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Swine represent the only livestock with an established invariant NKT (iNKT) cell–CD1d system. In this study, we exploited the fact that pig iNKT cells can be purified using a mouse CD1d tetramer reagent to establish their TCR repertoire by next generation sequencing. CD1d tetramer-positive pig cells predominantly expressed an invariant Vα–Jα rearrangement, without nontemplate nucleotide diversity, homologous to the Vα24–Jα18 and Vα14–Jα18 rearrangements of human and murine iNKT cells. The coexpressed β-chain used a Vβ segment homologous to the semivariant Vβ11 and Vβ8.2 segments of human and murine iNKT cell receptors. Molecular modeling found that contacts within CD1d and CDR1α that underlie fine specificity differences between mouse and human iNKT cells are conserved between pigs and humans, indicating that the response of porcine and human iNKT cells to CD1d-restricted Ags may be similar. Accordingly, pigs, which are an important species for diverse fields of biomedical research, may be useful for developing human-based iNKT cell therapies for cancer, infectious diseases, and other disorders. Our study also sequenced the expressed TCR repertoire of conventional porcine αβ T cells, which identified 48 Vα, 50 Jα, 18 Vβ, and 18 Jβ sequences, most of which correspond to human gene segments. These findings provide information on the αβ TCR usage of pigs, which is understudied and deserves further attention.

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 NKT (iNKT) cells are innate-like lymphocytes that recognize lipid Ags presented by CD1d. The prototypical Ag, α-galactosylceramide, strongly activates human and mouse iNKT cells, leading to the assumption that iNKT cell physiology in human and mouse is similar. In this article, we report the surprising finding that human, but not mouse, iNKT cells directly recognize myelin-derived sulfatide presented by CD1d. We propose that sulfatide is recognized only by human iNKT cells because of the unique positioning of the 3-O-sulfated β-galactose headgroup. Surface plasmon resonance shows that the affinity of human CD1d-sulfatide for the iNKT cell receptor is relatively low compared with CD1d–α-galactosylceramide (KD of 19–26 μM versus 1 μM). Apolipoprotein E isolated from human cerebrospinal fluid carries sulfatide that can be captured by APCs and presented by CD1d to iNKT cells. APCs from patients with metachromatic leukodystrophy, who accumulate sulfatides due to a deficiency in arylsulfatase-A, directly activate iNKT cells. Thus, we have identified sulfatide as a self-lipid recognized by human iNKT cells and propose that sulfatide recognition by innate T cells may be an important pathologic feature of neuroinflammatory disease and that sulfatide in APCs may contribute to the endogenous pathway of iNKT cell activation.

Invariant NKT (iNKT) cells are innate lymphocytes that respond to glycolipids presented by the MHC class Ib molecule CD1d and are rapidly activated to produce large quantities of cytokines and chemokines. iNKT cell development uniquely depends on interactions between double-positive thymocytes that provide key homotypic interactions between signaling lymphocyte activation molecule (SLAM) family members. However, the role of SLAM receptors in the differentiation of iNKT cell effector subsets and activation has not been explored. In this article, we show that C57BL/6 mice containing the New Zealand Black Slam locus have profound alterations in Ly108, CD150, and Ly9 expression that is associated with iNKT cell hyporesponsiveness. This loss of function was only apparent when dendritic cells and iNKT cells had a loss of SLAM receptor expression. Using small interfering RNA knockdowns and peptide-blocking strategies, we demonstrated that trans-Ly108 interactions between dendritic cells and iNKT cells are critical for robust activation. LY108 costimulation similarly increased human iNKT cell activation. Thus, in addition to its established role in iNKT cell ontogeny, Ly108 regulates iNKT cell function in mice and humans.

The human invariant NK (iNK) TCR is largely composed of the invariant TCR Vα24-Jα18 chain and semivariant TCR Vβ11 chains with variable CDR3β sequences. The direct role of CDR3β in Ag recognition has been studied extensively. Although it was noted that CDR3β can interact with CDR3α, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of Vβ11 CDR3 can encode an Arg or Ser residue as a result of somatic rearrangement. Clonotypic analysis of the two iNK TCR types with a single amino acid substitution revealed that the staining intensity by anti-Vα24 Abs depends on whether Ser or Arg is encoded. When stained with an anti–Vα24-Jα18 Ab, human primary invariant NKT cells could be divided into Vα24 low- and high-intensity subsets, and Arg-encoding TCR Vβ11 chains were more frequently isolated from the Vα24 low-intensity subpopulation compared with the Vα24 high-intensity subpopulation. The Arg/Ser substitution also influenced Ag recognition as determined by CD1d multimer staining and CD1d-restricted functional responses. Importantly, in silico modeling validated that this Ser-to-Arg mutation could alter the structure of the CDR3β loop, as well as the CDR3α loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3β residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

Invariant NKT (iNKT) cells are unconventional innate-like T cells demonstrating potent antitumor function in conventional mouse models. However, the iNKT cell ligands have had limited efficacy in human antitumor clinical trials, mostly due to the profound differences in the properties and compositions of iNKT cells between the two species, including the presence of a CD8+ subset of iNKT cells only in humans. To build reliable in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in. To further humanize the mouse model, we now introduced the human invariant NKT TCRα-chain (Vα24Jα18) into the hCD1d-knockin mice. Similar to humans, this humanized mouse model developed a subset of CD8αβ+ iNKT cells among other human-like iNKT subsets. The presence of the CD8αβ+ iNKT cells in the thymus suggests that these cells developed in the thymus. In the periphery, these NKT cells showed a strong Th1-biased cytokine response and potent cytotoxicity for syngeneic tumor cells upon activation, as do human CD8αβ+ iNKT cells. The low binding avidity of iNKT TCRs to the human CD1d/lipid complex and high prevalence of Vβ7 TCRβ among the CD8+ iNKT cells strongly point to a low avidity–based developmental program for these iNKT cells, which included the suppression of Th-POK and upregulation of eomesodermin transcriptional factors. Our establishment of this extensively humanized mouse model phenotypically and functionally reflecting the human CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell–based immunotherapies.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow–derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions −331 and −164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide–containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell–derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell–derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d+ cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d+ dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.

Invariant CD1d-restricted NKT (iNKT) cells play important roles in generating protective immune responses against infections. In this study, we have investigated the role of human iNKT cells in HSV-1 infection and their interaction with epidermal keratinocytes. These cells express CD1d and are the primary target of the virus. Keratinocytes loaded with α-galactosyl ceramide (α-GalCer) could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer–dependent and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1–infected cells. Notably, CD1d downregulation was not observed on infected keratinocytes, which were also found to inhibit TCR-independent iNKT cell activation. Further examination of the cytokine profile of iNKT–keratinocyte cocultures showed inhibition of IFN-γ, IL-5, IL-10, IL-13, and IL-17 secretion but upregulation of IL-4 and TNF-α after the infection. Moreover, cell-to-cell contact between infected keratinocytes and iNKT cells was required for the inhibition of activation, as the cell-free supernatants containing virus did not affect activation. Productive infection of iNKT cells was however not required for the inhibitory effect. After coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer–loaded CD1d-expressing cells. We found that exposure to HSV-1–infected cells resulted in impaired TCR signaling downstream of ZAP70. Additionally, infected cells upregulated the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of iNKT cell responses was independent of galectin-9. Thus, interference with activation of human iNKT cells by HSV-1 may represent a novel immunoevasive strategy used by the virus to avoid immune clearance.

CD23+CD21highCD1dhigh B cells in inflamed nodes (Bin cells) accumulate in the lymph nodes (LNs) draining inflamed joints of the TNF-α–transgenic mouse model of rheumatoid arthritis and are primarily involved in the significant histological and functional LN alterations that accompany disease exacerbation in this strain. In this study, we investigate the origin and function of Bin cells. We show that adoptively transferred GFP+ sorted mature follicular B (FoB) cells home preferentially to inflamed LNs of TNF-α–transgenic mice where they rapidly differentiate into Bin cells, with a close correlation with the endogenous Bin fraction. Bin cells are also induced in wild-type LNs after immunization with T-dependent Ags and display a germinal center phenotype at higher rates compared with FoB cells. Furthermore, we show that Bin cells can capture and process Ag-immune complexes in a CD21-dependent manner more efficiently than can FoB cells, and they express greater levels of MHC class II and costimulatory Ags CD80 and CD86. We propose that Bin cells are a previously unrecognized inflammation-induced B cell population with increased Ag capture and activation potential, which may facilitate normal immune responses but may contribute to autoimmunity when chronic inflammation causes their accumulation and persistence in affected LNs.

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate “off-the-shelf” therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting, it is crucial for these cells to “jump-start” adaptive antitumor immunity before their elimination by host alloreaction. In this study, we investigated whether CD1d upregulation in hESC-DCs may exploit invariant NKT (iNKT) cell adjuvant activity and boost antitumor immunity. Using a baculoviral vector carrying the CD1d gene, we produced CD1d-overexpressing hESC-DCs and demonstrated that the upregulated CD1d was functional in presenting α-galactosylceramide for iNKT cell expansion. Pulsed with melanoma Ag recognized by T cell 1 peptide, the CD1d-overexpressing hESC-DCs displayed enhanced capability to prime CD8+ T cells without relying on α-galactosylceramide loading. Blocking the CD1d with Ab reduced the immunogenicity, suggesting the importance of hESC–DC and iNKT cell interaction in this context. The CD1d-overexpressing hESC-DCs also induced a proinflammatory cytokine profile that may favor the T cell priming. Moreover, a similar immunostimulatory effect was observed when the CD1d upregulation strategy was applied in human monocyte-derived dendritic cells. Therefore, our study suggests that the upregulation of CD1d in hESC-DCs provides a novel strategy to enhance their immunogenicity. This approach holds potential for advancing the application of hESC-DCs into human cancer immunotherapy.

Type I NKT cells, or invariant NKT (iNKT) cells, express a semi-invariant TCR characterized by its unique Vα14-Jα18 usage (iVα14TCR). Upon interaction with glycolipid/CD1d complexes, the iVα14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the protein kinase Cθ-IκB kinase (IKK)α/β-NF-κB pathway, known to be crucial for iNKT development, as well as the RasGRP1–Ras-Erk1/2 pathway in T cells. DAG kinases play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. In this article, we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NF-κB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development.

NKT cells that express the semi-invariant TCR are innate-like lymphocytes whose functions are regulated by self and foreign glycolipid ligands presented by the Ag-presenting, MHC class I-like molecule CD1d. Activation of NKT cells in vivo results in rapid release of copious amounts of effector cytokines and chemokines with which they regulate innate and adaptive immune responses to pathogens, certain types of cancers, and self-antigens. The nature of CD1d-restricted ligands, the manner in which they are recognized, and the unique effector functions of NKT cells suggest an immunoregulatory role for this T cell subset. Their ability to respond fast and our ability to steer NKT cell cytokine response to altered lipid ligands make them an important target for vaccine design and immunotherapies against autoimmune diseases. This review summarizes our current understanding of CD1d-restricted ligand recognition by NKT cells and how these innate-like lymphocytes regulate inflammation.

Rare CD1d-α-galactosylceramide–specific T cells that do not express the invariant Vα24 chain of human NKT cells were recently identified after expansion in vitro with the lipid Ag, but their phenotype and frequency in vivo and lineage relationship with NKT cells could not be elucidated. By using a CD1d tetramer-based method to enrich these cells from fresh peripheral blood, we demonstrated their naive-like CD62LhighCD45RO−CD4+ phenotype and relatively high frequency of ∼10−5 in several healthy individuals. Notably, these cells expressed the NKT lineage-specific transcription promyelocytic leukemia zinc finger (PLZF), indicating a developmental relationship with NKT cells and ruling out the possibility that they were conventional MHC-restricted T cells cross-reacting against CD1d-α-galactosylceramide. Although PLZF is known to direct the effector program of NKT cells, we show in this study that the naive-like cells expressed it at a significantly lower amount than NKT cells. Further, we present mouse studies demonstrating a sharp PLZF expression threshold requirement for induction of the effector phenotype. These findings directly demonstrate in vivo the existence of naive-like CD1d-restricted human T cells marked by intermediate levels of PLZF.

Thymocytes expressing the NKT cell semi-invariant αβ TCR are thought to undergo agonist interactions with CD1d ligands prior to expressing promyelocytic leukemia zinc finger (PLZF), a broad complex, tramtrack, bric-a-brac, poxvirus, and zinc finger transcription factor that directs acquisition of the effector program of these innate-like T cells. Whether PLZF can mediate this effector conversion independently of agonist signaling has not been investigated. We demonstrated that transgenic (Tg) expression of PLZF under the CD4 promoter induced the innate effector program in two different MHC class II-restricted TCR-Tg Rag1−/− models examined. In CD4 thymocytes expressing a fixed Tg TCR β-chain, the associated TCRα sequences in wild-type and PLZF-Tg mice overlapped extensively, further demonstrating that PLZF could induce the effector program in most CD4 T cells that would normally be selected as naive cells. In contrast, PLZF altered the negative selection of thymocytes expressing TCR β-chains reactive against several retroviral superantigens. Thus, PLZF is remarkable in that it is a transcription factor capable of inducing an effector program in the absence of T cell agonist interactions or cell division. Its expression may also enhance the survival of agonist-signaled thymocytes.

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non–lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ–producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21+CD1d+ B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downreglation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

Cardiolipin (CL), a major phospholipid in bacterial cell walls, is sequestered from the immune system in mammalian mitochondria and is, therefore, a potential danger signal. Based on growing evidence that phospholipids constitute natural ligands for CD1 and that CD1d-restricted T cells recognize phospholipids, we hypothesized that CD1d binds and presents CL and that T cells in the normal immune repertoire respond to CL in a CD1d-restricted manner. We determined the murine CD1d-CL crystal structure at 2.3 Å resolution and established through additional lipid loading experiments that CL, a tetra-acylated phospholipid, binds to murine CD1d with two alkyl chains buried inside the CD1d binding groove and the remaining two exposed into the solvent. We furthermore demonstrate the functional stimulatory activity of CL, showing that splenic and hepatic γδ T cells from healthy mice proliferate in vitro in response to mammalian or bacterial CL in a dose-dependent and CD1d-restricted manner, rapidly secreting the cytokines IFN-γ and RANTES. Finally, we show that hepatic γδ T cells are activated in vivo by CD1d-bearing dendritic cells that have been pulsed with CL, but not phosphatidylcholine. Together, these findings demonstrate that CD1d is able to bind and present CL to a subset of CL-responsive γδ T cells that exist in the spleen and liver of healthy mice and suggest that these cells could play a role in host responses to bacterial lipids and, potentially, self-CL. We propose that CL-responsive γδ T cells play a role in immune surveillance during infection and tissue injury.

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40–CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag α-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

A protective and anti-inflammatory role for CD1d-dependent NKT cells (NKTs) has been reported in experimental and human autoimmune diseases. However, their role in arthritis has been unclear, with conflicting reports of CD1d-dependent NKTs acting both as regulatory and disease-promoting cells in arthritis. These differing modes of action might be due to genetic differences of inbred mice and incomplete backcrossing of gene-modified mice. We therefore put special emphasis on controlling the genetic backgrounds of the mice used. Additionally, we used two different murine arthritis models, Ag-induced arthritis (AIA) and collagen-induced arthritis (CIA), to evaluate acute and chronic arthritis in CD1d knockout mice and mice depleted of NK1.1+ cells. CD1d-deficient mice developed more severe AIA compared with wild-type littermates, with a higher degree of inflammation and proteoglycan depletion. Chronic arthritis in CIA was also worse in the absence of CD1d-dependent NKTs. Elevated levels of Ag-specific IFN-γ production accompanied these findings rather than changes in IL-17α. Depletion of NK1.1+ cells supported these findings in AIA and CIA. This report provides support for CD1d-dependent NKTs being suppressor cells in acute and chronic arthritis, likely via inhibition of arthritogenic Th1 cells. These results make CD1d-dependent NKTs an attractive target for therapeutic intervention.

NKT cells are considered to be innate-like regulatory cells. However, their regulatory functions in adaptive immune responses have not been studied in detail. In this study, we investigated the immunoregulatory functions of NKT cells during the secondary phase of an Ag-specific CD4+ T cell response. When compared with OVA-specific effector CD4+ T cells adoptively transferred into NKT cell-deficient naive CD1d−/− mice, the same T cells transferred into naive CD1d+/− mice exhibited substantially stronger immune responses on OVA challenge. The enhanced immune response of the transferred CD4+ T cells in the presence of NKT cells correlated with an increase in their proliferation in vivo. In addition, T cells transferred into CD1d+/− recipients showed enhanced cytokine productions relative to T cells in CD1d−/− recipients. To elucidate the physiological relevance of the regulatory role of NKT cells in a disease setting, OVA-specific asthma was induced in recipient mice after adoptive transfer of OVA-specific CD4+ T cells. CD1d+/− recipients showed stronger asthmatic phenotypes in all indications when compared with CD1d−/− recipients. Taken together, these results suggest that NKT cells are critical for the regulation of Ag-specific, conventional CD4+ T cells during the secondary phase of an adaptive immune response.

CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called NKT cells. The cytoplasmic tail of human CD1d possesses a tyrosine-based endosomal targeting motif (YXXZ). As such, these molecules traffic through the endocytic pathway, where it is believed that they are loaded with the antigenic lipid that stimulates NKT cells. In the current study, it was found that the T322 residue in the human CD1d tail is a major signal controlling transport to the cell surface and thus its functional expression. Mimicking the phosphorylation of this residue or removal of the entire cytoplasmic tail negates its ability to regulate CD1d trafficking, resulting in lysosomal targeting and degradation. These results demonstrate an important role of a heretofore unknown signal in the cytoplasmic tail of CD1d that may have relevance to other type I integral membrane proteins that traverse through the endocytic pathway.

B cells play critical roles in the pathogenesis of lupus. To examine the influence of B cells on disease pathogenesis in a murine lupus model, New Zealand Black and New Zealand White F1 hybrid (NZB/W) mice were generated that were deficient for CD19 (CD19−/− NZB/W mice), a B cell-specific cell surface molecule that is essential for optimal B cell signal transduction. The emergence of anti-nuclear Abs was significantly delayed in CD19−/− NZB/W mice compared with wild type NZB/W mice. However, the pathologic manifestations of nephritis appeared significantly earlier, and survival was significantly reduced in CD19−/− NZB/W mice compared with wild type mice. These results demonstrate both disease-promoting and protective roles for B cells in lupus pathogenesis. Recent studies have identified a potent regulatory B cell subset (B10 cells) within the rare CD1dhiCD5+ B cell subset of the spleen that regulates acute inflammation and autoimmunity through the production of IL-10. In wild type NZB/W mice, the CD1dhiCD5+B220+ B cell subset that includes B10 cells was increased by 2.5-fold during the disease course, whereas CD19−/− NZB/W mice lacked this CD1dhiCD5+ regulatory B cell subset. However, the transfer of splenic CD1dhiCD5+ B cells from wild type NZB/W mice into CD19−/− NZB/W recipients significantly prolonged their survival. Furthermore, regulatory T cells were significantly decreased in CD19−/− NZB/W mice, but the transfer of wild type CD1dhiCD5+ B cells induced T regulatory cell expansion in CD19−/− NZB/W mice. These results demonstrate an important protective role for regulatory B10 cells in this systemic autoimmune disease.

The T cell Ig domain and mucin domain (TIM)1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. However, the functional roles of TIM1 have not been examined in NKT cells. Therefore, we investigated the immunologic effects of TIM1 on NKT cells. We found that mouse NK1.1+TCR-β+, α-galactosyl ceramide/CD1d dimer+ NKT, and NKT hybridoma (DN32.D3) cells constitutively express TIM1 and TIM4 on their surface. Engagement of TIM1 on NKT cells by any of several anti-TIM1 mAbs suppressed the production of IFN-γ in the presence of TCR stimulation in vitro and in vivo, whereas the effects of such engagement on Th2 cytokine production by the NKT cells varied with the particular anti-TIM1 Ab clone. Moreover, in DN32.D3 TIM4-knockdown NKT hybridoma cells, TIM1 engagement by rTIM1 or TIM4 enhanced IL-4 production while inhibiting IFN-γ production in the presence of α-galactosyl ceramide stimulation. TIM1 engagement increased GATA-3 expression but reduced T-bet expression in NKT cells in the presence of TCR engagement. The adoptive transfer of NKT cells preincubated with anti-TIM1 mAbs into Jα18−/− mice aggravated bleomycin-induced pulmonary fibrosis by suppressing IFN-γ production. Taken together, these results suggest that TIM1 costimulation on NKT cells enhances the cellular production of IL-4 while inhibiting the production of IFN-γ. Thus, as a differential regulator of the immune response, TIM1 on NKT cells may be a useful therapeutic target for immune diseases.

Although B cells have been shown to possess a regulatory function, microenvironmental factors or cytokines involved in the induction of regulatory B cells remain largely uncharacterized. B cell-activating factor (BAFF), a member of TNF family cytokines, is a key regulator for B cell maturation and function. In this study, we detected significantly increased numbers of IL-10–producing B cells in BAFF-treated B cell cultures, an effect specifically abrogated by neutralization of BAFF with TACI-Fc. BAFF-induced IL-10–producing B cells showed a distinct CD1dhiCD5+ phenotype, which were mainly derived from marginal zone B cells. Moreover, BAFF activated transcription factor AP-1 for binding to IL-10 promoter. Notably, BAFF treatment in vivo increased the number of IL-10–producing B cells in marginal zone regions. Furthermore, BAFF-induced IL-10–producing B cells possess a regulatory function both in vitro and in vivo. Taken together, our findings identify a novel function of BAFF in the induction of IL-10–producing regulatory B cells.

The prevalence of asthma continues to increase in westernized countries, and optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted invariant NKT (iNKT) cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in animal models and are associated with asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway disease, mice were sensitized with OVA and treated with di-palmitoyl-phosphatidyl-ethanolamine polyethylene glycol (DPPE-PEG), a CD1d-binding lipid antagonist. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltration of lymphocytes upon OVA challenge, but had no effect on the development of OVA-specific Th2 responses. In addition, DPPE-PEG completely prevented the development of AHR after administration of α-galactosylceramide (α-GalCer) intranasally. Furthermore, we demonstrate that DPPE-PEG acts as antagonist to α-GalCer and competes with α-GalCer for binding to CD1d. Finally, we show that DPPE-PEG completely inhibits the α-GalCer–induced phosphorylation of ERK tyrosine kinase in iNKT cells, suggesting that DPPE-PEG specifically blocks TCR signaling and thus activation of iNKT cells. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel approach to treat iNKT cell-mediated diseases such as asthma.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10−/− B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgMhiCD1dhiCD21hiCD23hi in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-α, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnαr−/−) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnαr−/− mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN–producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Certain glycolipid Ags for Vα14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, α-galactosylceramide. Although the CD1d-exposed portions of OCH and α-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-γ, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.

Pulmonary mast cell progenitor (MCp) numbers increase dramatically in sensitized and aerosolized Ag-challenged mice. This increase depends on CD4+ T cells, as no MCp increase occurs in the lungs of sensitized wild-type (WT) mice after mAb depletion of CD4+ but not CD8+ cells before aerosol Ag challenge. Neither the genetic absence of IL-4, IL-4Rα chain, STAT-6, IFN-γ, or IL-12p40 nor mAb blockade of IFN-γ, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12p40, or IL-12p40Rβ1 before Ag challenge in WT mice reduces the pulmonary MCp increase. However, sensitized and Ag-challenged IL-9-deficient mice and sensitized WT mice given mAb to IL-9 just before Ag challenge show significant reductions in elicited lung MCp/106 mononuclear cells of 47 and 66%, respectively. CD1d-deficient mice and WT mice receiving anti-CD1d before Ag challenge also show significant reductions of 65 and 59%, respectively, in elicited lung MCp/106 mononuclear cells, revealing an additional requirement for MCp recruitment. However, in Jα18-deficient mice, which lack only type 1 or invariant NKT cells, the increase in the numbers of lung MCp with Ag challenge was intact, indicating that their recruitment must be mediated by type 2 NKT cells. Furthermore, anti-CD1d treatment of IL-9-deficient mice or anti-IL-9 treatment of CD1d-deficient mice does not further reduce the significant partial impairment of MCp recruitment occurring with a single deficiency. These findings implicate type 2 NKT cells and IL-9 as central regulators that function in the same pathway mediating the Ag-induced increase in numbers of pulmonary MCp.

C-glycoside analogues of α-galactosylceramide were shown to activate both human and mouse invariant NKT (iNKT) cells. Among these analogues, GCK152, which has an aromatic ring in the acyl chain, exhibited a stronger stimulatory activity against human iNKT cells and a much weaker activity against murine iNKT cells than GCK127 that has an almost identical fatty acyl chain as α-galactosylceramide. In this study, we have found that invariant TCR (invTCR) expressed by iNKT cells, but not CD1d expressed by APCs, command the species-specific preferential activity of C-glycosides, and that their preferential activity against human vs murine iNKT cells correlate with the binding affinity of glycolipid-CD1d complex to invTCR of respective iNKT cells rather than that of glycolipid to human or murine CD1d molecules. Overall, the structural difference of invTCR appears to supersede those of CD1d molecule in shaping the strength of the biological activity of C-glycoside analogues.

The requirement for TLR signaling in the initiation of an Ag-specific Ab response is controversial. In this report we show that a novel OVA-expressing recombinant Salmonella vaccine (Salmonella-OVA) elicits a Th1-biased cell-mediated and serum Ab response upon oral or i.p. immunization of C57BL/6 mice. In MyD88−/− mice, Th1-dependent Ab responses are greatly reduced while Th2-dependent Ab isotypes are elevated in response to oral and i.p., but not s.c. footpad, immunization. When the T effector response to oral vaccination is examined we find that activated, adoptively transferred Ag-specific CD4+ T cells accumulate in the draining lymph nodes, but fail to produce IFN-γ, in MyD88−/− mice. Moreover, CD1d tetramer staining shows that invariant NKT cells are activated in response to oral Salmonella-OVA vaccination in wild-type, but not MyD88−/−, mice. Treatment with neutralizing Ab to CD1d reduces the OVA-specific Ab response only in MyD88-sufficient wild-type mice, suggesting that both Ag-specific CD4 T cell and invariant NKT cell effector responses to Salmonella-OVA vaccination are MyD88 dependent. Taken together, our data indicate that the type of adaptive immune response generated to this live attenuated vaccine is regulated by both the presence of MyD88-mediated signals and vaccination route, which may have important implications for future vaccine design.

Invariant NK T (iNKT) cells are a separate lineage of T lymphocytes with innate effector functions. They express an invariant TCR specific for lipids presented by CD1d and their development and effector differentiation rely on a unique gene expression program. We asked whether this program includes microRNAs, small noncoding RNAs that regulate gene expression posttranscriptionally and play a key role in the control of cellular differentiation programs. To this aim, we investigated iNKT cell development in mice in which Dicer, the RNase III enzyme that generates functional microRNAs, is deleted in cortical thymocytes. We find that Dicer deletion results in a substantial reduction of iNKT cells in thymus and their disappearance from the periphery, unlike mainstream T cells. Without Dicer, iNKT cells do not complete their innate effector differentiation and display a defective homeostasis due to increased cell death. Differentiation and homeostasis of iNKT cells require Dicer in a cell-autonomous fashion. Furthermore, we identify a miRNA profile specific for iNKT cells, which exhibits features of activated/effector T lymphocytes, consistent with the idea that iNKT cells undergo agonist thymic selection. Together, these results define a critical role of the Dicer-dependent miRNA pathway in the physiology of iNKT cells.

CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.

The positive selection of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated Ag presentation by CD4+CD8+ thymocytes. Maturation of newly selected iNKT cells continues in the periphery and also involves CD1d expression. CD1d molecules acquire Ags for presentation in endosomal compartments, to which CD1d molecules have access through an intrinsic CD1d-encoded tyrosine motif and by association with the class II MHC chaperone, invariant chain. In this study, we report the generation of mice in which all CD1d is replaced by CD1d-enhanced yellow fluorescent fusion protein (EYFP). CD1d-EYFP molecules are stable, present lipid Ags, and have near normal subcellular distribution. CD1d-EYFP molecules mediated positive selection of Vα14 iNKT cell precursors at decreased efficiency, caused a delay in their terminal maturation, and did not invoke Vα14 iNKT cell effector function as wild-type CD1d could. Using these mice, we show that the intrinsic CD1d-encoded sorting motif mediates thymic selection and activation of Vα14 iNKT cells by professional APCs, while for peripheral terminal differentiation the intrinsic CD1d sorting motif is dispensable.

APCs, such as dendritic cells (DC), can present glycolipid Ags on CD1d molecules to NKT cells. This interaction activates DC and NKT cells, leading to release of cytokines and enhanced T cell responses. Thus, glycolipid Ags are currently being tested as adjuvants for immunotherapy. We were interested in the interaction of murine skin DC with NKT cells in skin-draining lymph nodes. We observed that all skin DC subsets expressed CD1d upon migration to the lymph nodes. Moreover, skin DC were able to present the synthetic glycolipid Ag α-galactosylceramide (α-GalCer) to the NKT cell hybridoma DN32.D3. Intradermally injected α-GalCer was presented by migratory skin DC and lymph node DC to NKT hybridoma cells in vitro. When we injected α-GalCer intradermally into the skin, the numbers of various leukocyte subsets in the draining lymph nodes did not change significantly. However, T and B cells as well as NKT cells up-regulated the activation marker CD69. Coapplication of α-GalCer with the tumor model Ag OVA induced strong cytolytic CD8+ T cell function that could inhibit the growth of B16 melanoma cells expressing OVA. However, mice that were devoid of migratory skin DC developed similar cytotoxic immune responses after intradermal immunization, indicating that skin DC are not required for the adjuvant properties of NKT cell activation and Ag presentation by this immunization route. In conclusion, migratory skin DC are able to interact with NKT cells; however, intradermally applied glycolipids are presented predominantly by lymph node DC to NKT cells.

Splenic marginal zone B (MZB) lymphocytes represent, along with dendritic cells (DC) a first line of defense against blood-borne pathogens. MZB cells express high levels of MHC class II and CD1d molecules but so far their ability to activate and orientate conventional and innate-like T lymphocytes, such as invariant NKT (iNKT) cells, is still elusive. In the present study, we show that murine MZB cells proliferate, mature phenotypically, and secrete cytokines in response to TLR (except TLR3) agonists. When pulsed with OVA peptide (but not whole OVA), MZB cells promote the release of IFN-γ and IL-4 by Ag-specific CD4+ T lymphocytes and their stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN), a potent MZB cell activator, biases them toward more Th1 inducers. Unlike DC, CpG ODN-stimulated MZB cells fail to stimulate iNKT cells. Although able to activate iNKT hybridomas, MZB cells sensitized with free α-galactosylceramide (α-GalCer), a CD1d-restricted glycolipid Ag, do not directly activate ex vivo sorted iNKT cells unless DC are added to the culture system. Interestingly, MZB cells amplify the DC-mediated activation of iNKT cells and depletion of MZB cells from total splenocytes strongly reduces iNKT cell activation (cytokine production) in response to α-GalCer. Thus, DC and MZB cells provide help to each other to optimize iNKT cell stimulation. Finally, in vivo transfer of α-GalCer-loaded MZB cells potently activates iNKT and NK cells. This study confirms and extends the concept that MZB cells are important players in immune responses, a property that might be exploited.

CD1d is an MHC class I-like membrane glycoprotein that presents lipid Ags to NKT cells. Despite intensive biochemical, genetic, and structural studies, the endogenous lipids associated with CD1d remain poorly defined because of the biochemical challenges posed by their hydrophobic nature. In this study, we report the generation of a protease-cleavable CD1d variant with a similar trafficking pattern to wild-type CD1d that can be purified in the absence of detergent and allows the characterization of the naturally associated lipids. In addition, we used soluble variants of CD1d that are secreted or retained in the endoplasmic reticulum (ER) to survey their acquired lipids. By using multiple mass spectrometry methods, we found that CD1d retained in the ER is predominantly loaded with the most abundant phospholipid in the cell, phosphatidyl choline, while the protease cleavable version of CD1d contains bound sphingomyelin and lysophospholipids in addition to phosphatidyl choline. The secreted soluble version of CD1d, in contrast, lacks detectable phosphatidyl choline and the only detectable associated lipid is sphingomyelin. The data suggest that, in the absence of infection or stress, CD1d molecules survey the ER, the secretory pathway, and the endocytic pathway, and accumulate the most abundantly available lipids present in these compartments.

Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.

Vα14 invariant (Vα14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4+CD8+ Vα14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4+CD8+ thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Vα14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Vα14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Vα14i NKT cell precursors are blocked at an immature CD44lowNK1.1− stage in a cell autonomous fashion. Residual c-Myc-deficient immature Vα14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Vα14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Vα14i NKT cell development to promote progression beyond the CD44lowNK1.1− precursor stage.

The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant NKT (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. We now report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFN-γ, which we demonstrate ameliorates the severity of murine Lyme carditis by at least two mechanisms. First, IFN-γ enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Second, IFN-γ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate the severity of murine Lyme carditis through the action of IFN-γ, which appears to self-renew through a positive feedback loop during infection.

T-bet−/− mice have been shown to have a profound deficiency in the ability to generate invariant NKT (iNKT) cells in the periphery due to a halt in terminal maturation, but despite this deficiency, T-bet−/− mice develop spontaneous airway hyperreactivity (AHR) and airway inflammation. Because in some situations the development of AHR requires the presence of iNKT cells, we sought to more clearly understand how AHR develops in T-bet−/− mice by examining T-bet−/− mice in several distinct mouse models of asthma, including spontaneous, OVA-induced and α-galactosylceramide (α-GalCer)-induced AHR. Surprisingly, we found that administration of α-GalCer, which very specifically activates iNKT cells, greatly increased the AHR response in the T-bet−/− mice. Moreover, in T-bet−/− mice, spontaneous AHR as well as AHR induced with OVA or α-GalCer were all eliminated by blocking CD1d, the restricting element of iNKT cells, using an anti-CD1d-blocking mAb. Although the number of the iNKT cells in T-bet−/− mice was reduced compared with that in wild-type mice, the remaining iNKT cells produced primarily IL-4 and IL-13, and only minimal amounts of IFN-γ. We conclude therefore that the AHR that develops in T-bet−/− mice is dependent on the presence of iNKT cells, and that whereas T-bet−/− have reduced numbers of iNKT cells, these are sufficient for the development of AHR.

Invariant NKT (iNKT) cells recognize glycolipid Ags, such as the marine sponge-derived glycosphingolipid α-galactosylceramide (αGalCer) presented by the CD1d protein. In vivo activation of iNKT cells with αGalCer results in robust cytokine production, followed by the acquisition of an anergic phenotype. Here we have investigated mechanisms responsible for the establishment of αGalCer-induced iNKT cell anergy. We found that αGalCer-activated iNKT cells rapidly up-regulated expression of the inhibitory costimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of αGalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of αGalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1/PD-L pathway failed to prevent bacterial- or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1/PD-L interactions enhanced the antimetastatic activities of αGalCer. Collectively, our findings reveal a critical role for the PD-1/PD-L costimulatory pathway in the αGalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.

NKT cells have been described as innate regulatory cells because of their rapid response to conserved glycolipids presented on CD1d via their invariant TCR. However, little is known about the contribution of the hepatic NKT cell to the development of a local and/or systemic immune response to acute septic challenge (cecal ligation and puncture (CLP)). We found not only that mice deficient in invariant NKT cells (Jα18−/−) had a marked attenuation in CLP-induced mortality, but also exhibited an oblation of the systemic inflammatory response (with little effect on splenic/peritoneal immune responsiveness). Flow cytometric data indicated that following CLP, there was a marked decline in the percentage of CD3+α-galactosylceramide CD1d tetramer+ cells in the mouse C57BL/6J and BALB/c liver nonparenchymal cell population. This was associated with the marked activation of these cells (increased expression of CD69 and CD25) as well as a rise in the frequency of NKT cells positive for both Th1 and Th2 intracellular cytokines. In this respect, when mice were pretreated in vivo with anti-CD1d-blocking Ab, we observed not only that this inhibited the systemic rise of IL-6 and IL-10 levels in septic mice and improved overall septic survival, but that the CLP-induced changes in liver macrophage IL-6 and IL-10 expressions were inversely effected by this treatment. Together, these findings suggest that the activation of hepatic invariant NKT cells plays a critical role in regulating the innate immune/systemic inflammatory response and survival in a model of acute septic shock.

NKT cells comprise a rare regulatory T cell population of limited TCR diversity, with most cells using a Valpha14 Jalpha18 TCR. These cells exhibit a critical dependence on the signaling adapter molecule, signaling lymphocytic activation molecule-associated protein (SAP), for their ontogeny, an aspect not seen in conventional alphabeta T cells. Prior studies demonstrate that SAP enhances TCR-induced activation of NF-kappaB in CD4(+) T cells. Because NF-kappaB is required for NKT cell development, SAP might promote the ontogeny of this lineage by signaling to NF-kappaB. In this study, we demonstrate that forced expression of the NF-kappaB target gene, Bcl-x(L), or inhibitory NF-kappaB kinase beta, a catalytic subunit of the IkappaB kinase complex essential for NF-kappaB activation, fails to restore NKT cell development in sap(-/-) mice, suggesting that SAP mediates NKT cell development independently of NF-kappaB. To examine the role of SAP in NKT cell function, we generated NKT cells in sap(-/-) mice by expressing a transgene encoding the Valpha14 Jalpha18 component of the invariant TCR. These cells bound alpha-galactosylceramide-loaded CD1d tetramers, but exhibited a very immature CD24(+)NK1.1(-) phenotype. Although sap(-/-) tetramer-reactive cells proliferated in response to TCR activation, they did not produce appreciable levels of IL-4 or IFN-gamma. The reduction in cytokine production correlated with the near absence of GATA-3 and T-bet, key transcription factors regulating cytokine expression and maturation of NKT cells. Ectopic expression of GATA-3 partially restored IL-4 production by the NKT cells. Collectively, these data suggest that by promoting GATA-3 and T-bet expression, SAP exerts control over NKT cell development and mature NKT cell cytokine production.