Harnessing the Versatility of Invariant NKT Cells in a Stepwise Approach to Sepsis Immunotherapy.

Sepsis results from a heavy-handed response to infection that may culminate in organ failure and death. Many patients who survive acute sepsis become immunosuppressed and succumb to opportunistic infections. Therefore, to be successful, sepsis immunotherapies must target both the initial and the protracted phase of the syndrome to relieve early immunopathology and late immunosuppression, respectively. Invariant NKT (iNKT) cells are attractive therapeutic targets in sepsis. However, repeated treatments with α-galactosylceramide, the prototypic glycolipid ligand of iNKT cells, result in anergy. We designed a double-hit treatment that allows iNKT cells to escape anergy and exert beneficial effects in biphasic sepsis. We tested the efficacy of this approach in the sublethal cecal ligation and puncture mouse model, which mirrors polymicrobial sepsis with progression to an immunosuppressed state. Septic mice were treated with [(C2S, 3S, 4R)-1-O-(α-d-galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol] (OCH), a TH2-polarizing iNKT cell agonist, before they received α-galactosylceramide. This regimen reduced the morbidity and mortality of cecal ligation and puncture, induced a transient but robust IFN-γ burst within a proinflammatory cytokine/chemokine landscape, transactivated NK cells, increased MHC class II expression on macrophages, and restored delayed-type hypersensitivity to a model hapten, consistent with recovery of immunocompetence in protracted sepsis. Structurally distinct TH2-polarizing agonists varied in their ability to replace OCH as the initial hit, with their lipid chain length being a determinant of efficacy. The proposed approach effectively exploits iNKT cells' versatility in biphasic sepsis and may have translational potentials in the development of new therapies.

Immune Networks and Therapeutic Targeting of iNKT Cells in Cancer.

One of the primary goals in tumor immunotherapy is to reset the immune system from tolerogenic to immunogenic - a process in which invariant natural killer T (iNKT) cells are implicated. iNKT cells develop in the thymus and perform immunosurveillance against tumor cells peripherally. When optimally stimulated, iNKT cells differentiate and display more efficient immune functions. Some cells survive and act as effector memory cells. We discuss the putative roles of iNKT cells in antitumor immunity, and posit that it may be possible to develop novel therapeutic strategies to treat cancers using iNKT cells. In particular, we highlight the challenge of uniquely energizing iNKT cell-licensed dendritic cells to serve as effective immunoadjuvants for both arms of the immune system, thus coupling immunological networks.

CD1d expression in glioblastoma is a promising target for NKT cell-based cancer immunotherapy.

Glioblastoma is the most common and aggressive type of brain tumor with high recurrence and fatality rates. Although various therapeutic strategies have been explored, there is currently no effective treatment for glioblastoma. Recently, the number of immunotherapeutic strategies has been tested for malignant brain tumors. Invariant natural killer T (iNKT) cells play an important role in anti-tumor immunity. To address if iNKT cells can target glioblastoma to exert anti-tumor activity, we assessed the expression of CD1d, an antigen-presenting molecule for iNKT cells, on glioblastoma cells. Glioblastoma cells from 10 of 15 patients expressed CD1d, and CD1d-positive glioblastoma cells pulsed with glycolipid ligand induced iNKT cell-mediated cytotoxicity in vitro. Although CD1d expression was low on glioblastoma stem-like cells, retinoic acid, which is the most common differentiating agent, upregulated CD1d expression in these cells and induced iNKT cell-mediated cytotoxicity. Moreover, intracranial administration of human iNKT cells induced tumor regression of CD1d-positive glioblastoma in orthotopic xenografts in NOD/Shi-scid IL-2RγKO (NOG) mice. Thus, CD1d expression represents a novel target for NKT cell-based immunotherapy for glioblastoma patients.

Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production.

Invariant natural killer T (iNKT) cells are evolutionarily conserved innate T cells that influence inflammatory responses. We have shown that iNKT cells, previously thought to be rare in humans, were highly enriched in human and murine adipose tissue, and that as adipose tissue expanded in obesity, iNKT cells were depleted, correlating with proinflammatory macrophage infiltration. iNKT cell numbers were restored in mice and humans after weight loss. Mice lacking iNKT cells had enhanced weight gain, larger adipocytes, fatty livers, and insulin resistance on a high-fat diet. Adoptive transfer of iNKT cells into obese mice or in vivo activation of iNKT cells via their lipid ligand, alpha-galactocylceramide, decreased body fat, triglyceride levels, leptin, and fatty liver and improved insulin sensitivity through anti-inflammatory cytokine production by adipose-derived iNKT cells. This finding highlights the potential of iNKT cell-targeted therapies, previously proven to be safe in humans, in the management of obesity and its consequences.

IL-21 Selectively Protects CD62L+ NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy.

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L+ NKTs persist longer and have more potent antilymphoma activity than CD62L- cells. However, the conditions governing preservation of CD62L+ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory-like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L+ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L- cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L+ cells versus IL-2 alone. Gene expression analysis comparing CD62L+ and CD62L- cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L+ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21-expanded NKTs upregulated granzyme B expression and produced more TH1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti-CD19-CAR-transduced NKTs against CD1d+ and CD19+ lymphoma cells, respectively. Further, IL-2/IL-21-expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2-expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell-based immunotherapies targeting lymphoma and other malignancies.

Invariant natural killer T cells ameliorate murine chronic GVHD by expanding donor regulatory T cells.

Chronic graft-versus-host-disease (cGVHD) can cause multiorgan system disease, typically with autoimmune-like features, resulting in high mortality and morbidity caused by treatment limitations. Invariant natural killer T cells (iNKTs), a small population characterized by expression of a semi-invariant T-cell receptor, rapidly produce copious amounts of diverse cytokines on activation that exert potent immune regulatory function. Here, we show that iNKTs are significantly reduced in a cGVHD murine model that recapitulates several aspects of autoimmunity and organ fibrosis observed in patients with cGVHD. Low iNKT infused doses effectively prevented and, importantly, reversed established cGVHD, as did third-party iNKTs. iNKTs suppressed the autoimmune response by reducing the germinal center (GC) reaction, which was associated with an increase in total Tregs and follicular Tregs (Tfr) that control the GC reaction, along with pathogenic antibody production. Treg depletion during iNKT infusions completely abolished iNKT efficacy in treating cGVHD. iNKT cell interleukin 4 production and GC migration were critical to cGVHD reversal. In vivo stimulation of iNKT cells by α-galactosyl-ceramide was effective in both preventing and treating cGVHD. Together, this study demonstrates iNKT deficiency in cGVHD mice and highlights the key role of iNKTs in regulating cGVHD pathogenesis and as a potentially novel prophylactic and therapeutic option for patients with cGVHD.

iNKT cells ameliorate human autoimmunity: Lessons from alopecia areata.

Alopecia areata (AA) is understood to be a CD8+/NKG2D+ T cell-dependent autoimmune disease. Here, we demonstrate that human AA pathogenesis of is also affected by iNKT10 cells, an unconventional T cell subtype whose number is significantly increased in AA compared to healthy human skin. AA lesions can be rapidly induced in healthy human scalp skin xenotransplants on Beige-SCID mice by intradermal injections of autologous healthy-donor PBMCs pre-activated with IL-2. We show that in this in vivo model, the development of AA lesions is prevented by recognized the iNKT cell activator, α-galactosylceramide (α-GalCer), which stimulates iNKT cells to expand and produce IL-10. Moreover, in pre-established humanized mouse AA lesions, hair regrowth is promoted by α-GalCer treatment through a process requiring both effector-memory iNKT cells, which can interact directly with CD8+/NKG2D+ T cells, and IL-10. This provides the first in vivo evidence in a humanized model of autoimmune disease that iNKT10 cells are key disease-protective lymphocytes. Since these regulatory NKT cells can both prevent the development of AA lesions and promote hair re-growth in established AA lesions, targeting iNKT10 cells may have preventive and therapeutic potential also in other autoimmune disorders related to AA.

Dissecting CD8+ NKT Cell Responses to Listeria Infection Reveals a Component of Innate Resistance.

A small pool of NK1.1(+) CD8(+) T cells is harbored among the conventional CD8(+) T cell compartment. Conclusions drawn from the analysis of immune responses mediated by cytotoxic CD8(+) T cells are often based on the total population, which includes these contaminating NK1.1(+) CD8(+) T cells. An unresolved question is whether NK1.1(+) CD8(+) cells are conventional T cells that acquire NK1.1 expression upon activation or delineation into memory phenotype or whether they are a distinct cell population that induces immune responses in a different manner than conventional T cells. To address this question, we used the Listeria monocytogenes model of infection and followed CD8(+) NK1.1(+) T cells and NK1.1(-) CD8(+) T cells during each phase of the immune response: innate, effector, and memory. Our central finding is that CD8(+) NK1.1(+) cells and conventional NK1.1(-) CD8(+) T cells both contribute to the adaptive immune response to Listeria, but only CD8(+) NK1.1(+) cells were equipped with the ability to provide a rapid innate immune response, as demonstrated by early and Ag-independent IFN-γ production, granzyme B expression, and degranulation. More importantly, purified conventional CD8(+) T cells alone, in the absence of any contaminating CD8(+) NK1.1(+) cells, were not sufficient to provide early protection to lethally infected mice. These results highlight the role of CD8(+) NK1.1(+) T cells in mounting early innate responses that are important for host defense and support the therapeutic potential of this subset to improve the effectiveness of protective immunity.

IL-9 Expression by Invariant NKT Cells Is Not Imprinted during Thymic Development.

Invariant NKT (iNKT) cell thymic development can lead to distinct committed effector lineages, namely NKT1, NKT2, and NKT17. However, following identification of IL-9-producing iNKT cells involved in mucosal inflammation, their development remains unaddressed. In this study, we report that although thymic iNKT cells from naive mice do not express IL-9, iNKT cell activation in the presence of TGF-ß and IL-4 induces IL-9 secretion in murine and human iNKT cells. Acquisition of IL-9 production was observed in different iNKT subsets defined by CD4, NK1.1, and neuropilin-1, indicating that distinct functional subpopulations are receptive to IL-9 polarization. Transcription factor expression kinetics suggest that regulatory mechanisms of IL-9 expression are shared by iNKT and CD4 T cells, with Irf4 and Batf deficiency deeply affecting IL-9 production. Importantly, adoptive transfer of an enriched IL-9(+) iNKT cell population leads to exacerbated allergic inflammation in the airways upon intranasal immunization with house dust mite, confirming the ability of IL-9-producing iNKT cells to mediate proinflammatory effects in vivo, as previously reported. Taken together, our data show that peripheral iNKT cells retain the capacity of shaping their function in response to environmental cues, namely TGF-ß and IL-4, adopting an IL-9-producing NKT cell phenotype able to mediate proinflammatory effects in vivo, namely granulocyte and mast cell recruitment to the lungs.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.

Tuberous sclerosis 1 promotes invariant NKT cell anergy and inhibits invariant NKT cell-mediated antitumor immunity.

Development of effective immune therapies for cancer patients requires better understanding of hurdles that prevent the generation of effective antitumor immune responses. Administration of α-galactosylceramide (α-GalCer) in animals enhances antitumor immunity via activation of the invariant NKT (iNKT) cells. However, repeated injections of α-GalCer result in long-term unresponsiveness or anergy of iNKT cells, severely limiting its efficacy in tumor eradication. The mechanisms leading to iNKT cell anergy remain poorly understood. We report in this study that the tuberous sclerosis 1 (TSC1), a negative regulator of mTOR signaling, plays a crucial role in iNKT cell anergy. Deficiency of TSC1 in iNKT cells results in resistance to α-GalCer-induced anergy, manifested by increased expansion of and cytokine production by iNKT cells in response to secondary Ag stimulation. It is correlated with impaired upregulation of programmed death-1, Egr2, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a melanoma lung metastasis model. Our data suggest targeting TSC1/2 as a strategy for boosting antitumor immune therapy.

Critical role of TCR specificity in the development of Vγ1Vδ6.3+ innate NKTγδ cells.

A large fraction of innate NKTγδ T cells uses TCRs composed of a semi-invariant Vδ6.3/6.4-Dδ2-Jδ1 chain together with more diverse Vγ1-Jγ4 chains. To address the role of γδTCR specificity in their generation, we analyzed their development in mice transgenic (Tg) for a Vγ1-Jγ4 chain frequently expressed by NKTγδ cells (Tg-γ) and in mice Tg for the same Vγ1-Jγ4 chain together with a Vδ6BDδ2Jδ1 chain not usually found among NKTγδ cells (Tg-γδ). Surprisingly, both promyelocytic leukemia zinc finger (PLZF)(+) and NK1.1(+) NKTγδ cells were found in the thymus of Tg-γδ albeit at lower numbers than in Tg-γ mice, and virtually all of them expressed the Tg TCR. However, the PLZF(+) subset, but not the NK1.1(+) subset, also expressed an endogenous Vδ6.3/6.4 chain, and its size was severely reduced in TCRδ(-/-) Tg-γδ mice. These results could suggest that the PLZF(+) and the NK1.1(+) subsets are developmentally unrelated. However, PLZF(+) and NK1.1(+) NKTγδ cells express identical Vδ6.3/6.4 chains, and NK1.1(+) cells can be obtained upon intrathymic injection of sorted PLZF(+) cells, thus indicating their developmental relationship. In fact, the NK1.1(+) γδ thymocytes present in Tg-γδ mice correspond to a small subset of NK1.1(+) γδ thymocytes in wild-type animals, which express a more diverse repertoire of TCRs and can be recognized by the expression of the CD62L Ag. Collectively, our data demonstrated that TCR specificity is essential for the development of most NKTγδ T cells and revealed a developmental heterogeneity in γδ T cells expressing the NK1.1 marker.

Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation.

Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4(-) iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4(-) iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4(-) iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4(-) iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4(-) iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

NKG2D CAR T-cell therapy inhibits the growth of NKG2D ligand heterogeneous tumors.

Tumor heterogeneity presents a substantial barrier to increasing clinical responses mediated by targeted therapies. Broadening the immune response elicited by treatments that target a single antigen is necessary for the elimination of tumor variants that fail to express the targeted antigen. In this study, it is shown that adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) inhibited the growth of target-expressing and -deficient tumor cells within ovarian and lymphoma tumors. Mice bearing the ID8 ovarian or RMA lymphoma tumors were treated with T cells transduced with a NKG2D-based CAR (chNKG2D). NKG2D CAR T-cell therapy protected mice from heterogeneous RMA tumors. Moreover, adoptive transfer of chNKG2D T cells mediated tumor protection against highly heterogeneous ovarian tumors in which 50, 20 or only 7% of tumor cells expressed significant amounts of NKG2D ligands. CAR T cells did not mediate an in vivo response against tumor cells that did not express sufficient amounts of NKG2D ligands, and the number of ligand-expressing tumor cells correlated with therapeutic efficacy. In addition, tumor-free surviving mice were protected against a tumor re-challenge with NKG2D ligand-negative ovarian tumor cells. These data indicate that NKG2D CAR T-cell treatment can be an effective therapy against heterogeneous tumors and induce tumor-specific immunity against ligand-deficient tumor cells.

Role of invariant natural killer T cells in lipopolysaccharide-induced pregnancy loss.

We aimed to investigate the role of invariant natural killer T (iNKT) cells in infection-associated pregnancy loss. Wild-type (WT) C57BL/6 mice and iNKT cell-deficient Jα18(-/-) mice were treated with lipopolysaccharide (LPS). Embryo resorption rates (ERRs), decidual costimulatory molecule and activation molecule expression, and cytokine production were determined. WT and Jα18(-/-) mice were adoptively transferred with purified iNKT cells. ERRs, decidual costimulatory molecule and activation molecule expression, and cytokine production were assessed. LPS-treated Jα18(-/-) mice showed markedly reduced ERRs, decreased CD40, CD80, CD86, and CD69 expression, and reduced Th1 cytokine production at the maternal-fetal interface compared with WT mice. ERRs, expression of CD40, CD80, CD86, and CD69, and Th1 cytokine production in LPS-injected Jα18(-/-) mice following iNKT cell adoptive transfer were remarkably upregulated compared with control mice that did not receive adoptively transferred iNKT cells. Our results suggest that iNKT cells play an important role in LPS-induced pregnancy loss.

Invariant NKT cell reconstitution in pediatric leukemia patients given HLA-haploidentical stem cell transplantation defines distinct CD4+ and CD4- subset dynamics and correlates with remission state.

Immune reconstitution plays a crucial role on the outcome of patients given T cell-depleted HLA-haploidentical hematopoietic stem cell transplantation (hHSCT) for hematological malignancies. CD1d-restricted invariant NKT (iNKT) cells are innate-like, lipid-reactive T lymphocytes controlling infections, cancer, and autoimmunity. Adult mature iNKT cells are divided in two functionally distinct CD4(+) and CD4(-) subsets that express the NK receptor CD161 and derive from thymic CD4(+)CD161(-) precursors. We investigated iNKT cell reconstitution dynamics in 33 pediatric patients given hHSCT for hematological malignancies, with a follow-up reaching 6 y posttransplantation, and correlated their emergence with disease relapse. iNKT cells fully reconstitute and rapidly convert into IFN-γ-expressing effectors in the 25 patients maintaining remission. CD4(+) cells emerge earlier than the CD4(-) ones, both displaying CD161(-) immature phenotypes. CD4(-) cells expand more slowly than CD4(+) cells, though they mature with significantly faster kinetics, reaching full maturation by 18 mo post-hHSCT. Between 4 and 6 y post-hHSCT, mature CD4(-) iNKT cells undergo a substantial expansion burst, resulting in a CD4(+)

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma.

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.

CD1d-restricted IFN-γ-secreting NKT cells promote immune complex-induced acute lung injury by regulating macrophage-inflammatory protein-1α production and activation of macrophages and dendritic cells.

Immune complex-induced acute lung injury (IC-ALI) has been implicated in various pulmonary disease states. However, the role of NKT cells in IC-ALI remains unknown. Therefore, we explored NKT cell functions in IC-ALI using chicken egg albumin and anti-chicken egg albumin IgG. The bronchoalveolar lavage fluid of CD1d(-/-) and Jα18(-/-) mice contained few Ly6G(+)CD11b(+) granulocytes, whereas levels in B6 mice were greater and were increased further by α-galactosyl ceramide. IFN-γ and MIP-1α production in the lungs was greater in B6 than CD1d(-/-) mice. Adoptive transfer of wild type (WT) but not IFN-γ-, MIP-1α-, or FcγR-deficient NKT cells into CD1d(-/-) mice caused recruitment of inflammatory cells to the lungs. Moreover, adoptive transfer of IFN-γR-deficient NKT cells enhanced MIP-1α production and cell recruitment in the lungs of CD1d(-/-) or CD1d(-/-)IFN-γ(-/-) mice, but to a lesser extent than WT NKT cells. This suggests that IFN-γ-producing NKT cells enhance MIP-1α production in both an autocrine and a paracrine manner. IFN-γ-deficient NKT cells induced less IL-1ß and TNF-α production by alveolar macrophages and dendritic cells in CD1d(-/-) mice than did WT NKT cells. Taken together, these data suggest that CD1d-restricted IFN-γ-producing NKT cells promote IC-ALI by producing MIP-1α and enhancing proinflammatory cytokine production by alveolar macrophages and dendritic cells.

A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.