Liver iNKT Cells Research Articles

A pivotal role for antigen-presenting cells overexpressing SOCS3 in controlling invariant NKT cell responses during collagen-induced arthritis

ObjectiveSuppressor of cytokine signalling (SOCS) proteins constitute a class of intracellular proteins that are key physiological regulators of immune cell function. It has previously been shown that antigen-presenting cells (APCs)...

TCR-Dependent and -Independent Activation Underlie Liver-Specific Regulation of NKT Cells

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

BackgroundInvariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported.ResultsWe used an interferon (IFN)-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice.ConclusionsThis is the first report that supplies direct evidence for explicit activation events of NKT cells in vivo and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.

Plasticity of Invariant NKT Cell Regulation of Allergic Airway Disease Is Dependent on IFN-γ Production

Invariant NKT cells (iNKT cells) play a pivotal role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation. However, it is unclear what role they play in the initiation (sensitization) phase as opposed to the effector (challenge) phase. The role of iNKT cells during sensitization was examined by determining the response of mice to intratracheal transfer of OVA-pulsed or OVA-alpha-galactosylceramide (OVA/alphaGalCer)-pulsed bone marrow-derived dendritic cells (BMDCs) prior to allergen challenge. Wild-type (WT) recipients of OVA-BMDCs developed AHR, increased airway eosinophilia, and increased levels of Th2 cytokines in bronchoalveolar lavage fluid, whereas recipients of OVA/alphaGalCer BMDCs failed to do so. In contrast, transfer of these same OVA/alphaGalCer BMDCs into IFN-gamma-deficient (IFN-gamma(-/-)) mice enhanced the development of these lung allergic responses, which was reversed by exogenous IFN-gamma treatment following OVA-BMDC transfer. Further, Jalpha18-deficient recipients, which lack iNKT cells, developed the full spectrum of lung allergic responses following reconstitution with highly purified WT liver or spleen iNKT cells and transfer of OVA-BMDCs, whereas reconstituted recipients of OVA/alphaGalCer BMDCs failed to do so. Transfer of iNKT cells from IFN-gamma(-/-) mice restored the development of these responses in Jalpha18-deficient recipients following OVA-BMDC transfer; the responses were enhanced following OVA/alphaGalCer BMDC transfer. iNKT cells from these IFN-gamma(-/-) mice produced higher levels of IL-13 in vitro compared with WT iNKT cells. These data identify IFN-gamma as playing a critical role in dictating the consequences of iNKT cell activation in the initiation phase of the development of AHR and airway inflammation.

NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-γ and CXCR3 chemokines

AbstractIschemia-reperfusion injury (IRI) triggers an inflammatory cascade that is initiated by the activation of CD1d-restricted iNKT cells. In sickle cell disease (SCD), misshapen erythrocytes evoke repeated transient bouts of microvascular IRI. Compared with C57BL/6 controls, NY1DD mice have more numerous and activated (CD69+, interferon-γ+ [IFN-γ+]) lung, liver, and spleen iNKT cells that are hyperresponsive to hypoxia/reoxygenation. NY1DD mice have increased pulmonary levels of IFN-γ, IFN-γ–inducible chemokines (CXCL9, CXCL10), and elevated numbers of lymphocytes expressing the chemokine receptor CXCR3. Treating NY1DD mice with anti-CD1d antibody to inhibit iNKT cell activation reverses baseline pulmonary dysfunction manifested as elevated vascular permeability, decreased arterial oxygen saturation, and increased numbers of activated leukocytes. Anti-CD1d antibodies decrease pulmonary levels of IFN-γ and CXCR3 chemokines. Neutralization of CXCR3 receptors ameliorates pulmonary dysfunction. Crossing NY1DD to lymphocyte-deficient Rag1−/− mice decreases pulmonary dysfunction. This is counteracted by the adoptive transfer of 1 million NKT cells. Like mice, people with SCD have increased numbers of activated circulating iNKT cells expressing CXCR3. Together, these data indicate that iNKT cells play a pivotal role in sustaining inflammation in SCD mice by a pathway involving IFN-γ and production of chemotactic CXCR3 chemokines and that this mechanism may translate to human disease.

Role for hedgehog pathway in regulating growth and function of invariant NKT cells

Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of NKT cells, which comprise a substantial sub-population of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of pro-fibrogenic iNKT cell populations during certain types of fibrosing liver damage.

Enrichment of Human CD4+ Vα24/Vβ11 Invariant NKT Cells in Intrahepatic Malignant Tumors

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

Acute inflammation with IL-18/IL-12 or α-GalCer treatment induces liver iNKT cell apoptosis and repopulation from peripheral tissues, whereas chronic inflammation ablates systemic iNKT cells with thymic-dependent repopulation (134.24)

Abstract We previously showed that IL-18/IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells. We now demonstrate that acute treatment with IL-18/IL-12 initially decreased the number of NK1.1(+) iNKT cells while α-GalCer decreased both NK1.1(+) and NK1.1(-) iNKT cells in the liver. Genomic qPCR analysis of iNKT cell's restricted Vα14Jα18 TCR confirmed the loss of cells at 24hrs in the liver rather than receptor down modulation. A small or no decrease in the genomic levels of Vα14Jα18 from other lymphoid tissues indicates the loss of iNKT cells from IL-18/IL-12 or α-GalCer treatment of mice was regulated by the liver microenvironment. Furthermore, an increase in AnnexinV was detected on liver iNKT cells following treatment with IL-18/IL-12 and an increase in cells that were tunnel positive was observed in the liver following α-GalCer treatment of mice. Although iNKT cells initially (24hrs) decrease in the liver following acute administration of α-GalCer or IL-18/IL-12, these cells subsequently (72hrs) reappear and expand in the liver. To study the expansion of iNKT cells in the liver, we administered BrdU to mice 24hrs after acute treatment with IL-18/IL-12 or α-GalCer and found a high percentage of proliferating liver iNKT cells were recent emigrating NK1.1(-) iNKT cells.

NKT activation together with blockade of PD-L1 pathway lead to enhanced tumor responses (40.32)

Abstract Invariant NKT cells (iNKT cells) are a unique population of lymphocytes that have a biased TCR gene expression, are restriction by CD1d molecules, and rapidly produce a spectrum of cytokines following activation. Various studies have shown that positive costimulatory pathways can influence the activation of iNKT cells. However, little is understood about the regulation of NKT cells by negative costimulatory pathways. Here we show that PD-L1 and PD-1. but not PD-L2, are expressed on iNKT cells in spleen, liver and thymus. In vivo activation with α-GalCer lead to increased cytokine production and expansion of iNKT cells in the absence of the PD-L1 pathway. To study whether PD-L1 deficiency on NKT cells would affect antigen specific T cell responses we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 T cells after adoptive transfer into wild type recipients. However, this expansion was significantly augmented when OT-1 T cells were adoptively transferred into PD-L1-/- recipients. In a B16 melanoma metastasis model, there was an increased clearance of lung nodules when PD-L1-/- recipients were given dendritic cells loaded with α-GalCer and TRP-2 peptide. These data demonstrate that blocking PDL1:PD-1 interactions between NKT cells and cells of the adaptive immune system are important in mounting an effective anti-tumor immune response.

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably α-galactosylceramide (α-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver α-GalCer/CD1d tetramer-reactive (α-GalCer/CD1d +) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas α-GalCer/CD1d +NK1.1 + T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.

Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection

The invariant (i) natural killer (NK)T cells consistently express the Vα14 chain of the T cell receptor (TCR) and recognize α-galactosylceramide (α-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1 − iNKT cell population contracted, whereas NK1.1 + iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following α-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1 − iNKT cells from NK1.1 + cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.

4 SPLF Étude de l’implication des cellules inkt dans l’asthme allergique expérimental : mise au point de nouveaux protocoles thérapeutiques

The invariant (i) natural killer (NK)T cells consistently express the Vα14 chain of the T cell receptor (TCR) and recognize α-galactosylceramide (α-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1− iNKT cell population contracted, whereas NK1.1+ iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following α-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1− iNKT cells from NK1.1+ cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.