 60 mg subcutaneously twice weekly as switch maintenance after first line induction therapy in mCRC. Methods: The trial was prematurely closed after randomization of 59 patients. The final analysis showed a superior effect of MGN1703 compared to placebo with hazard ratios (HR) for the primary endpoint PFS on maintenance of 0.55 (p=0.041) and 0.56 (p=0.070) by local investigator assessment or independent radiological review, respectively. Exploratory PFS analyses of pretreatment characteristics identified patients with normalized CEA, objective response, and the presence of activated NKT cells at the end of induction chemotherapy to benefit the most from maintenance with MGN1703. We analysed the impact of these factors on the secondary endpoint overall survival (OS). Results: At time of final study analysis, OS data were not mature. Only 35% of MGN1703 patients had an event, as opposed to 50% of placebo patients. The HR for OS of whole study population was 0.63 (median 22.6 vs. 15.1 months, p=NS). The subgroup of patients randomized into the study with confirmed RECIST response had a HR of 0.40 (median 24.5 vs. 15.1 months, p=NS), suggesting this may be the population with greater benefit. HR for patients with normalized CEA or with activated NKT cells were 0.69 and 0.43, respectively. Based on this evidence, in the phase III IMPALA study patients with objective response after standard induction therapy are randomized to standard treatment or switch maintenance with MGN1703. CEA and activated NKT cells are stratification factors for the study and will be prospectively assessed. Conclusions: The pretreatment characteristics predictive of a PFS benefit in the IMPACT study seem to retain their value also in exploratory analyses for OS. This information has been used to design the phase III IMPALA study, currently recruiting patients. Clinical trial information: NCT01208194.

Mesenchymal stem cells: a friend or foe in immune-mediated diseases.

Mesenchymal stem cells (MSCs) are adult, self-renewable, multipotent cells that can be found in almost all postnatal tissues. Because of their capacity for self-renewal and differentiation into tissues of mesodermal origin and due to their immunomodulatory ability, MSCs are used in many preclinical and clinical studies as possible new therapeutic agents for the autoimmune or degenerative diseases treatment. In dependence of inflammatory environment to which they are exposed to, MSCs adopt immunosuppressive or pro-inflammatory phenotype. In the presence of high levels of pro-inflammatory cytokines or through activation of Toll-like receptor (TLR)-3, MSCs adopt an immune-suppressive phenotype and suppress the proliferation, activation and effector function of professional antigen presenting cells (dendritic cells, macrophages, B lymphocytes), T lymphocytes, NK cells, NKT cells, and neutrophils. During the early phase of inflammation, through TLR4 activation and in the presence of low levels of inflammatory cytokines, MSCs adopt a pro-inflammatory phenotype, promote neutrophil and T cell activation and enhance immune response. Here we review the current findings on the immunoregulatory plasticity of MSCs involved in regulation of immune response.

Mucosal Immune Responses Associated with NKT Cell Activation and Dendritic Cell Expansion by Nasal administration of α-galactosylceramide in the Nasopharynx

Objective: Intranasal immunization is an effective method to induce mucosal immune responses in the upper respiratory tract.α-galactosylceramide (α-GalCer) is considered as one of the most potent candidates for a mucosal adjuvant. In the present study, mucosal immune responses in the nasopharynx associated with NKT cell activation by nasal administration of α-GalCer were examined for inducing protective immunity in the nasopharynx, with the ultimate goal of developing a mucosal vaccine for preventing upper respiratory infectious diseases. Methods: Mice were administered α-GalCer intranasally as a ligand for NKT cells, without any antigen, weekly total three times. One week after the final administration of α-GalCer, mice were killed, and nasal immune responses were examined. Dendritic cells (DCs) in nasal-associated lymphoid tissue (NALT), a mucosal inductive site, were examined by immunohistochemistry. DCs, NKT cells, and B cells in NALT and nasal passage (NP) were examined by flow cytometry. Cytokine-producing CD4+ T cells were also examined by flow cytometry. Quantification of immunoglobulin (Ig)-producing cells was examined by enzyme-linked immunospot (ELISPOT) assay. In addition, bacterial challenges with live Haemophilus influenzae (Hi) and Streptococcus pneumoniae (Sp) were performed, and bacterial clearance from nasopharynx was examined. Results: After nasal immunization of α -GalCer, DCs increased in NALT. Antibody-producing cells, mainly those that produce IgA, significantly increased in the NP, a mucosal effector site. Interleukin (IL)-17-producing Th 17 cells were also induced in the NP. Bacterial challenges with live Hi and Sp resulted in enhanced clearances of both bacterial species from the nasopharynx. It is interesting that bacterial clearance was impaired by IL-17 neutralization. Conclusion: Nasal vaccination is effective for the induction of protective immunity against upper respiratory infection. The results of the present study demonstrated that nasal administration of α-GalCer could activate NKT cells in the nasopharynx, followed by the maturation of DCs, B cells, and some cytokine-producing CD4+ T cells. These findings suggest that the activation of NKT cells by nasal administration of α-GalCer induced protective immunity in the nasopharynx, possibly involving the interaction with DCs and induction of Th17 cells.

NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production.

Background. Natural killer (NK) and natural killer T (NKT) cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 105 cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham); pretreated with isotype control antibody (CON) group; pretreated with anti-asialo GM1 antibody (NKd) group; and pretreated with anti-CD1d monoclonal antibody (NKTd) group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3−/NK1.1+) cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3−/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

Protein S exacerbates alcoholic hepatitis by stimulating liver natural killer T cells

Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown. This study investigated the role of PS in acute alcoholic hepatitis. A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol. The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls. The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells.

Highly purified mycobacterial phosphatidylinositol mannosides drive cell-mediated responses and activate NKT cells in cattle.

Mycobacterial lipids play an important role in the modulation of the immune response upon contact with the host. Using novel methods, we have isolated highly purified phosphatidylinositol mannoside (PIM) molecules (phosphatidylinositol dimannoside [PIM2], acylphosphatidylinositol dimannoside [AcPIM2], diacyl-phosphatidylinositol dimannoside [Ac2PIM2], acylphosphatidylinositol hexamannoside [AcPIM6], and diacylphosphatidylinositol hexamannoside [Ac2PIM6]) from virulent Mycobacterium tuberculosis to assess their potential to stimulate peripheral blood mononuclear cell (PBMC) responses in Mycobacterium bovis-infected cattle. Of these molecules, one (AcPIM6) induced significant levels of gamma interferon (IFN-γ) in bovine PBMCs. Three PIM molecules (AcPIM6, Ac2PIM2, and Ac2PIM6) were shown to drive significant proliferation in bovine PBMCs. AcPIM6 was subsequently used to phenotype the proliferating cells by flow cytometry. This analysis demonstrated that AcPIM6 was predominantly recognized by CD3(+) CD335(+) NKT cells. In conclusion, we have identified PIM lipid molecules that interact with bovine lymphocyte populations, and these lipids may be useful as future subunit vaccines or diagnostic reagents. Further, these data demonstrate, for the first time, lipid-specific NKT activation in cattle.

Identification of Previously Unrecognized CD1d-Restricted Peripheral T Cell Lymphomas (PTCLs) in Mouse and Human Reveals Blocking Anti-CD1d Monoclonal Antibodies As a New Therapeutic Possibility in PTCLs

Background. Peripheral T-cell lymphomas (PTCLs) originate from post-thymic T cells but compared to B-cell lymphomas the exact cell of origin is usually unknown except for angioimmunoblastic T-cell lymphoma arising from a follicular helper T-cell. Furthermore, no recurrent cytogenetic or molecular abnormalities are identified in PTCLs. Recently, recurrent impairment of the p53 pathway has been pointed out in PTCLs. However, p53 knockout (KO) mice are known to develop immature thymic T-cell lymphomas and solid tumors but surprisingly PTCLs have not been reported for more than 20 years in those mice.NKT cells are a T-cell subset responsive to glycolipids presented by CD1d, a major histocompatibility complex (MHC) class I-like antigen-presenting molecule, in contrast to conventional T cells, which recognize peptide antigens. Two types of NKT cells have been described so far: type I or invariant NKT cells (iNKT) that express a Valpha14-Jalpha18 (in mice) or Valpha24-Jalpha18 (in humans) constant chain and type II NKT cells that express a variable TCR but are CD1d-dependent as well. Most type II NKT cells are of alpha/beta phenotype but CD1d-restricted gamma/delta T cells have also been described in mice and humans.Methods. The development of PTCLs in p53 KO mice (B6.129S2-Trp53tm1Tyj/J) was studied. Identification of PTCLs was made by immunohistochemistry and flow cytometry analysis. Gene expression profile analysis (GeneChip Mouse Genome 430 2.0 array, Affymetrix) was performed to characterize lymphomas developed in the mouse model. Transfer experiments were done by intravenously retro-orbital injection into syngeneic, immunocompetent C57Bl/6J WT animals or immunocompromised CD3ε-/- mice. Therapeutic trials in mice were performed with the use of blocking anti-CD1d monoclonal antibodies (mAb) (clone HB323; BioXcell).Results. We found that p53 KO mice developed well-known and characterized thymic T-cell lymphomas and solid tumors as previously described. However, about 20% of p53 KO mice spontaneously developed a previously unrecognized entity of PTCLs originating from CD1d-restricted iNKT cells (ie type I NKT cells) referred to as NKTLs for NKT lymphomas thereafter. Both alpha-galactosylceramide-CD1d tetramer staining and unique Valpha14-Jalpha18 TCR rearrangement confirmed the iNKT nature of these lymphomas.Chronic injection of Streptococcus pneumoniae (Spn), reported to express glycolipid antigens activating NKT cells, significantly increased the incidence of these NKTLs compared to a control group of p53 KO mice injected with PBS (P=0.03). Gene expression profile analysis indicated a significant down-regulation of genes in the TCR signaling pathway of NKTLs (false discovery rate q-value=0.01 by gene set enrichment analysis) suggesting an underlying antigenic chronic stimulation as previously reported in chronically activated T cells (Figure 1). Moreover, NKTLs were characterized by upregulation of PD-1 and loss of NK1.1 expression compared to resting NKT cells (P<0.01 for both), which are features of activated and anergic iNKT cells. Altogether, those data indicate that NKTLs in mice could arise from chronically activated iNKT cells by endogenous or exogenous glycolipids. Furthermore, in vivo TCR/CD1d interactions were required for NKTLs survival after transfer in recipient mice and the use of blocking anti-CD1d mAb significantly prolonged mice overall survival (logrank P<0.001, Figure 2).We did not identify human PTCLs arising from type I iNKT cells by using alphaGalCer-CD1d tetramer staining. However, using sulfatide-loaded CD1d tetramers (ie another type of glycolipid-CD1d tetramer identifying type II NKT cells), we identified CD1d-restricted human PTCLs among gamma/delta hepatosplenic T-cell lymphomas (HSTLs) and PTCL-not otherwise specified (PTCL-NOS) expressing the Vd1 TCR but not the Vd2 TCR (Figure 3).Conclusion. This demonstrates for the first time the existence of human PTCLs arising from gamma/delta CD1d-restricted type II NKT cells. These results refine the classification of PTCLs in humans by identifying a new cell of origin and pave the way for the development of blocking anti-CD1d antibodies for therapeutic purposes. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.