iNKT Cell Numbers Research Articles

Common variable immunodeficiency is associated with a functional deficiency of invariant natural killer T cells

Common variable immunodeficiency (CVID) is the commonest symptomatic primary antibody disorder, with monogenic causes identified in less than 10% of all cases. X-linked proliferative disease is a monogenic disorder that is associated with hypogammaglobulinemia and characterized by a deficiency of invariant NKT (iNKT) cells. We sought to evaluate whether a defect in iNKT cell number or function was associated with CVID. An evaluation of the function and number of iNKT cells in CVID. Six-color flow cytometry enumerated iNKT cells in 36 patients with CVID and 50 healthy controls. Their proliferative capacity and cytokine production (IFN-γ, IL-13, IL-17) was then investigated following activation with CD1d ligand alpha-galactosylceramide. A reduction in the number of iNKT cells (31 iNKT cells/10(5) T cells) in patients with CVID compared with healthy controls (100 iNKT cells/10(5) T cells) was observed (P < .0001). Two cohorts could be discerned within the CVID group: group 1 with an abnormal number of iNKT cells (n = 28) and group 2 with a normal number of iNKT cells (n = 8). This segregation coassociated with the proliferative capacity of iNKT cells between the 2 groups. However, differences in the function of iNKT cells were noted in group 2, in which an increase in IFN-γ (P = .0016) and a decrease in IL-17 (P = .0002) production was observed between patients with CVID and controls. Finally, a significant association was seen between the number of iNKT cells and the percentage of class-switched memory B cells and propensity to lymphoproliferation (P = .002) in patients with CVID. iNKT cells are deficient and/or functionally impaired in most of the patients with CVID.

Mechanistic target of rapamycin complex 1 is critical for invariant natural killer T-cell development and effector function.

The mechanisms that control invariant natural killer T (iNKT)-cell development and function are still poorly understood. The mechanistic or mammalian target of rapamycin (mTOR) integrates various environmental signals/cues to regulate cell growth, proliferation, metabolism, and survival. We report here that ablation of mTOR complex 1 (mTORC1) signaling by conditionally deleting Raptor causes severe defects in iNKT-cell development at early stages, leading to drastic reductions in iNKT-cell numbers in the thymus and periphery. In addition, loss of Raptor impairs iNKT-cell proliferation and production of cytokines upon α-galactosylceramide stimulation in vitro and in vivo, and inhibits liver inflammation in an iNKT cell-mediated hepatitis model. Furthermore, Raptor deficiency and rapamycin treatment lead to aberrant intracellular localization and functional impairment of promyelocytic leukemia zinc-finger, a transcription factor critical for iNKT-cell development and effector programs. Our findings define an essential role of mTORC1 to direct iNKT-cell lineage development and effector function.

Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

Increased Th2-like Invariant Natural Killer T cells in Peripheral Blood From Patients With Asthma.

PurposeInvariant natural killer T (iNKT) cells might play an important role in asthma pathogenesis in humans. Our previous study found no difference in the number of blood iNKT cells between asthma patients and controls. However, few studies have examined the function of blood iNKT cells in human asthma.MethodsTwenty asthma patients and eight controls were included in this study. Blood iNKT cells were identified using double staining with anti-Vα24 and anti-Vβ11 monoclonal antibodies (mAbs) or with 6B11 and anti-Vβ11 mAbs. Intracellular IL-4, IL-10, and IFN-γ cytokines were stained in blood iNKT cells using their respective mAbs and isotypes. In addition, their relationships with clinical parameters were analyzed.ResultsThe number of Vα24+Vβ11+ iNKT cells or 6B11+Vβ11+ iNKT cells did not differ between asthma patients and controls. However, among Vα24+Vβ11+iNKT cells, the proportion of IL-4+iNKT cells was increased in asthma patients compared to controls (7.0±3.0% vs 0.5%±0.4%, P<0.05). There were no differences in the proportions of IL-10+or IFN-γ+iNKT cells between the groups. The proportion of IL-4+ cells among 6B11+Vβ11+iNKT cells inversely correlated with FEV1, expressed as a percentage predicted value in asthma patients (Rs=-0.64, P<0.05, n=19).ConclusionsBlood iNKT cells are thought to be Th2-like, and IL-4-producing iNKT cells may be associated with lung function in human asthma.

Sphingolipids from a Symbiotic Microbe Regulate Homeostasis of Host Intestinal Natural Killer T Cells

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

A divergent response of innate regulatory T-cells to sepsis in humans: Circulating invariant natural killer T-cells are preserved

BackgroundSepsis is associated with severe immunosuppression, evidenced by loss and dysfunction of CD3+ lymphocytes and γδ-TCR+ T-cells. There is limited data addressing changes in the invariant natural killer T-(iNKT) cell population with sepsis, and whether such changes correlate with clinical outcomes. Specifically, septic geriatric patients have marked mortality. How γδ-TCR+ T-cells and iNKT-cells are altered in the settings of sepsis and advanced age, and how these changes correlate with mortality are unknown. Methods49 young (18–50years) and 55 geriatric (>65years) ICU patients with confirmed sepsis were enrolled. Blood was stained with antibodies to detect the percentage and absolute number of CD3+ (T-cells), γδ-TCR+ T-cell, TCR-Vα-24+ (iNKT-cells), and CD69+ (marker of cell activation). Blood from 10 healthy controls was also collected. ResultsSeptic patients displayed marked leukocytosis, decreased CD3+ lymphocytes, and γδ-TCR+ T-cells, and increased percentage and number of iNKT-cells. Young and geriatric patients had similar degree of leukocytosis, along with percentage, number, and %CD69+ CD3+ T-cell and γδ-TCR+ T-cells; however, percentage, number, and %CD69+iNKT-cells were most markedly elevated in geriatric patients. Geriatric non-survivors had higher percentage and number of, but decreased %CD69+, iNKT-cells vs survivors. ConclusionsiNKT-cells are increased in sepsis, suggesting that they typify an evolving morbid state. This is most pronounced in geriatric non-survivors, a group demonstrating dysfunctional regulatory iNKT-cell phenotype.

Effect of invariant natural killer T cells with IL-5 and activated IL-6 receptor in ventilator-associated lung injury in mice

ABSTRACTMechanical ventilation (MV) is well known to potentially cause ventilator-associated lung injury (VALI). It has also been reported recently that activation of invariant natural killer T (iNKT) cells is involved in the onset/progression of airway inflammation. We analyzed the roles of inflammatory cells, including iNKT cells, and cytokines/chemokines in a mouse model of VALI. C57BL/6 and Vα14+NKT cell-deficient (Jα18KO) female mice were subjected to MV for 5 hours. The MV induced lung injury in the mice, with severe histological abnormalities, elevation in the percentages of neutrophils in the bronchoalveolar lavage fluid (BALF), and increase in the number of iNKT cells in the lung. Jα18KO mice subjected to MV for 5 hours also showed lung injury, with decrease of the PaO2/FiO2 ratio (P/F ratio) and elevation of the levels of total protein, IL-5, IL-6, IL-12p40, and keratinocyte-derived cytokine (KC) in the BALF. Intranasal administration of anti-IL-5 monoclonal antibody (mAb) or anti-IL-6 receptor (IL-6R) mAb into the Jα18KO mice prior to the start of MV resulted in significant improvement in the blood oxygenation. In addition, the anti-IL-5 mAb administration was associated with a decrease in the levels of IL-5, IL-9, and IL-6R in the BALF, and anti-IL-6R mAb administration suppressed the mRNA expressions of IL-5, IL-6, IL-6R, and KC. These results suggest that iNKT cells may play a role in attenuating the inflammatory caused by ventilation through IL-5 and IL-6R.

Combined deletion of Id2 and Id3 genes reveals multiple roles for E proteins in invariant NKT cell development and expansion.

The invariant NKT (iNKT) cells represent a unique group of αβ T cells that have been classified based on their exclusive usage of the invariant Vα14Jα18 TCRα-chain and their innate-like effector function. Thus far, the transcriptional programs that control Vα14Jα18 TCRα rearrangements and the population size of iNKT cells are still incompletely defined. E protein transcription factors have been shown to play necessary roles in the development of multiple T cell lineages, including iNKT cells. In this study, we examined E protein functions in T cell development through combined deletion of genes encoding E protein inhibitors Id2 and Id3. Deletion of Id2 and Id3 in T cell progenitors resulted in a partial block at the pre-TCR selection checkpoint and a dramatic increase in numbers of iNKT cells. The increase in iNKT cells is accompanied with a biased rearrangement involving Vα14 to Jα18 recombination at the double-positive stage and enhanced proliferation of iNKT cells. We further demonstrate that a 50% reduction of E proteins can cause a dramatic switch from iNKT to innate-like γδ T cell fate in Id2- and Id3-deficient mice. Collectively, these findings suggest that Id2- and Id3-mediated inhibition of E proteins controls iNKT development by restricting lineage choice and population expansion.

A Phase I Single Ascending Dose Study Of NKTT120 In Stable Adult Sickle Cell Patients

▪ BackgroundMultiple lines of evidence suggest that invariant NKT (iNKT) cells generate an inflammatory cascade that promotes and sustains sickle cell vaso-occlusion. In prior studies of mouse models and patients with sickle cell disease (SCD), iNKT cells are increased in number and more likely to be activated compared to controls. Depleting iNKT cells in a mouse model of SCD decreases inflammation and prevents end-organ injury. NKTT120 is a humanized monoclonal antibody that specifically depletes iNKT cells. Preclinical studies show that NKTT120 has high affinity and specificity for iNKT cells. NKTT120 depletes iNKT cells in a dose-dependent manner. Return of iNKT cells to the peripheral circulation following NKTT120 administration occurs in a dose- and time-dependent manner. Our global hypothesis is that NKTT120 will deplete iNKT cells, reduce inflammation and prevent painful vaso-occlusive crises. In this phase 1 dose-escalation study, we will examine the safety of NKTT120 in steady state adults with SCD. ObjectiveTo determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in steady state adults with SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. MethodsPhase 1 study utilizing a 3+3 design to evaluate single doses escalated over a range from 0.001 mg/kg to 0.1 mg/kg (0.001, 0.003, 0.01, 0.03, and 0.10 mg/kg). Primary outcome measure is safety. Secondary outcomes include pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects will maintain a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires will be administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. ResultsOne month of follow-up data on iNKT cell numbers is available for the first four patients in the study (Figure 1). Three subjects received the lowest dose of 0.001 mg/kg and 1 subject received 0.003 mg/kg. All 4 subjects showed a reduction in the iNKT cell percent of CD3+ T cells 6 hours after NKTT120 administration. Three subjects have completed the study with iNKT cells returning to pre-dosing levels at day 7 while the remaining subject is awaiting iNKT cell recovery five weeks after dosing. The effects of NKTT120 were specific to iNKT cells as T cell, B cell and NK cell percent of lymphocytes was not affected. NKTT120 has been well tolerated with no adverse events reported. [Display omitted] ConclusionsIn steady state adults with SCD, NKTT120 administered at the lowest dose of 0.001 mg/kg specifically reduces iNKT cells without toxicity. The dose of 0.003 mg/kg is currently being evaluated in this ongoing trial and higher doses of NKTT120 are anticipated to further deplete iNKT cells in the blood and tissue with longer times to recovery. This reduction of iNKT cells should result in a suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures:Field:NKT Therapeutics: Consultancy. Eaton:NKT Therapeutics: Employment, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy.

Antitumor Impact of Interferon-γ Producing CD1d-restricted NKT Cells in Murine Malignant Mesothelioma

CD1d-restricted natural killer T (iNKT) cells have been shown to provide adjuvant activity against cancer by producing interferon (IFN)-γ. However, the role of invariant NKT (iNKT) cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the antitumor effect of iNKT cells in the tumor microenvironment by using an intrathoracic murine malignant pleural mesothelioma model that we had previously developed and to provide pleural effusion as a good surrogate of the tumor microenvironment. We found that the number of iNKT cells increased dramatically in the pleural effusion after intrathoracic tumor cell injection at an earlier phase compared with accumulation of CD8 T cells. These iNKT cells showed increased expression of CD25 and increased ratio of cells positive for IFN-γ intracellular staining. iNKT cells sorted from pleural effusion of tumor burden mice produced larger amount of IFN-γ compared with naive mice. Mice pretreated in vivo with anti-CD1d-blocking Ab showed increased amount of pleural effusion and decreased ratio of total and effector-type CD8 T cells as well as decreased intracellular IFN-γ expression of CD8T-cell in the pleural effusion. In vivo administration of α-galactosylceramide (α-GalCer) showed prolonged survival associated with less pleural effusion and increased ratio of IFN-γ-positive iNKT cells and CD8 T cells in the pleural effusion. Therefore, this study suggests that iNKT cells accumulating in the tumor microenvironment play an antitumor effect by producing IFN-γ and enhancing subsequent CD8 T-cell response. Furthermore, in vivo administration of α-GalCer could suppress mesothelioma growth by activating iNKT cells.

Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr(-/-)) background and control Ldlr(-/-) mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr(-/-) mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.

AB0190 Synovial fluid derived invariant natural killer T cells in chronic arthritides show an increased programmed death-1 expression and anergic phenotype

BackgroundOne potential factor for chronicity in arthritides such as rheumatoid arthritis (RA) and spondyloarthritis (SpA) is a suboptimal feedback by regulatory T cells. invariant Natural Killer T (iNKT) cells are...

SP0086 Regulatory B cells in healthy and in patients with SLE

Recent advances have demonstrated the existence of a B cell subset with regulatory function named (Bregs). Bregs have been shown to restrain the severity of several autoimmune disease and contribute...

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

BATF regulates the development and function of IL-17 producing iNKT cells

BackgroundBATF plays important roles in the function of the immune system. Batf null mice are deficient in both CD4+ Th17 cells and T follicular helper cells and possess an intrinsic B cell defect that leads to the complete absence of class switched Ig. In this study, Tg mice overexpressing BATF in T cells were used together with Batf null mice to investigate how altering levels of BATF expression in T cells impacts the development and function of a recently characterized population of iNKT cells expressing IL-17 (iNKT-17).ResultsBATF has a direct impact on IL-17 expression by iNKT cells. However, in contrast to the Th17 lineage where BATF activates IL-17 expression and leads to the expansion of the lineage, BATF overexpression restricts overall iNKT cell numbers while skewing the compartment in vivo and in vitro toward an iNKT-17 phenotype.ConclusionsThis work is the first to demonstrate that BATF joins RORγt as the molecular signature for all IL-17 producing cells in vivo and identifies BATF as a component of the nuclear protein network that could be targeted to regulate IL-17-mediated disease. Interestingly, these studies also reveal that while the Il17a gene is a common target for BATF regulation in Th17 and iNKT-17 cells, this regulation is accompanied by opposite effects on the growth and expansion of these two cell lineages.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2(-/-) mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

Invariant natural killer T cells are phenotypically and functionally altered in Fabry disease

Fabry disease is a lysosomal storage disease belonging to the group of sphingolipidoses.In Fabry disease there is accumulation of mainly globotriaosylceramide due to deficiency of the lysosomal enzyme α-galactosidase A. The lysosome is an important compartment for the activity of invariant natural killer T (iNKT) cells. iNKT cells are lipid-specific T cells that were shown to be important in infection, autoimmunity and tumor surveillance. In several mouse models of lysosomal storage disorders there is a decrease in iNKT cell numbers. Furthermore, alterations on iNKT cell subsets have been recently described in the Fabry disease mouse model. Herein, we analyzed iNKT cells and their subsets in Fabry disease patients. Although there were no differences in the percentage of iNKT cells between Fabry disease patients and control subjects, Fabry disease patients presented a reduction in the iNKT CD4+ cells accompanied by an increase in the iNKT DN cells. Since iNKT cell subsets produce different quantities of pro-inflammatory and anti-inflammatory cytokines, we analyzed IFN-γ and IL-4 production by iNKT cells of Fabry disease patients and mice.We found a significant reduction in the production of IL-4 by mice splenic iNKT cells and human iNKT cell subsets, but no significant alterations in the production of IFN-γ. Altogether, our results suggest a bias towards a pro-inflammatory phenotype in Fabry disease iNKT cells.

Direct identification of rat iNKT cells reveals remarkable similarities to human iNKT cells and a profound deficiency in LEW rats

iNKT cells are a particular lymphocyte population with potent immunomodulatory capa-city; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α-chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d-dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αβ T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ-specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell-based therapies and of iNKT-cell biology.

Commensal Bacteria Play an Important Role in Ulcerative Colitis via Bacterial Sphingolipids-Mediated Regulation of Host Invariant Natural Killer T Cells

Our group has shown previously that microbiota can impact ulcerative colitis disease phenotypes by modulating invariant natural killer T (iNKT) cell functions. We further found that chemokine CXCL16 is responsible for this modulation, with germ-free (GF) mice having high CXCL16 level, large iNKT cell numbers and severe experimental ulcerative colitis outcome. In this study, we hypothesized that in addition to CXCL16 regulated functions, intestinal microbiota can directly impact iNKT cells by providing bacterial antigen, such as glycosphingolipids. We generated a Bacteroides fragilis mutant (del SPT) that no longer produces any sphingolipids. We then mono-colonized GF mice using wild-type or del SPT mutant. Theses mice were further challenged with an experimental ulcerative colitis model—oxazolone colitis model and the disease phenotypes were evaluated. Results: We found that the wild-type B. fragilis mono-colonized mice have significantly lower colonic iNKT cell numbers than the mutant mono-colonized mice, which are similar to the GF mice. CXCL16 levels in both of the mono-colonized mice are as low as that of the conventional mice, indicating the existence of a new pathway for colonic iNKT cell regulation. When challenged in the oxazolone colitis model, the mutant-colonized mice were much more susceptible than the wild-type-colonized mice. These phenotypes were diminished when the mice were treated with CD1d antibody, indicating iNKT cell-specific functions were involved. Our study demonstrated that microbiota regulate colonic iNKT cells via a new and unknown pathway. This pathway uses components in bacterial sphingolipid biosynthesis and it points to a new direction for therapeutic intervention of ulcerative colitis.