Stephane Sidobre

Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity

Using natural killer T (NKT) cell–deficient mice, we show here that allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma, does not develop in the absence of Vα14i NKT cells. The failure of NKT cell–deficient mice to develop AHR is not due to an inability of these mice to produce type 2 T-helper (Th2) responses because NKT cell–deficient mice that are immunized subcutaneously at non-mucosal sites produce normal Th2-biased responses. The failure to develop AHR can be reversed by the adoptive transfer of tetramer-purified NKT cells producing interleukin (IL)-4 and IL-13 to Ja281−/− mice, which lack the invariant T-cell receptor (TCR) of NKT cells, or by the administration to Cd1d−/− mice of recombinant IL-13, which directly affects airway smooth muscle cells. Thus, pulmonary Vα14i NKT cells crucially regulate the development of asthma and Th2-biased respiratory immunity against nominal exogenous antigens. Therapies that target Vα14i NKT cells may be clinically effective in limiting the development of AHR and asthma.

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6+ cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

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.

Schistosoma mansoni antigens modulate the activity of the innate immune response and prevent onset of type 1 diabetes

Infection with Schistosoma mansoni (S. mansoni) or exposure to eggs from this helminth inhibits the development of type 1 diabetes in NOD mice. In this study we show that soluble extracts of S. mansoni worm or egg completely prevent onset of type 1 diabetes in these mice but only if injection is started at 4 weeks of age. T cells from diabetes‐protected mice make IL‐10 in recall responses to parasite antigens. These cells are furthermore impaired in their ability to transfer diabetes to NOD‐SCID recipients. Bone marrow dendritic cells derived from NOD mice are found to make more IL‐10 and less IL‐12 following culture with S. mansoni soluble egg antigens in conjunction with lipopolysaccharides. NOD mice are deficient in NKT cells. Soluble worm and egg antigens increase the numbers of Vα14i NKT cells in NOD mice. These effects of schistosome antigens on the innate immune system provide a mechanism for their ability to prevent type 1 diabetes in NOD mice.

Cross-presentation of Disialoganglioside GD3 to Natural Killer T Cells

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2− CD1−) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of ∼1:2,000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-γ followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1− tumors.

Glycolipid Antigen Drives Rapid Expansion and Sustained Cytokine Production by NK T Cells

NKT cells are enigmatic lymphocytes that respond to glycolipid Ags presented by CD1d. Although they are key immunoregulatory cells, with a critical role in immunity to cancer, infection, and autoimmune diseases, little is known about how they respond to antigenic challenge. Current theories suggest that NKT cells die within hours of stimulation, implying that their direct impact on the immune system derives from the initial cytokine burst released before their death. Here we show that NKT cell disappearance results from TCR down-regulation rather than apoptosis, and that they expand to many times their normal number in peripheral tissues within 2–3 days of stimulation, before contracting to normal numbers over subsequent days. This expansion is associated with ongoing cytokine production, biased toward a Th1 (IFN-γ+ IL-4−) phenotype, in contrast to their initial Th0 (IFN-γ+IL-4+) phenotype. This study provides critical new insight into how NKT cells can have such a major impact on immune responses, lasting many days beyond the initial stimulation of these cells.

Mouse Vα14i natural killer T cells are resistant to cytokine polarization in vivo

Under different circumstances, natural killer T (NKT) cells can cause a T helper (Th) 1 or a Th2 polarization of immune responses. We show here, however, that mouse NKT cells with an invariant Vα14 rearrangement (Vα14i NKT cells) rapidly produce both IL-4 and IFN-γ, and this pattern could not be altered by methods that polarize naive CD4+ T cells. Surprisingly, although cytokine protein was detected only after activation, resting Vα14i NKT cells contained IL-4 and IFN-γ mRNAs. Despite this finding, in vivo priming of mice with the glycolipid antigen recognized by Vα14i NKT cells resulted in a more Th2-oriented response upon antigen re-exposure. The Vα14i NKT cells from primed mice retain the ability to produce IL-4 and IFN-γ, but they are less effective at activating NK cells to produce IFN-γ. Our data therefore indicate that Vα14i NKT cells have a relatively inflexible immediate cytokine response, but that changes in their ability to induce IFN-γ secretion by NK cells may determine the extent to which they promote Th1 responses.

Homeostasis of V alpha 14i NKT cells.

CD1d-reactive natural killer T (NKT) cells with an invariant Vα14 rearrangement (Vα14i) are a distinct subset of T lymphocytes that likely have important immune-regulatory functions. Little is known regarding the factors responsible for their peripheral survival. Using α-galactosylceramide–containing CD1d tetramers to detect Vα14i NKT cells, we show here that the expansion of Vα14i NKT cells in lymphopenic mice was not dependent on CD1d expression and was unaffected by the presence of host NKT cells. Additionally, we found that IL-15 was important in the expansion and/or survival of Vα14i NKT cells, with IL-7 playing a lesser role. These results demonstrate that the homeostatic requirements for CD1d-restricted NKT cells, which are CD4+ or CD4−CD8−, resemble those of CD8+ memory T cells. We propose that this expansion and/or survival in the periphery of Vα14i NKT cells is affected by competition for IL-15, and that IL-15–requiring cells—such as NK cells and CD8+ memory cells—may define the Vα14i NKT cell niche.

Cutaneous Immunization Rapidly Activates Liver Invariant Vα14 NKT Cells Stimulating B-1 B Cells to Initiate T Cell Recruitment for Elicitation of Contact Sensitivity

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Vα14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jα18−/− and CD1d−/− NKT cell–deficient mice and is reconstituted by populations enriched for Vα14i NKT cells. Transfers are not effective if cells are derived from IL-4−/− mice. Staining with specific tetramers directly showed that hepatic Vα14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell–deficient mice. The B-1 cells act downstream of the Vα14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jα18−/− or CD1d−/− NKT cell–deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jα18−/− mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Vα14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

Activation of natural killer T cells in NZB/W mice induces Th1-type immune responses exacerbating lupus.

In vivo treatment of mice with the natural killer T (NKT) cell ligand, alpha-galactosylceramide (alphaGalCer), ameliorates autoimmune diabetes and experimental autoimmune encephalomyelitis (EAE) by shifting pathogenic Th1-type immune responses to nonpathogenic Th2-type responses. In the current study, in vivo activation of NKT cells in adult NZB/W mice by multiple injections of alphaGalCer induced an abnormal Th1-type immune response as compared with the Th2-type response observed in nonautoimmune C57BL/6 mice. This resulted in decreased serum levels of IgE, increased levels of IgG2a and IgG2a anti-double-stranded DNA (anti-dsDNA) Abs, and exacerbated lupus. Conversely, treatment of NZB/W mice with blocking anti-CD1d mAb augmented Th2-type responses, increased serum levels of IgE, decreased levels of IgG2a and IgG2a anti-dsDNA Abs, and ameliorated lupus. While total CD4+ T cells markedly augmented in vitro IgM anti-dsDNA Ab secretion by splenic B cells, the non-CD1d-reactive (CD1d-alphaGalCer tetramer-negative) CD4+ T cells (accounting for 95% of all CD4+ T cells) failed to augment Ab secretion. The CD1d-reactive tetramer-positive CD4+ T cells augmented anti-dsDNA Ab secretion about tenfold. In conclusion, activation of NKT cells augments Th1-type immune responses and autoantibody secretion that contribute to lupus development in adult NZB/W mice, and anti-CD1d mAb might be useful for treating lupus.