iNKT Cell Function Research Articles

Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-γ-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-γ expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-γ production, but also the protective function of iNKT cells in arthritis.

Tissue-specific functions of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are an innate-like T cell subset that expresses an invariant T cell receptor (TCR) α-chain and recognizes lipids presented on CD1d. They secrete diverse cytokines and can influence many types of immune responses. Despite having highly similar TCR specificities, iNKT cells differentiate in the thymus into distinct subsets that are analogous to T helper 1 (TH1), TH2 and TH17 cell subsets. Additional iNKT cell subsets that may require peripheral activation have also been described, including one that produces IL-10. In general, iNKT cells are non-circulating, tissue-resident lymphocytes, but the prevalence of different iNKT cell subsets differs markedly between tissues. Here, we summarize the functions of iNKT cells in four tissues in which they are prevalent, namely, the liver, the lungs, adipose tissue and the intestine. Importantly, we explain how local iNKT cell responses at each site contribute to tissue homeostasis and protection from infection but can also contribute to tissue inflammation and damage.

Insights Into Mucosal-Associated Invariant T Cell Biology From Studies of Invariant Natural Killer T Cells.

Mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells are innate-like T cells that function at the interface between innate and adaptive immunity. They express semi-invariant T cell receptors (TCRs) and recognize unconventional non-peptide ligands bound to the MHC Class I-like molecules MR1 and CD1d, respectively. MAIT cells and iNKT cells exhibit an effector-memory phenotype and are enriched within the liver and at mucosal sites. In humans, MAIT cell frequencies dwarf those of iNKT cells, while in laboratory mouse strains the opposite is true. Upon activation via TCR- or cytokine-dependent pathways, MAIT cells and iNKT cells rapidly produce cytokines and show direct cytotoxic activity. Consequently, they are essential for effective immunity, and alterations in their frequency and function are associated with numerous infectious, inflammatory, and malignant diseases. Due to their abundance in mice and the earlier development of reagents, iNKT cells have been more extensively studied than MAIT cells. This has led to the routine use of iNKT cells as a reference population for the study of MAIT cells, and such an approach has proven very fruitful. However, MAIT cells and iNKT cells show important phenotypic, functional, and developmental differences that are often overlooked. With the recent availability of new tools, most importantly MR1 tetramers, it is now possible to directly study MAIT cells to understand their biology. Therefore, it is timely to compare the phenotype, development, and function of MAIT cells and iNKT cells. In this review, we highlight key areas where MAIT cells show similarity or difference to iNKT cells. In addition, we discuss important avenues for future research within the MAIT cell field, especially where comparison to iNKT cells has proven less informative.

Regulation of invariant NKT cell development and function by a 0.14 Mbp locus on chromosome 1: a possible role for Fcgr3.

Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a 5-fold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

Functions of CD1d-Restricted Invariant Natural Killer T Cells in Antimicrobial Immunity and Potential Applications for Infection Control.

CD1d-restricted invariant natural killer T (iNKT) cells are innate-type lymphocytes that express a T-cell receptor (TCR) containing an invariant α chain encoded by the Vα14 gene in mice and Vα24 gene in humans. These iNKT cells recognize endogenous, microbial, and synthetic glycolipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule CD1d. Upon TCR stimulation by glycolipid antigens, iNKT cells rapidly produce large amounts of cytokines, including interferon-γ (IFNγ) and interleukin-4 (IL-4). Activated iNKT cells contribute to host protection against a broad spectrum of microbial pathogens, and glycolipid-mediated stimulation of iNKT cells ameliorates many microbial infections by augmenting innate and acquired immunity. In some cases, however, antigen-activated iNKT cells exacerbate microbial infections by promoting pathogenic inflammation. Therefore, it is important to identify appropriate microbial targets for the application of iNKT cell activation as a treatment or vaccine adjuvant. Many studies have found that iNKT cell activation induces potent adjuvant activities promoting protective vaccine effects. In this review, we summarize the functions of CD1d-restricted iNKT cells in immune responses against microbial pathogens and describe the potential applications of glycolipid-mediated iNKT cell activation for preventing and controlling microbial infections.

Orchestrating liver repair: A newly discovered function of hepatic iNKT cells.

Orchestrating liver repair: A newly discovered function of hepatic iNKT cells.

Critical Contribution of NK Group 2 Member D Expressed on Invariant Natural Killer T Cells in Concanavalin A-Induced Liver Hepatitis in Mice

Natural killer group 2D (NKG2D) is a well-characterized activating receptor expressed on many immune cells, including invariant natural killer T (iNKT) cells. These cells were shown to be responsible of liver injury in the model of concanavalin A (Con A)-induced hepatitis, considered to be an experimental model of human autoimmune hepatitis. In this study, we investigated whether NKG2D plays a role in the hepatitis induced by iNKT cell-mediated immune response to Con A. By using killer cell lectin-like receptor subfamily K, member 1 deficient (Klrk1−/−) mice, we found that the absence of NKG2D reduced the hepatic injury upon Con A administration. This was not due to an intrinsic functional defect of NKG2D-deficient iNKT cells as mice missing NKG2D have normal distribution and function of iNKT cells. Furthermore, increased resistance to Con A-induced hepatitis was confirmed using neutralizing anti-NKG2D antibodies. The reduced pathogenic effect of Con A in the absence of NKG2D correlates with a reduction in pathogenic cytokine production and FAS-Ligand (FAS-L) expression by iNKT cells. We also found that Con A administration led to an increase in the retinoic acid early inducible (RAE-1) surface expression on wild-type hepatocytes. Finally, we found that Con A has no direct action on FAS-L expression or cytokine production by iNKT cells and thus propose that NKG2D-L expression on stressed hepatocytes promote cytotoxic activity of iNKT cells via its interaction with NKG2D contributing to hepatic injury. In conclusion, our results highlight NKG2D as an essential receptor required for the activation of iNKT cells in Con A-induced hepatitis and indicate that it represents a potential drug target for prevention of autoimmune hepatitis.

Elevated Hepatic CD1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection.

Activation of invariant NKT (iNKT) cells manifests antiviral immune responses in vivo. However, clinical trials have failed to show consistent hepatitis B virus (HBV) DNA reduction postadministration of iNKT cell-specific agonist α-galactosylceramide (α-GalCer). In this study, we aimed to investigate HBV infection-related iNKT cell defects and explore iNKT cell-based therapeutic potential for chronic hepatitis B (CHB). Liver specimens from 30 HBV-infected hepatocellular carcinoma patients were collected for CD1d/hepatitis B surface Ag (HBsAg) staining and/or intrahepatic iNKT cell assay. Two hundred and six chronic HBV-infected patients (including 130 CHB patients) were enrolled in the study of circulating iNKT cell frequency and function. We found that liver and hepatoma tissue that positively stained for HBsAg had higher CD1d expression as compared with HBsAg negatively stained counterparts. The elevated CD1d expression in infected tissue is supposed to facilitate the iNKT cell-based antiviral effects locally. However, iNKT cell defects that related with disease progression suggested iNKT cells attenuated their effects during chronic HBV infection. The residual iNKT cells in CHB patients showed aberrant activation and hyporesponsiveness to α-GalCer. Exogenous IL-2 fully rescued α-GalCer-induced expansion of iNKT cells from CHB patients, and synergistic effects of IL-2 and IL-15 helped to recover the CD1d-dependent IFN-γ production. In conclusion, our results highlight the increased CD1d expression in HBV-infected liver and differential iNKT cell defects associated with disease progression during chronic HBV infection. The reversibility of iNKT cell defects suggests protective immune responses could be partially recovered in CHB.

The immune adaptor ADAP plays a pivotal role invariant natural killer T cell functions

Abstract Invariant natural killer T cells (iNKTs) comprise a sub-lineage of T lymphocytes that express an “invariant” T cell receptor (TCR) with specificity for glycolipid antigens (Ag) such as a-galactosyl ceramide (aGC), when presented in complex with the MHC class I molecule CD1d. Following TCR engagement, iNKTs rapidly secrete cytokines and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells as well as directly kill tumor cells. Based on their dual functions as potent cytokine producers and tumor killers, iNKTs serve as a powerful tool for use in cell-based immunotherapies for cancer. Although TCR-CD1d interactions are generally required for iNKT cell functions, the signaling mechanisms that co-operate with the TCR to promote maximal iNKT cell responses remain poorly understood. Our previous studies demonstrate that the adaptor protein SAP and its signaling partner, Fyn (a Src-tyrosine kinase) is critical for iNKT cell functions. We recently investigated the role of ADAP (adhesion and degranulation promoting adaptor protein), a Fyn-binding protein in iNKT functions. We observe that aGC-induced iNKT cell activation, cytokine production, and bystander immune cell activation as well as anti-tumor response are remarkably reduced in ADAP-deficient (Adap−/−) mice. These data suggest that ADAP is a critical regulator of Ag-induced iNKT cell functions. Studies are currently underway to elucidate the mechanism(s) by which ADAP regulates iNKT cell functions. These studies will have an impact by establishing new paradigms for iNKT cell signaling and offering insights into how iNKTs can be best activated to enhance host immunity and treat cancer.

Opposing roles of CD2 and 2B4 in iNKT cell cytotoxic responses

Abstract Invariant natural killer T cells (iNKTs) comprise a unique lineage of innate-type lipid-reactive T lymphocytes with important roles in host immunity, including defense against specific pathogens and cancers. iNKTs rapidly produce cytokines and mount potent cytotoxic responses following T cell receptor (TCR) engagement, but the mechanisms that control iNKT cell functions remain poorly understood. CD2 and 2B4 are receptors on iNKTs that both bind to CD48 on target cells and modulate immune activation by facilitating cell-cell interactions and transducing intracellular signals. To determine their roles during iNKT cell activation, we examined the TCR-induced killing activity of murine iNKTs that lacked expression of CD2 or 2B4. Through these studies, we observe that Cd2−/− iNKTs fail to kill target cells while 2b4−/− iNKTs exhibit significantly enhanced cytolysis, both in vitro as well as in vivo. These novel findings suggest that CD2 is a positive and 2B4 a negative regulator of TCR-induced iNKT cell functions. We are currently investigating whether 2B4 exerts its inhibitory role by directly competing with CD2 for CD48 binding and/or by inducing a negative signal. As CD1d (the ligand for the invariant TCR) and CD48 (the ligand for CD2 and 2B4) are both widely expressed on hematopoietic tumors, further elucidation of the mechanisms by which CD2 and 2B4 regulate TCR-induced iNKT cell anti-tumor activity is of significant scientific and clinical importance. The results obtained from these studies will have an impact by establishing new paradigms for iNKT signaling and offering insights into how iNKTs can be best activated to enhance host immunity and treat hematopoietic cancers such as leukemia and lymphoma.

Glucocorticoid receptor signaling during prolonged psychological stress compromises the ability of invariant NKT cells to participate in antitumor immune surveillance

Abstract The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by psychological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by glycolipid antigens and/or select environmental cues such as inflammatory cytokines, participate in antitumor immune responses. However, how mediators of stress may impact iNKT cell functions in this context remains unexplored. We subjected BALB/c and/or C57BL/6 mice to prolonged psychological stress, in a well-established model of physical restraint, before administering α-GalCer (a glycolipid agonist of iNKT cells) or a combination of IL-12 and IL-18. We report that stress abrogates IFN-γ production by activated iNKT cells in vivo, an effect that is reversible by blockade of glucocorticoid receptors. Accordingly, α-GalCer-stimulated iNKT cells in stressed mice failed to protect against pulmonary metastasis of B16-F10 melanoma. We also observed that otherwise α-GalCer-transactivated NK cells from stressed mice had a diminished capacity to lyse YAC-1 lymphoma cells. Finally, the suppressive effect of stress on iNKT cells was not due to their apoptotic death. In fact, unlike conventional T cells, iNKT cells were resistant to glucocorticoid-induced apoptosis. Taken together, our findings define a novel mechanism underlying stress-induced immunosuppression, which involves innate-like but not conventional T lymphocytes.

Tim-3 blockade promotes iNKT cell function to inhibit HBV replication.

Increased expression of T cell immunoglobulin and mucin domain‐3 (Tim‐3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim‐3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim‐3 was up‐regulated on hepatic iNKT cells from HBV‐transgenic (Tg) mice or iNKT cells stimulated with α‐galactosylceramide (α‐Galcer). Compared with Tim‐3− iNKT cells, Tim‐3+ iNKT cells expressed more IFN‐γ, IL‐4 and CD107a, indicating a strong relationship between Tim‐3 and iNKT cell activation. Constantly, treatment of Tim‐3 blocking antibodies significantly enhanced the production of IFN‐γ, TNF‐α, IL‐4 and CD107a in iNKT cells both in vivo and in vitro. This Tim‐3− mediated suppression of iNKT cells was further confirmed in Tim‐3 knockout (KO) mice. Moreover, Tim‐3 blockade promoted α‐Galcer‐triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down‐regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim‐3 blockade in promoting iNKT cell‐mediated HBV inhibition. Therefore, combination of α‐Galcer with Tim‐3 blockade might be a promising approach in chronic hepatitis B therapy.

Short-term Oral Antibiotics Treatment Promotes Inflammatory Activation of Colonic Invariant Natural Killer T and Conventional CD4+ T Cells.

The gut mucosa is continuously exposed to a vast community of microorganisms, collectively defined as microbiota, establishing a mutualistic relationship with the host and contributing to shape the immune system. Gut microbiota is acquired at birth, and its composition is relatively stable during the entire adult life. Intestinal dysbiosis, defined as a microbial imbalance of gut bacterial communities, can be caused by several factors, including bacterial infections and antibiotic use, and has been associated with an increased risk to develop or exacerbate immune-mediated pathologies, such as allergic reactions, asthma, and inflammatory bowel diseases. Still, the mechanisms by which antibiotic-induced gut dysbiosis may lead to development of mucosal inflammation are still matter of debate. To this end, we aimed to evaluate the impact of antibiotic treatment on phenotype and functions of intestinal immune cell populations, including invariant natural killer T (iNKT) cells, a subset of lipid-specific T cells profoundly influenced by alterations on the commensal microbiota. To this aim, a cocktail of broad-spectrum antibiotics was administered for 2 weeks to otherwise healthy mice before re-colonization of the intestinal microbial community with oral gavage of eubiotic or dysbiotic mucosa-associated bacteria and luminal colonic content, followed or not by intestinal inflammation induction. Here. we showed that short-term antibiotic treatment alters frequency and functions of intestinal iNKT cells, even in the absence of intestinal inflammation. The presence of a dysbiotic microbiota after antibiotic treatment imprints colonic iNKT and CD4+ T cells toward a pro-inflammatory phenotype that collectively contributes to aggravate intestinal inflammation. Nonetheless, the inflammatory potential of the dysbiotic microbiota decreases over time opening the possibility to temporally intervene on the microbial composition to re-equilibrate dysbiosis, thus controlling concomitantly mucosal immune T cell activations.

Id Proteins Suppress E2A-Driven Invariant Natural Killer T Cell Development prior to TCR Selection.

A family of transcription factors known as E proteins, and their antagonists, Id proteins, regulate T cell differentiation at critical developmental checkpoints. Id proteins promote the differentiation of conventional αβ T cells and suppress the expansion of innate-like αβ T cells known as invariant natural killer T (iNKT) cells. However, it remains to be determined whether Id proteins differentially regulate these distinct lineage choices in early stages of T cell development. In this manuscript, we report that in Id-deficient mice, uninhibited activity of the E protein family member E2A mediates activation of genes that support iNKT cell development and function. There is also biased rearrangement in Id-deficient DP cells that promotes selection into the iNKT lineage in these mice. The observed expansion of iNKT cells is not abrogated by blocking pre-TCR signaling, which is required for conventional αβ T cell development. Finally, E2A is found to be a key transcriptional regulator of both iNKT and γδNKT lineages, which appear to have shared lineage history. Therefore, our study reveals a previously unappreciated role of E2A in coordinating the development of the iNKT lineage at an early stage, prior to their TCR-mediated selection alongside conventional αβ T cells.

Sodium chloride inhibits IFN-γ,but not IL-4, production by invariant NKT cells.

Invariant NKT (iNKT) cells are a distinct subset of T cells that exert Janus-like functions in vivo by producing IFN-γ and IL-4. Sodium chloride modulates the functions of various immune cells, including conventional CD4+ T cells and macrophages. However, it is not known whether sodium chloride affects iNKT cell function, so we addressed this issue. Sodium chloride inhibited IFN-γ, but not IL-4, production by iNKT cells upon TCR or TCR-independent (IL-12 and IL-18) stimulation in a dose-dependent manner. Consistently, sodium chloride reduced the expression level of tbx21, but not gata-3, in iNKT cells stimulated with TCR engagement or IL-12 + IL-18. Sodium chloride increased phosphorylated p38 expression in iNKT cells and inhibitors of p38, NFAT5, SGK1, and TCF-1 restored IFN-γ production by iNKT cells stimulated with sodium chloride and TCR engagement. Furthermore, adoptive transfer of iNKT cells pretreated with sodium chloride restored antibody-induced joint inflammation to a lesser extent than for untreated iNKT cells in Jα18 knockout mice. These findings suggest that sodium chloride inhibits IFN-γ production by iNKT cells in TCR-dependent and TCR-independent manners, which is dependent on p38, NFAT5, SGK1, and TCF-1. These findings highlight the functional role of sodium chloride in iNKT cell-mediated inflammatory diseases.

Intracellular IL-4 and IFN-γ expression in iNKT cells from patients with chronic lymphocytic leukemia.

Malignant B cells in chronic lymphocytic leukemia serve an essential role in the whole immune response, so their interactions with other immune cells are more complex than observed in solid tumors. The latest study results indicate that the immune dysregulation in chronic lymphocytic leukemia (CLL) also affects a small population of invariant natural killer T cells (iNKT). Using peripheral blood iNKT cells obtained from patients with CLL, the objective of the present study was to assess the intracellular expression of typical cytokines involved in the Th1 (IFN-γ) and Th2 (IL-4) response pathways following stimulation with the iNKT-specific ligand α-galactosylceramide. iNKT cells from patients with CLL exhibited upregulated IL-4 and IFN-γ expression in comparison to those from HVs. No significant association between the ability of iNKT cells to produce IL-4 or IFN-γ and the expression of CD1d on leukemic B lymphocytes or monocytes was identified. However, the function of iNKT cells was compromised in patients with CLL by a strong Th2 bias (high IL-4 and low IFN-γ expression). The ratio of iNKT+IFN-γ+:iNKT+IL-4+ was significantly decreased in the CLL group when compared with HVs, and this decreased further as the disease progressed. This change may result in the promotion of leukemic B lymphocyte survival. Therefore, in the pathogenesis of CLL, Th2 bias may delay the antitumor response that relies on stimulation of the Th1 immune response.

Mixed Signals: Co-Stimulation in Invariant Natural Killer T Cell-Mediated Cancer Immunotherapy.

Invariant natural killer T (iNKT) cells are an integral component of the immune system and play an important role in antitumor immunity. Upon activation, iNKT cells can directly kill malignant cells as well as rapidly produce cytokines that stimulate other immune cells, making them a front line defense against tumorigenesis. Unfortunately, iNKT cell number and activity are reduced in multiple cancer types. This anergy is often associated with upregulation of co-inhibitory markers such as programmed death-1. Similar to conventional T cells, iNKT cells are influenced by the conditions of their activation. Conventional T cells receive signals through the following three types of receptors: (1) T cell receptor (TCR), (2) co-stimulation molecules, and (3) cytokine receptors. Unlike conventional T cells, which recognize peptide antigen presented by MHC class I or II, the TCRs of iNKT cells recognize lipid antigen in the context of the antigen presentation molecule CD1d (Signal 1). Co-stimulatory molecules can positively and negatively influence iNKT cell activation and function and skew the immune response (Signal 2). This study will review the background of iNKT cells and their co-stimulatory requirements for general function and in antitumor immunity. We will explore the impact of monoclonal antibody administration for both blocking inhibitory pathways and engaging stimulatory pathways on iNKT cell-mediated antitumor immunity. This review will highlight the incorporation of co-stimulatory molecules in antitumor dendritic cell vaccine strategies. The use of co-stimulatory intracellular signaling domains in chimeric antigen receptor-iNKT therapy will be assessed. Finally, we will explore the influence of innate-like receptors and modification of immunosuppressive cytokines (Signal 3) on cancer immunotherapy.

Invariant Natural Killer T Cells in Immune Regulation of Blood Cancers: Harnessing Their Potential in Immunotherapies.

Invariant natural killer T (iNKT) cells are a unique innate T lymphocyte population that possess cytolytic properties and profound immunoregulatory activities. iNKT cells play an important role in the immune surveillance of blood cancers. They predominantly recognize glycolipid antigens presented on CD1d, but their activation and cytolytic activities are not confined to CD1d expressing cells. iNKT cell stimulation and subsequent production of immunomodulatory cytokines serve to enhance the overall antitumor immune response. Crucially, the activation of iNKT cells in cancer often precedes the activation and priming of other immune effector cells, such as NK cells and T cells, thereby influencing the generation and outcome of the antitumor immune response. Blood cancers can evade or dampen iNKT cell responses by downregulating expression of recognition receptors or by actively suppressing or diverting iNKT cell functions. This review will discuss literature on iNKT cell activity and associated dysregulation in blood cancers as well as highlight some of the strategies designed to harness and enhance iNKT cell functions against blood cancers.

A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease

iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.

Invariant natural killer T-cell development and function with loss of microRNA-155.

Invariant natural killer T (iNKT) cells are adaptive T cells with innate-like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR-155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon-γ expression. We examined the role of miR-155 in the development and function of iNKT cells. Using germline-deficient miR-155 mice, we showed that loss of miR-155 resulted in unchanged iNKT cell frequency and cell number. Although miR-155 was up-regulated in iNKT cells upon activation with α-galactosylceramide, loss of miR-155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR-155 expression.