Michel Dy

Cutting Edge: Invariant Vα14 NKT Cells Are Required for Allergen-Induced Airway Inflammation and Hyperreactivity in an Experimental Asthma Model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jα18−/− mice, which are exclusively deficient in the invariant Vα14+ (iVα14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iVα14 NKT cells fully reconstitutes the capacity of Jα18−/− mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69+) iVα14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iVα14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iVα14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.

Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells.

NKT cells are a remarkably versatile population whose functional capacities are determined by cytokines present in their microenvironment. In this study, we provide evidence for a new immunoregulatory effect of the proinflammatory cytokine IL-18 on NKT cells. We found that IL-18, mainly known for its involvement in NK cell activation and in Th 1 immune responses, substantially enhanced IL-4 production as well as the percentage of IL-4+ cells among NKT lymphocytes activated by their specific ligand α-galactosylceramide (α-GalCer). The effect of IL-18 on IL-4 production by activated NKT cells took place both in vivo and in vitro and was not affected by IL-12 which increased IFN-γ secretion in the same conditions. We show that NKT cells are the main targets for IL-18-induced IL-4 production since it occurred neither in NKT-deficient mice nor after stimulation of Th2 lymphocytes. Finally, we provide evidence that the IL-4 promptly generated by NKT cells in response to IL-18 plus α-galactosylceramide in vivo can effectively contribute to the adaptive Th2 immune response by up-regulating the early activation marker CD69 on B cells. Our data support the notion that, in contrast to the exclusive IFN-γ inducer IL-12, IL-18 acts in a more subtle manner as a costimulatory factor in both pro-Th1 and pro-Th2 responses depending on the nature of the stimulation and the target cells.

Fas/Fas Ligand Interactions Promote Activation-Induced Cell Death of NK T Lymphocytes

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-γ production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, α-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.

α‐Galactosylceramide‐induced iNKT cells suppress experimental allergic asthma in sensitized mice: Role of IFN‐γ

Allergic asthma is a multifaceted syndrome consisting of eosinophil‐rich airway inflammation, bronchospasm, and airway hyper‐responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of α‐galactosylceramide (α‐GalCer), 1 h before the first airway allergen challenge of OVA‐sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL‐4 and IL‐5 production in bronchoalveolar lavage fluid, and specific anti‐OVA IgE antibodies. Further, α‐GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. α‐GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro‐asthmatic IL‐4 to a protective IFN‐γ production. The role of IFN‐γ from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA‐sensitized and α‐GalCer‐treated mice which protected immunized recipients from manifesting asthma by an IFN‐γ‐dependent pathway. Our findings demonstrate for the first time that α‐GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell‐ and IFN‐γ‐dependent pathway.

IL‐4‐producing NK T cells are biased towards IFN‐γ production by IL‐12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL‐4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL‐4 or IFN‐γ. In agreement with our previous reports showing that their IL‐4‐producing capacity is strikingly dependent on IL‐7, CD4− CD8− TCRα β+ NK T lymphocytes, obtained after expansion with IL‐1 plus granulocyte‐macrophage colony‐stimulating factor, produced almost undetectable amounts of IL‐4 or IFN‐γ in response to TCR/CD3 cross‐linking. However, the capacity of these T cells to produce IFN‐γ is strikingly enhanced when IL‐12 is added either during their expansion or the anti‐CD3 stimulation, while IL‐4 secretion is always absent. A similar effect of IL‐12 on IFN‐γ production was observed when NK T lymphocytes were obtained after expansion with IL‐7. It is noteworthy that whatever cytokines are used for their expansion, IL‐12 stimulation, in the absence of TCR/CD3 cross‐linking, promotes consistent IFN‐γ secretion by NK T cells without detectable IL‐4 production. Experiments in vivo demonstrated a significant up‐regulation of the capacity of NK T cells to produce IFN‐γ after anti‐CD3 mAb injection when mice were previously treated with IL‐12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.

Recruitment of STAT3 for Production of IL-10 by Colon Carcinoma Cells Induced by Macrophage-Derived IL-6

The immunosuppressive cytokine IL-10 is associated with poor prognosis in colon cancer. Although macrophages are involved in antitumor defenses, production of IL-10 by tumor cells may permit malignant cells escape to cell-mediated immune defenses. To investigate interactions between macrophages and tumor cells in humans, we cultured macrophages isolated from patients and tested the effect of these macrophages on the production of IL-10 by several tumor cell lines. Macrophages were isolated from pleural effusions of patients with malignancy and from noncancer control patients. We demonstrated that culture supernatants of macrophages from both sources strongly stimulated IL-10 production by the three different human colon adenocarcinoma cell lines, Colo 205, Colo 320, and HT29. Recombinant IL-6, but not IL-10, TNF-α, and IFN-α, stimulated the secretion of IL-10 by colon tumor cells. mAbs against IL-6 and IL-6R prevented the effect of macrophage culture supernatants and of rIL-6, respectively, on the production of IL-10 by the three cell lines. Cocultures of macrophages and colon cancer cells showed that these tumor cells first stimulated macrophages to produce IL-6, which was then followed by IL-6-induced IL-10 production by colon cancer cells. Finally, we showed that IL-10 gene regulation was mediated by STAT3, which was phosphorylated after the binding of IL-6 to IL-6R. This is the first demonstration that IL-6, secreted by macrophages, can induce a STAT3-mediated IL-10 production by colon tumor cells.

Proinflammatory Environment Dictates the IL-17–Producing Capacity of Human Invariant NKT Cells

CD1d-reactive invariant NKT (iNKT) cells have been implicated in a number of experimental models of human pathologies. Given the scope of their immunoregulatory activities mediated through distinct cytokine patterns, it has been proposed that this functional diversity originates from distinct iNKT subpopulations. In this study, we report that human CD161+ iNKT cells are intrinsically endowed with the capacity to generate IL-17, but require TGF-β, IL-1β, and IL-23 to carry out this potential. IL-17–producing iNKT cells are already present in cord blood but, in contrast to peripheral blood iNKT cells, they cannot generate IFN-γ. These IL-17 producers respond to aryl hydrocarbon receptor stimulation and express IL-23 receptor and retinoic acid-related orphan receptor C, similar to conventional T helper 17 cells, from which they differ by their restricted ability to coproduce IL-22. In conclusion, IL-17 production by human iNKT cells depends on two critical parameters, namely an intrinsic program and a proinflammatory environment.

Early posttransplantation donor-derived invariant natural killer T-cell recovery predicts the occurrence of acute graft-versus-host disease and overall survival

Invariant natural killer T (iNKT) cells can experimentally dissociate GVL from graft-versus-host-disease (GVHD). Their role in human conventional allogeneic hematopoietic stem cell transplantation (HSCT) is unknown. Here, we analyzed the post-HSCT recovery of iNKT cells in 71 adult allografted patients. Results were compared with conventional T- and NK-cell recovery and correlated to the occurrence of GVHD, relapse, and survival. We observed that posttransplantation iNKT cells, likely of donor origin, recovered independently of T and NK cells in the first 90 days after HSCT and reached greater levels in recipient younger than 45 years (P = .003) and after a reduced-intensity conditioning regimen (P = .03). Low posttransplantation iNKT/T ratios (ie, < 10(-3)) were an independent factor associated with the occurrence of acute GVHD (aGVHD; P = .001). Inversely, reaching iNKT/T ratios > 10(-3) before day 90 was associated with reduced nonrelapse mortality (P = .009) without increased risk of relapse and appeared as an independent predictive factor of an improved overall survival (P = .028). Furthermore, an iNKT/T ratio on day 15 > 0.58 × 10(-3) was associated with a 94% risk reduction of aGVHD. These findings provide a proof of concept that early postallogeneic HSCT iNKT cell recovery can predict the occurrence of aGVHD and an improved overall survival.

Cutting Edge: Histamine Receptor H4 Activation Positively Regulates In Vivo IL-4 and IFN-γ Production by Invariant NKT Cells

Histamine (HA) is a biogenic amine with multiple activities in the immune system. In this study we demonstrate that histamine-free histidine decarboxylase-deficient (HDC−/−) mice present a numerical and functional deficit in invariant NK T (iNKT) cells as evidenced by a drastic decrease of IL-4 and IFN-γ production. This deficiency was established both by measuring cytokine levels in the serum and intracellularly among gated iNKT cells. It resulted from the lack of HA, because a single injection of this amine into HDC−/− mice sufficed to restore normal IL-4 and IFN-γ production. HA-induced functional recovery was mediated mainly through the H4 histamine receptor (H4R), as assessed by its abrogation after a single injection of a selective H4R antagonist and the demonstration of a similar iNKT cell deficit in H4R−/− mice. Our findings identify a novel function of HA through its H4R and suggest that it might become instrumental in modulating iNKT cell functions.