Maria C. Leite-de-Moraes

Activation of natural killer T cells by α -galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6–9). Thus, given that NKT cells respond to the α-galactosylceramide (α-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by α-GalCer might prevent the onset and/or recurrence of T1D. Here we show that α-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, α-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to α-GalCer. Protection from T1D by α-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet β cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility thatα-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

Identification of an IL-17–producing NK1.1neg iNKT cell population involved in airway neutrophilia

Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1neg) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-γ and IL-4. NK1.1neg iNKT cells produce IL-17 upon synthetic (α-galactosylceramide [α-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1neg iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by α-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell–deficient Jα18−/− mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before α-GalCer administration. Collectively, our findings reveal that NK1.1neg iNKT lymphocytes represent a new population of IL-17–producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion.

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.

Critical role of ROR-γt in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation

Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon-γ (IFN-γ), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramerposCD44posNK1.1negCD4neg cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR-γt is critical for the thymic differentiation of this subset because only ROR-γtpos iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramerposCD44posNK1.1negCD4neg thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR-γtneg iNKT cell precursors give rise to progeny, but acquire neither ROR-γt expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR-γt expression.

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.

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.

Invariant and Noninvariant Natural Killer T Cells Exert Opposite Regulatory Functions on the Immune Response during Murine Schistosomiasis

ABSTRACT CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) α chain (Vα14Jα18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jα18−/− (exclusively deficient in iNKT cells) and CD1d−/− (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response.

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.

Invariant Vα14+ NKT Cells Participate in the Early Response to Enteric Listeria monocytogenes Infection

Invariant Vα14+ NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Vα14+ NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid α-galactosylceramide (CD1d/αGC), we found that splenic and hepatic Vα14+ NKT cells in C57BL/6 mice were early producers of IFN-γ (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Vα14+ NKT cells derived from TCRα° Vα14-Jα18 transgenic (TCRα°Vα14Tg) mice into alymphoid Rag°γc° mice demonstrated that Vα14+ NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-γ and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jα18° mice, but not TCRα° or TCRα°Vα14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Vα14+ NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-γ production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.