Maturation Of iNKT Cells Research Articles

The TNF superfamily cytokine receptor Death Receptor 3 is a costimulatory molecule for iNKT cells

Abstract Invariant NKT (iNKT) cells are a small population of thymus-generated T lymphocytes with innate-like phenotype and function that play a disproportionally important role in immune regulation and surveillance. iNKT cells are generated upon strong agonistic TCR signaling by glycolipid-loaded CD1d molecules, which differs from conventional T cells that are positively selected by weak TCR engagement with peptide-loaded MHC molecules. Whether iNKT cells are also distinct in their costimulatory requirement, however, has remained largely unresolved. Here, we report that the TNF superfamily receptor death receptor 3 (DR3) plays a previously unappreciated costimulatory role in peripheral iNKT cell activation. We found that the co-injection of agonistic anti-DR3 antibodies significantly augmented the activation and proliferation of mature iNKT cells in α-GalCer injected mice. Moreover, iNKT cells of α-GalCer and anti-DR3 co-injected mice dramatically increased their expression of proinflammatory cytokines, resulting in severe thymic atrophy. The DR3 costimulatory effect was strictly dependent on iNKT cells, because iNKT-deficient Traj18−/−mice were refractory to α-GalCer and anti-DR3 costimulation-induced inflammation. Because DR3 ligation alone did not suffice to activate iNKT cells, and DR3 exerted its effects only in the context of TCR costimulation, these results strongly support DR3 being a new bona fidecostimulatory molecule for iNKT cells. Considering the interest in utilizing iNKT cells in adoptive T cell therapies and as anti-tumor bioreagents, employing DR3 as a tool to boost iNKT cell immunity will open new venues in translational and clinical research. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Cullin 3-Mediated Regulation of Intracellular Iron Homeostasis Promotes Thymic Invariant NKT Cell Maturation.

The E3 ubiquitin ligase cullin 3 (Cul3) is critical for invariant NKT (iNKT) cell development, as iNKT cells lacking Cul3 accumulate in the immature developmental stages. However, the mechanisms by which Cul3 mediates iNKT cell development remain unknown. In this study, we investigated the role of Cul3 in both immature and mature thymic iNKT cells using a mouse model with a T cell-specific deletion of Cul3. We found that mature iNKT cells lacking Cul3 proliferated and died more than wild-type cells did. These cells also displayed increased glucose metabolism and autophagy. Interestingly, we found that tight regulation of iron homeostasis is critical for iNKT cell development. Without Cul3, mature iNKT cells harbored higher levels of cytosolic iron, a phenotype associated with increased cell death. Taken together, our data suggest that Cul3 promotes iNKT cell development partially through intracellular iron homeostasis.

METTL14-dependent m6A modification controls iNKT cell development and function.

N6-methyladenosine (m6A), the most common form of RNA modification, controls CD4+ T cell homeostasis by targeting the IL-7/STAT5/SOCS signaling pathways. The role of m6A modification in unconventional T cell development remains unknown. Using mice with T cell-specific deletion of RNA methyltransferase METTL14 (T-Mettl14−/−), we demonstrate that m6A modification is indispensable for iNKT cell homeostasis. Loss of METTL14-dependent m6A modification leads to the upregulation of apoptosis in double-positive thymocytes, which in turn decreases Vα14-Jα18 gene rearrangements, resulting in drastic reduction of iNKT numbers in the thymus and periphery. Residual T-Mettl14−/− iNKT cells exhibit increased apoptosis, impaired maturation, and decreased responsiveness to IL-2/IL-15 and TCR stimulation. Furthermore, METTL14 knockdown in mature iNKT cells diminishes their cytokine production, correlating with increased Cish expression and decreased TCR signaling. Collectively, our study highlights a critical role for METTL14-dependent-m6A modification in iNKT cell development and function.

A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs.

Many aspects of the porcine immune system remain poorly characterized, which poses a barrier to improving swine health and utilizing pigs as preclinical models. Here, we employ single-cell RNA sequencing (scRNA-seq) to create a cell atlas of the early-adolescent pig thymus. Our data show conserved features as well as species-specific differences in cell states and cell types compared with human thymocytes. We also describe several unconventional Tcell types with gene expression profiles associated with innate effector functions. This includes a cell census of more than 11,000 differentiating invariant natural killer T (iNKT) cells, which reveals that the functional diversity of pig iNKT cells differs substantially from the iNKT0/1/2/17 subset differentiation paradigm established in mice. Our data characterize key differentiation events in porcine thymopoiesis and iNKT cell maturation and provide important insights into pig Tcell development.

Mettl14-dependent m6A modification controls iNKT cells development and function

Abstract N6-Methyladenosine (m6A), the most common form of RNA modification, controls CD4+ T cell homeostasis by targeting the IL-7/STAT5/SOCS signaling pathways. The role of m6A modification in unconventional T cell development remains unknown. Using mice with T cell-specific deletion of RNA methyltransferase METTL14 (T-Mettl14 −/−), we demonstrated that m6A modification was indispensable for iNKT cell homeostasis. Loss of METTL14-dependent m6A modification led to the upregulation of p53-mediated apoptosis in double positive thymocytes, which in turn decreased distal Va-Ja gene rearrangements, including the invariant Va14-Ja18 TCR, resulting in a drastic reduction of iNKT numbers in the thymus and periphery. Residual T-Mettl14−/− iNKT cells exhibited increased apoptosis, impaired maturation, and decreased responsiveness to IL-2/IL-15 and TCR stimulation. Furthermore, METTL14 knockdown in mature iNKT cells diminished their cytokine production, correlating with increased Cish expression and decreased TCR signaling. Collectively, our study highlights a critical role for METTL14-dependent-m6A modification in iNKT cell development and function. This work was supported by NIH grants R01 AI41083 and R01 AI057460 to C.-R.W.

Effects of TNF-α deletion on iNKT cell development, activation, and maturation in the steady-state and chronic alcohol-consuming mice.

Cytokines play critical roles in regulating iNKT cell development, activation, and maturation. TNF-α co-occurs with iNKT cells in steady-state and many disease conditions. How TNF-α affects iNKT cell function has not been thoroughly investigated. It was found that chronic alcohol consumption enhanced iNKT cell activation and maturation. The underlying mechanism is not known. Herein, a TNF-α KO mouse model was used to address these issues. It was found that the depletion of TNF-α mitigated alcohol consumption-enhanced iNKT cell activation and maturation. In steady-state, depletion of TNF-α did not affect the frequency of iNKT cells in the thymus and spleen but decreased iNKT cells in the liver and increased liver iNKT cell apoptosis. The portion of stage-2 immature iNKT cells increased, stage-3 mature iNKT cells decreased in the thymus of TNF-α KO mice, suggesting that depletion of TNF-α impairs iNKT cell development and maturation. The percentage of CD69+ iNKT cells was significantly lower in the thymus, spleen, and liver of TNF-α KO mice compared to their wild-type littermates, suggesting that depletion of TNF-α inhibits iNKT cell activation. Moreover, the percentage of splenic IL-4- and IFN-γ-producing iNKT cells was significantly lower in TNF-α KO mice than in their wild-type littermates. The depletion of TNF-α increased PLZF+ iNKT cells in the thymus and down-regulated the expression of CD122 on iNKT cells. Collectively, these results support that TNF-α plays a vital role in the regulation of iNKT cell development, activation, and maturation, and alcohol consumption enhances iNKT cell activation and maturation through TNF-α.

Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of "innate-like" T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1-/- ), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1-/- mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1-/- mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Regulation of the terminal maturation of iNKT cells by mediator complex subunit 23

Invariant natural killer T cells (iNKT cells) are a specific subset of T cells that recognize glycolipid antigens and upon activation rapidly exert effector functions. This unique function is established during iNKT cell development; the detailed mechanisms of this process, however, remain to be elucidated. Here the authors show that deletion of the mediator subunit Med23 in CD4+CD8+ double positive (DP) thymocytes completely blocks iNKT cell development at stage 2. This dysregulation is accompanied by a bias in the expression of genes related to the regulation of transcription and metabolism, and functional impairment of the cells including the loss of NK cell characteristics, reduced ability to secrete cytokines and attenuated recruitment capacity upon activation. Moreover, Med23-deficient iNKT cells exhibit impaired anti-tumor activity. Our study identifies Med23 as an essential transcriptional regulator that controls iNKT cell differentiation and terminal maturation.

IAP Antagonists Enhance Cytokine Production from Mouse and Human iNKT Cells.

Inhibitor of apoptosis protein (IAP) antagonists are in clinical trials for a variety of cancers, and mouse models show synergism between IAP antagonists and anti-PD-1 immunotherapy. Although IAP antagonists affect the intrinsic signaling of tumor cells, their most pronounced effects are on immune cells and the generation of antitumor immunity. Here, we examined the effects of IAP antagonism on T-cell development using mouse fetal thymic organ culture and observed a selective loss of iNKT cells, an effector cell type of potential importance for cancer immunotherapy. Thymic iNKT-cell development probably failed due to increased strength of TCR signal leading to negative selection, given that mature iNKT cells treated with IAP antagonists were not depleted, but had enhanced cytokine production in both mouse and human ex vivo cultures. Consistent with this, mature mouse primary iNKT cells and iNKT hybridomas increased production of effector cytokines in the presence of IAP antagonists. In vivo administration of IAP antagonists and α-GalCer resulted in increased IFNγ and IL-2 production from iNKT cells and decreased tumor burden in a mouse model of melanoma lung metastasis. Human iNKT cells also proliferated and increased IFNγ production dramatically in the presence of IAP antagonists, demonstrating the utility of these compounds in adoptive therapy of iNKT cells. Cancer Immunol Res; 6(1); 25-35. ©2017 AACR.

Critical Role for Very-Long Chain Sphingolipids in Invariant Natural Killer T Cell Development and Homeostasis.

The role of sphingolipids (SLs) in the immune system has come under increasing scrutiny recently due to the emerging contributions that these important membrane components play in regulating a variety of immunological processes. The acyl chain length of SLs appears particularly critical in determining SL function. Here, we show a role for very-long acyl chain SLs (VLC-SLs) in invariant natural killer T (iNKT) cell maturation in the thymus and homeostasis in the liver. Ceramide synthase 2-null mice, which lack VLC-SLs, were susceptible to a hepatotropic strain of lymphocytic choriomeningitis virus, which is due to a reduction in the number of iNKT cells. Bone marrow chimera experiments indicated that hematopoietic-derived VLC-SLs are essential for maturation of iNKT cells in the thymus, whereas parenchymal-derived VLC-SLs are crucial for iNKT cell survival and maintenance in the liver. Our findings suggest a critical role for VLC-SL in iNKT cell physiology.

MiR150 over-expression impairs iNKT cell development, maturation, and function

Abstract CD1d-restricted invariant natural killer T (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNAs have been shown to be critically involved in iNKT cell development, maturation, and function, but the roles of specific miRNAs in it remain not completely understood. We had previously shown the dynamic upregulation of miR-150 during thymus iNKT cell maturation and identified the interrupted iNKT final stage maturation and elevated IFNγ production in miR-150KO mice. To determine the requirement of dynamic expressional changes of miR150 during iNKT cell development, we generated thymus-specific miR150 knock in mice (miR-150KI). MiR-150 over expression resulted in reduced overall iNKT cell number in both thymus and periphery, which could be related to increased apoptosis. MiR-150KI thymus iNKT cells showed the blockade of iNKT maturation at multiple maturation transition points. Splenic iNKT with miR-150KI displayed interrupted maturation and reduced cytokine production, including IFNγ, IL-4, and TNFα, upon in vitro stimulation. Furthermore, Bone marrow chimera study confirmed the effect of miR-150KI on iNKT cell development, maturation, and function are cell autonomous. Altogether, our results indicated that the tight control of miR150 expression is required in iNKT cell development and function. Detailed molecular mechanisms of miR-150 mediated iNKT cell regulation in different developmental stages and activation status are currently under investigation.

Temporal regulation of Wnt/β-catenin signaling is important for invariant NKT cell development and terminal maturation.

The Wnt/β-catenin signaling pathway plays important roles during various cellular functions including survival and proliferation of immune cells. The critical role of this pathway in conventional T cell development is established but little is known about its contributions to innate T cell development. In this study, we found that β-catenin level, an indication of the strength of Wnt/β-catenin signaling, is regulated during invariant NKT (iNKT) cell development. β-catenin levels were greatly increased during iNKT cell selection from double positive thymocytes to Stage 0 of iNKT cell development and during subsequent development to Stage 1. Thereafter, β-catenin levels decrease from Stage 2, which is essential for the terminal maturation of iNKT cells. Failure to dampen Wnt/β-catenin signaling as in mice expressing a stabilized active form of β-catenin (CATtg) resulted in increased Stage 2 and decreased Stage 3 iNKT cells. Inefficient transition from Stage 2 to 3 in CATtg iNKT cells seems to be contributed by poor expression of IL-15R (CD122) and transcription factor T-bet, both of which are necessary for terminal maturation of iNKT cells in the thymus. Consequently, IFN-γ+ iNKT cells were greatly reduced in CATtg mice. Together, our findings reveal that proper regulation of β-catenin and in turn Wnt signaling plays an important role in the terminal maturation and function of iNKT cells.

MTORC2 regulates multiple aspects of NKT-cell development and function.

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN-γ, IL-4 and IL-17A, iNKT-cells are classified as NKT-1, NKT-2, and NKT-17 subsets, respectively. The molecular pathways regulating iNKT-cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT-cell subsets, however these reports are conflicting. To resolve these questions, we used Rictorfl/fl CD4cre+ mice and found that Rictor is required for NKT-17 cell development and normal iNKT-cell cytolytic function. Conversely, Rictor is not absolutely required for IL-4 and IFN-γ production as peripheral iNKT-cells make copious amounts of these cytokines. Overall iNKT-cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT-cells. Thus, mTORC2 regulates multiple aspects of iNKT-cell development and function.

MicroRNA-183/96/182 cluster regulates iNKT cell development and function (IRM7P.706)

Abstract MicroRNAs (miRNAs), a class of non-coding small RNAs. Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function (PNAS, 2009). However, the roles of specific miRNA in iNKT cell development and function remain unclear. The miR-183/96/182 cluster, composed of 3 miRNA genes, is significantly upregulated upon T cell activation. In this study, we investigate the role of miR-183/96/182 cluster on development and function of iNKT cells, using miR-183/96/182 knockout (KO) mice. We found that the frequency of immature iNKT cells significantly increased, while mature iNKT cells significantly decreased in thymus of KO mice compared to littermate control (P<0.05), indicating the blockage of iNKT cell development in thymus. Furthermore, lack of miR-183/96/182 resulted in a substantial reduction of iNKT cell number (p<0.05) in the thymus and in spleen. The mutant iNKT cells displayed defective cytokine production, including IL-4 and IFNγ, compared to iNKT cells from littermate control (P<0.05). Finally, bone marrow chimera transferring experiments further suggested the miR-183/96/182 cluster controlled iNKT cell development and function by cell-intrinsic characteristics. Thus, our data define a specific role of miR-183/96/182 cluster in the development and function of iNKT cells.

STAT3 regulates iNKT cell development and function but is not required for NKT17 cell development (LYM6P.715)

Abstract Signal transducer and activator of transcription 3 (STAT3) controls the expansion of T help 17 cell (Th17) lineage through induction of transcription factor orphan nuclear receptor (RORγt). CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses and have three sublineages, NKT1, NKT2, and NKT17 cells. However, it still remains unclear how to regulate NKT17 cell development. Using lymphocyte-specific STAT3 hyperactivation mice, we found that overactivation of STAT3 universally suppressed iNKT cell cytokine secretion, including IL-17, while it enhanced conventional Th17 cell development. Consistently, STAT3 hyperactivation reduces the RORγt expression of iNKT cells. Surprisingly, bone marrow chimera experiments revealed that STAT3 overactivation disturbed iNKT cell maturation and increased IFNγ- and IL-4-secreting iNKT cells while it did not affect IL-17 secreting iNKT cells and did not alter RORγt expression of iNKT cells. Our results demonstrate that STAT3 regulates the development and function of iNKT cells, but unlike conventional Th17 cells, STAT3 is not required for IL-17 production and RORγt expression in iNKT cells.

Regulated Expression of miR-155 is Required for iNKT Cell Development.

Invariant natural killer T cells (iNKT cells) are CD1d-restricted, lipid antigen-reactive T lymphocytes with immunoregulatory functions. iNKT cell development in the thymus proceeds through subsequent stages, defined by the expression of CD44 and NK1.1, and is dictated by a unique gene expression program, including microRNAs. Here, we investigated whether miR-155, a microRNA involved in differentiation of most hematopoietic cells, played any role in iNKT cell development. To this end, we assessed the expression of miR-155 along iNKT cell maturation in the thymus, and studied the effects of miR-155 on iNKT cell development using Lck-miR-155 transgenic mice, which over express miR-155 in T cell lineage under the lymphocyte-specific protein tyrosine kinase (Lck) promoter. We show that miR-155 is expressed by newly selected immature wild-type iNKT cells and turned off along iNKT cells differentiation. In transgenic mice, miR-155 over-expression resulted in a substantial block of iNKT cell maturation at Stage 2, in the thymus toward an overall reduction of peripheral iNKT cells, unlike mainstream T cells. Furthermore, the effects of miR-155 over-expression on iNKT cell differentiation were cell autonomous. Finally, we identified Ets1 and ITK transcripts as relevant targets of miR-155 in iNKT cell differentiation. Altogether, these results demonstrate that a tight control of miR-155 expression is required for the development of iNKT cells.

Chronic alcohol consumption enhances iNKT cell maturation and activation

Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1− iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1+CD44hi mature iNKT cells but does not alter the number of NK1.1− immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1− iNKT cells, especially the NK1.1−CD44lo Stage I iNKT cells. The percentage of NKG2A+ iNKT cells increases in all of the tissues and organs examined; whereas CXCR3+ iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response.

Jarid2 is induced by TCR signalling and controls iNKT cell maturation

Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.

The Differential Roles of mTOR, ERK, and JNK Pathways in Invariant Natural Killer T-cell Function and Survival

Invariant natural killer T (iNKT) cell is a critical element for both innate and adaptive immunity. The quick responses of mature iNKT cells to TCR stimulation require activation of several different signaling pathways. However, the roles of these signaling pathways in mature iNKT cell biology remain incompletely understood. To address this issue, single signaling pathway was blocked with inhibitor in iNKT cells, and the roles of these signaling pathways were estimated. Results showed that mammalian target of rapamycin (mTOR) plays an essential role for cytokine production and survival in iNKT cells. In contrast, ERK and JNK are more important for iNKT cell effector function, but not survival. Our findings delineate the distinct roles of different signaling pathways in mature iNKT cells and therefore shed new light for modulating iNKT cell function in disease conditions.

The transcriptional repressor NKAP is required for iNKT cell development and maturation (HEM3P.274)

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.