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While the transcription factor GATA-3 is well-established for its crucial role in T cell development, its specific influence on invariant natural killer T (iNKT) cells remains relatively unexplored. Using flow cytometry and single-cell transcriptomic analysis, we demonstrated that GATA-3 deficiency in mice leads to the absence of iNKT2 and iNKT17 cell subsets, as well as an altered distribution of iNKT1 cells. Thymic iNKT cells lacking GATA-3 exhibited diminished expression of PLZF and T-bet, key transcription factors involved in iNKT cell differentiation, and reduced production of Th2, Th17, and cytotoxic effector molecules. Single-cell transcriptomics revealed a comprehensive absence of iNKT17 cells, a substantial reduction in iNKT2 cells, and an increase in iNKT1 cells in GATA-3-deficient thymi. Differential expression analysis highlighted the regulatory role of GATA-3 in T cell activation signaling and altered expression of genes critical for iNKT cell differentiation, such as Icos , Cd127 , Eomes , and Zbtb16 . Notably, restoration of Icos, but not Cd127, expression could rescue iNKT cell development in GATA-3-deficient mice. In conclusion, our study demonstrates the pivotal role of GATA-3 in orchestrating iNKT cell effector lineage differentiation through the regulation of T cell activation pathways and Icos expression, providing insights into the molecular mechanisms governing iNKT cell development and function.

Patients with colorectal cancer frequently develop liver metastases after, and perhaps as a consequence of, lifesaving surgical resection of the primary tumor. This creates a potential opportunity for prophylactic metastatic treatment with novel immunostimulatory molecules. Here, we used state-of-the-art intravital imaging of an experimental liver metastasis model to visualize the early behavior and function of invariant natural killer T (iNKT) cells stimulated with α-galactosylceramide (α-GalCer). Intravenous α-GalCer prior to tumor cell seeding in the liver significantly inhibited tumor growth. However, some seeding tumor cells survived. A multiple dosing regimen reduced tumor burden and prolonged the life of mice, whereas tumors returned within 5 days after a single dose of α-GalCer. With multiple doses of α-GalCer, iNKT cells increased in number and granularity (as did NK cells). As a result, the total number of contacts and time in contact with tumors increased substantially. In the absence of iNKT cells, the beneficial effect of α-GalCer was lost. Robust cytokine production dissipated over time. Repeated therapy, even after cytokine dissipation, led to reduced tumor burden and prolonged survival. Serial transplantation of tumors exposed to α-GalCer-activated iNKT cells did not induce greater resistance, suggesting no obvious epigenetic or genetic immunoediting in tumors exposed to activated iNKT cells. Very few tumor cells expressed CD1d in this model, and as such, adding monomers of CD1d-α-GalCer further reduced tumor growth. The data suggest early and repeated stimulation of iNKT cells with α-GalCer could have direct therapeutic benefit for patients with colorectal cancer who develop metastatic liver disease., (©2021 American Association for Cancer Research.)

iNKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their unique functional characteristics are induced by a transcriptional program initiated by positive selection mediated by CD1d expressed by CD4 + CD8 + (double positive, DP) thymocytes. Here, using a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24 hi CD44 lo NKT cells, we examined transcriptional events in four immature thymic iNKT cell subsets. A transcriptional regulatory network approach identified transcriptional changes in proximal components of the TCR signalling cascade in DP NKT cells. Subsequently, positive and negative selection, and lineage commitment, occurred at the transition from DP NKT to CD4 NKT. Thus, this study introduces previously unrecognised steps in early NKT cell development, and separates the events associated with modulation of the T cell signalling cascade prior to changes associated with positive selection and lineage commitment., (© 2021. The Author(s).)

Background: Invariant Natural Killer T (iNKT) cells are innate lymphocytes bridging the innate and adaptive immune systems and are critical first responders against cancer and infectious diseases. iNKT cell phenotype and functionality are studied using in vitro stimulation assays assessing cytokine response and proliferation capabilities. The most common stimulant is the glycolipid α-Galactosyl Ceramide (α-GalCer), which stimulates iNKT cells when presented by CD1d, an MHC class I-like molecule expressed by antigen-presenting cells (APC). Another stimulant used is α-GalCer-loaded DimerX, a CD1d-Ig fusion protein which stimulates iNKT cells in an APC-independent fashion. Here, we demonstrate use of the PBS-57-loaded CD1d-tetramer as an APC-independent stimulant, where PBS-57 is an α-GalCer analogue., Methods: Using healthy fresh (n = 4) and frozen (n = 7) peripheral blood mononuclear cells (PBMCs), 10-h cytokine response (measuring IFN-γ production) and 10-day proliferation assays were performed assessing iNKT functionality using α-GalCer, CD1d-tetramer and DimerX stimulants., Results: All stimulants effectively induced IFN-γ production in both fresh and frozen PBMC. After the 10-h activation, CD1d-tetramer was significantly more effective than α-GalCer (p = 0.032) in inducing IFN-γ production in fresh PBMC and significantly more effective than both α-GalCer (p = 0.004) and DimerX (p = 0.021) in frozen PBMC. Similarly, all stimulants induced strong proliferation responses in all samples, although this was only significant in the frozen PBMC. No significant differences in proliferation were observed between stimulants., Significance: This study supports PBS-57-loaded CD1d-tetramer as an effective in vitro APC-independent iNKT cell stimulant, which is comparable to or even more effective than α-GalCer and DimerX. As CD1d is downregulated during infectious disease and cancer as evasion strategies, in vitro assays which are APC-independent can assist in providing objective insight to iNKT activation by not relying on CD1d expression by APCs. Overall, the novel CD1d-tetramer stimulation equips researchers with an expanded "toolkit" to successfully assess iNKT cell function., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation 1 . However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (α-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescence-activated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of α-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the α-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the α-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and  8, respectively. In α-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.

Natural killer T (NKT) cells constitute a unique subset of T lymphocytes characterized by specifically interacting with antigenic glycolipids conjugated to the CD1d receptor on antigen-presenting cells. Functionally, NKT cells are capable of performing either effector or suppressor immune responses, depending on their production of proinflammatory or anti-inflammatory cytokines, respectively. Effector NKT cells are subdivided into three subsets, termed NKT1, NKT2, and NKT17, based on the cytokines they produce and their similarity to the cytokine profile produced by Th1, Th2, and Th17 lymphocytes, respectively. Recently, a new subgroup of NKT cells termed NKT10 has been described, which cooperates and interacts with other immune cells to promote immunoregulatory responses. Although the tissue-specific functions of NKT cells have not been fully elucidated, their activity has been associated with the pathogenesis of different inflammatory diseases with immunopathogenic similarities to periodontitis, including osteolytic pathologies such as rheumatoid arthritis and osteoporosis. In the present review, we revise and discuss the pathogenic characteristics of NKT cells in these diseases and their role in the pathogenesis of periodontitis; particularly, we analyze the potential regulatory role of the IL-10-producing NKT10 cells., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Samanta Melgar-Rodríguez et al.)

Natural killer T (NKT) cells are a unique subset of lymphocytes that recognize lipid antigens in the context of the non-classical class I MHC molecule, CD1d, and serve as a link between the innate and adaptive immune system through their expeditious release of cytokines. Whereas NKT have well-established roles in mitigating a number of human diseases, herein, we focus on their role in cancer. NKT cells have been shown to directly and indirectly mediate anti-tumor immunity and manipulating their effector functions can have therapeutic significances in treatment of cancer. In this review, we highlight several therapeutic strategies that have been used to harness the effector functions of NKT cells to target different types of solid tumors. We also discuss several barriers to the successful utilization of NKT cells and summarize effective strategies being developed to harness the unique strengths of this potent population of T cells. Collectively, studies investigating the therapeutic potential of NKT cells serve not only to advance our understanding of this powerful immune cell subset, but also pave the way for future treatments focused on the modulation of NKT cell responses to enhance cancer immunotherapy.

Invariant NKT ( i NKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature i NKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for i NKT cell generation, cytokines are proposed to contribute to i NKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic i NKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of i NKT cells, surprisingly, premature IL-2Rβ expression on immature i NKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control i NKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic i NKT cell differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Won, Kim, Crossman, Awasthi, Gress and Park.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

The field of flow cytometry has witnessed rapid technological advancements in the last few decades. While the founding principles of fluorescent detection on cells (or particles) within a uniform fluid stream remains largely unchanged, the availability more sensitive cytometers with the ability to multiplex more and more florescent signals has resulted in very complex high-order assays. This results in the co-use of fluorophores with increased levels of emission overlap and/or spillover spreading than in years past and thus requires careful and well thought out planning for flow cytometry assay development. As an example, we present the development of a large 18-color (20 parameter) flow cytometry assay designed to take an in depth analysis of effector lymphocyte phenotypes, with careful attention to assay controls and panel design.

CD1d-restricted invariant NKT (iNKT) cells are innate-like T cells that respond to glycolipids, a class of Ags that are invisible to conventional T cells. iNKT cells develop in the thymus where they receive strong "agonist" TCR signals. During their ontogeny, iNKT cells differentiate into discrete iNKT1, iNKT2, and iNKT17 effector subsets akin to helper CD4 T cells. In this study, we found that transgenic (Tg) expression of the canonical Vα14-Jα18 TCRα-chain at the double-positive thymocyte stage led to premature iNKT cell development and a cell-intrinsic bias toward iNKT2 cells, due to increased TCR signaling upon selection. Consistent with the strong iNKT2 bias, innate memory CD8 + T cells were found in greater numbers in Vα14 Tg mice, whereas the prevalence of mucosa-associated invariant T cells was reduced. iNKT cells from Vα14 Tg mice were hyporesponsive to stimulation by their cognate Ag α-galactosylceramide. Finally, Vα14 Tg mice displayed increased B16F10 melanoma tumor growth compared with wild-type mice. This study reveals some of the limitations of Vα14 Tg mice and warrants the cautious interpretation of past and future findings using this mouse model., (Copyright © 2020 The Authors.)

Most T lymphocytes leave the thymus as naïve cells with limited functionality. However, unique populations of innate-like T cells differentiate into functionally distinct effector subsets during their development in the thymus. Here, we profiled >10,000 differentiating thymic invariant natural killer T (iNKT) cells using single-cell RNA sequencing to produce a comprehensive transcriptional landscape that highlights their maturation, function, and fate decisions at homeostasis. Our results reveal transcriptional profiles that are broadly shared between iNKT and mucosal-associated invariant T (MAIT) cells, illustrating a common core developmental program. We further unmask a mutual requirement for Hivep3, a zinc finger transcription factor and adapter protein. Hivep3 is expressed in early precursors and regulates the post-selection proliferative burst, differentiation and functions of iNKT cells. Altogether, our results highlight the common requirements for the development of innate-like T cells with a focus on how Hivep3 impacts the maturation of these lymphocytes.

CD1d-dependent type I NKT cells, which are activated by lipid antigen, are known to play important roles in innate and adaptive immunity, as are a portion of type II NKT cells. However, the heterogeneity of NKT cells, especially NKT-like cells, remains largely unknown. Here, we report the profiling of NKT (NK1.1 + CD3e + ) cells in livers from wild type (WT), Jα18-deficient and CD1d-deficient mice by single-cell RNA sequencing. Unbiased transcriptional clustering revealed distinct cell subsets. The transcriptomic profiles identified the well-known CD1d-dependent NKT cells and defined two CD1d-independent NKT cell subsets. In addition, validation of marker genes revealed the differential organ distribution and landscape of NKT cell subsets during liver tumor progression. More importantly, we found that CD1d-independent Sca-1 - CD62L + NKT cells showed a strong ability to secrete IFN-γ after costimulation with IL-2, IL-12 and IL-18 in vitro. Collectively, our findings provide a comprehensive characterization of NKT cell heterogeneity and unveil a previously undefined functional NKT cell subset.

Invariant natural killer T (iNKT) cells acquire effector functions during development by mechanisms that remain poorly understood. Here, we show that the Hippo kinases Mst1 and Mst2 act as molecular rheostats for the terminal maturation and effector differentiation programs of iNKT cells. Loss of Mst1 alone or together with Mst2 impedes iNKT cell development, associated with defective IL-15-dependent cell survival. Mechanistically, Mst1 enforces iNKT cellular and transcriptional quiescence associated with maturation and commitment to iNKT1 cells by suppressing proliferation and Opa1-related mitochondrial metabolism that are dynamically regulated during iNKT cell development. Furthermore, Mst1 shapes the reciprocal fate decisions between iNKT1 and iNKT17 effector cells, which respectively depend upon mitochondrial dynamics and ICOS-mTORC2 signaling. Collectively, these findings establish Mst1 as a crucial regulator of mitochondrial homeostasis and quiescence in iNKT cell development and effector lineage differentiation and highlight that establishment of quiescence programs underlies iNKT cell development and effector maturation., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Raynor et al.)

The thymus supports multiple αβ T cell lineages that are functionally distinct, but mechanisms that control this multifaceted development are poorly understood. Here we examine medullary thymic epithelial cell (mTEC) heterogeneity and its influence on CD1d-restricted iNKT cells. We find three distinct mTEC low subsets distinguished by surface, intracellular and secreted molecules, and identify LTβR as a cell-autonomous controller of their development. Importantly, this mTEC heterogeneity enables the thymus to differentially control iNKT sublineages possessing distinct effector properties. mTEC expression of LTβR is essential for the development thymic tuft cells which regulate NKT2 via IL-25, while LTβR controls CD104 + CCL21 + mTEC low that are capable of IL-15-transpresentation for regulating NKT1 and NKT17. Finally, mTECs regulate both iNKT-mediated activation of thymic dendritic cells, and iNKT availability in extrathymic sites. In conclusion, mTEC specialization controls intrathymic iNKT cell development and function, and determines iNKT pool size in peripheral tissues.

Thymic natural killer T (NKT)2 cells are a subset of invariant NKT cells with PLZF hi GATA3 hi IL-4 + . The differentiation of NKT2 cells is not fully understood. In the present study, we report an important role of TRAF3-interacting protein 3 (TRAF3IP3) in the functional maturation and expansion of committed NKT2s in thymic medulla. Mice with T-cell-specific deletion of TRAF3IP3 had decreased thymic NKT2 cells, decreased IL-4-producing peripheral iNKTs, and defects in response to α-galactosylceramide. Positive selection and high PLZF expression in CD24 + CD44 - and CCR7 + CD44 - immature iNKTs were not affected. Only CD44 hi NK1.1 - iNKTs in Traf3ip3 -/- mice showed reduced expression of Egr2, PLZF, and IL-17RB, decreased proliferation, and reduced IL-4 production upon stimulation. This Egr2 and IL-4 expression was augmented by MEK1/ERK activation in iNKTs, and TRAF3IP3 at the trans-Golgi network recruited MEK1 and facilitated ERK phosphorylation and nuclear translocation. LTβR-regulated bone marrow-derived nonlymphoid cells in the medullary thymic microenvironment were required for MEK/ERK activation and NKT2 maturation. These data demonstrate an important functional maturation process in NKT2 differentiation that is regulated by MEK/ERK signaling at the trans-Golgi network.

Co-expression of Yin Yang 1 (YY1) is required for the full function of the transcription factor, PLZF, which is essential for the development of natural killer T cell (NKT cell) effector functions. Discordant expression of YY1 and PLZF, therefore, might define NKT cell subsets with distinct effector functions. A subset of NKT cells was identified that expressed low levels of YY1. YY1 lo NKT cells were found in all tissues, had a mature phenotype and, distinct from other NKT cells, expressed almost no ThPOK or Tbet. When activated, YY1 lo NKT cells produced little IL-4 or IFN-γ. YY1 lo NKT cells were found to constitutively transcribe IL-10 mRNA and, accordingly, produced IL-10 upon primary activation. Finally, we find that tumor infiltrating NKT cells are highly enriched for the YY1 lo subset. Low YY1 expression, therefore, defines a previously unrecognized NKT cell subset that is committed to producing IL-10.

Novel therapies are urgently needed for ovarian cancer, the deadliest gynecologic malignancy. Ovarian cancer has thus far been refractory to immunotherapies that stimulate the host immune system to recognize and kill cancer cells. This may be because of a suppressive tumor immune microenvironment and lack of recruitment and activation of immune cells that kill cancer cells. Our previous work showed that epigenetic drugs including DNA methyltransferase inhibitors and histone deacetylase 6 inhibitors (DNMTis and HDAC6is) individually increase immune signaling in cancer cells. We find that combining DNMTi and HDAC6i results in an amplified type I interferon response, leading to increased cytokine and chemokine expression and higher expression of the MHC I antigen presentation complex in human and mouse ovarian cancer cell lines. Treating mice bearing ID8 Trp53-/- ovarian cancer with HDAC6i/DNMTi led to an increase in tumor-killing cells such as IFNg+ CD8, NK, and NKT cells and a reversal of the immunosuppressive tumor microenvironment with a decrease in MDSCs and PD-1 hi CD4 T cells, corresponding with an increase in survival. Thus combining the epigenetic modulators DNMTi and HDAC6i increases anti-tumor immune signaling from cancer cells and has beneficial effects on the ovarian tumor immune microenvironment.

Recent studies suggest that murine invariant natural killer T (iNKT) cell development culminates in three terminally differentiated iNKT cell subsets denoted as NKT1, 2, and 17 cells. Although these studies corroborate the significance of the subset division model, less is known about the factors driving subset commitment in iNKT cell progenitors. In this review, we discuss the latest findings in iNKT cell development, focusing in particular on how T-cell receptor signal strength steers iNKT cell progenitors toward specific subsets and how early progenitor cells can be identified. In addition, we will discuss the essential factors for their sustenance and functionality. A picture is emerging wherein the majority of thymic iNKT cells are mature effector cells retained in the organ rather than developing precursors., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2020 Hogquist K and Georgiev H.)

The development, differentiation and function of invariant NKT (iNKT) cells require a well-defined set of transcription factors, but how these factors are integrated to each other and the detailed signaling networks remain poorly understood. Using a Dicer-deletion mouse model, our previous studies have demonstrated the critical involvement of microRNAs (miRNAs) in iNKT cell development and function, but the role played by individual miRNAs in iNKT cell development and function is still not clear. In this study, we show the dynamic changes of miRNA 183 cluster (miR-183C) expression during iNKT cell development. Mice with miR-183C deletion showed a defective iNKT cell development, sublineage differentiation, and cytokine secretion function. miRNA target identification assays indicate the involvement of multiple target molecules. Our study not only confirmed the role of miR-183C in iNKT cell development and function but also demonstrated that miR-183C achieved the regulation of iNKT cells through integrated targeting of multiple signaling molecules and pathways., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The invariant NKT ( i NKT) cells recognize glycolipid antigens presented by the non-classical MHC like molecule CD1d. They represent an innate T-cell lineage with the ability to rapidly produce a variety of cytokines in response to agonist stimulation to bridge innate and adaptive immunity. In thymus, most i NKT cells complete their maturation and differentiate to multiple effector lineages such as i NKT-1, i NKT-2, and i NKT-17 cells that possess the capability to produce IFNγ, IL-4, and IL-17A, respectively, and play distinct roles in immune responses and diseases. Mechanisms that control i NKT lineage fate decisions are still not well understood. Evidence has revealed critical roles of Foxo1 of the forkhead box O1 subfamily of transcription factors in the immune system. However, its role in i NKT cells has been unknown. In this report, we demonstrate that deletion of Foxo1 causes severe decreases of i NKT cell total numbers due to impairment of late but not early i NKT cell development. Deficiency of Foxo1 results in decreases of i NKT-1 but increases of i NKT-17 cells. Our data reveal that Foxo1 controls i NKT effector lineage fate decision by promoting i NKT-1 but suppressing i NKT-17 lineages., (Copyright © 2019 Tao, Li, Gao, Wang and Zhong.)

Invariant natural killer T (iNKT) cells are a unique T cell lineage that develop in the thymus and emerge with a memory-like phenotype. Accordingly, following antigenic stimulation, they can rapidly produce copious amounts of Th1 and Th2 cytokines and mediate activation of several immune cells. Thus, it is not surprising that iNKT cells play diverse roles in a broad range of diseases. Given their pivotal roles in host immunity, it is crucial that we understand the mechanisms that govern iNKT cell development and effector functions. Over the last two decades, several studies have contributed to the current knowledge of iNKT cell biology and activity. Collectively, these studies reveal that the thymic development of iNKT cells, their lineage expansion, and functional properties are tightly regulated by a complex network of transcription factors and signaling molecules. While prior studies have clearly established the importance of the SLAM-SAP-Fyn signaling axis in iNKT cell ontogenesis, recent studies provide exciting mechanistic insights into the role of this signaling cascade in iNKT cell development, lineage fate decisions, and functions. Here we summarize the previous literature and discuss the more recent studies that guide our understanding of iNKT cell development and functional responses.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells conserved across mammalian species, including mice and humans. By sequencing RNA from sorted MR1-5-OP-RU tetramer + cells derived from either human blood or murine lungs, we define the basic transcriptome of an activated MAIT cell in both species and demonstrate how this profile changes during the resolution of infection and during reinfection. We observe strong similarities between MAIT cells in humans and mice. In both species, activation leads to strong expression of pro-inflammatory cytokines and chemokines as well as a strong tissue repair signature, recently described in murine commensal-specific H2-M3-restricted T cells. Transcriptomes of MAIT cells and H2-M3-specific CD8+ T cells displayed the most similarities to invariant natural killer T (iNKT) cells when activated, but to γδ T cells after the resolution of infection. These data define the requirements for and consequences of MAIT cell activation, revealing a tissue repair phenotype expressed upon MAIT cell activation in both species., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Invariant natural killer T1 (iNKT1) cells are characterized by the preferential expression of T-box transcription factor T-bet (encoded by Tbx21) and the production of cytokine IFN-γ, but the relationship between the developmental process and iNKT1 lineage diversification in the thymus remains elusive. We report in the present study a crucial role of programmed cell death 5 (PDCD5) in iNKT cell terminal maturation and iNKT1 fate determination. Mice with T cell-specific deletion of PDCD5 had decreased numbers of thymic and peripheral iNKT cells with a predominantly immature phenotype and defects in response to α-galactosylceramide. Loss of PDCD5 also selectively abolished the iNKT1 lineage by reducing T-bet expression in iNKT cells at an early thymic developmental stage (before CD44 upregulation). We further demonstrated that TOX2, one of the high mobility group proteins that was highly expressed in iNKT cells at stage 1 and could be stabilized by PDCD5, promoted the permissive histone H3K4me3 modification in the promoter region of Tbx21. These data indicate a pivotal and unique role of PDCD5/TOX2 in iNKT1 lineage determination. They also suggest that the fate of iNKT1 may be programmed at the developmental stage of iNKT cells in the thymus.

Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis., Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity., Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes., Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

A 26-color staining panel was developed to profile human antigen-specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal-associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross-validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

It is established that iNKT cells are a cell type that require strong TCR signal for their proper development and represent a model for thymic agonist selection. The nature of the signal perceived by iNKT cells promoting their specification is not well understood. To address this question, we analyzed iNKT cell development in relevant TCR Vα14-Jα18 alpha chain transgenic mice (Vα14Tg). In CD4-Vα14Tg mice, where the transgene is driven by CD4 promoter, we identified a block in iNKT cell development at early developmental stages due to a reduced expression of key transcription factors accompanied with a reduced TCR expression levels. This indicates that TCR signal strength control iNKT cell differentiation. Importantly, we found in WT mice that early precursors of iNKT cells express higher TCR levels compared to positively selected precursors of mainstream T cells showing that TCR levels could contribute to the strength of iNKT cell TCR signaling. Overall, our study highlights TCR signal strength associated with a higher TCR density as an important regulator of iNKT cell lineage specification., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Eomes regulates the differentiation of CD8 + T cells into effector and memory phases. However, its role in invariant (i)NKT cells remains unknown. Here, we show the impact of Eomes on iNKT cells in the thymus and peripheral tissue using conditional knockout (Eomes-cKO) mice. In the thymus, CD1d-tetramer + CD24 + CD44 - NK1.1 - CD69 + stage 0 iNKT cells express higher levels of Eomes than the other iNKT stages. We also found that Eomes regulates NKT1 cell differentiation predominantly. Interestingly, the expression of Eomes in the steady state is low, but can be upregulated after TCR stimulation. We also showed epigenetic changes in the Eomes locus after activation. In addition, vaccination of C57BL/6, but not Eomes-cKO mice with iNKT ligand-loaded dendritic cells generated KLRG1 + iNKT cells in lung, characterized as effector memory phenotype by transcriptome profiling. Thus, Eomes regulates not only the differentiation of NKT1 cells in the thymus, but also their differentiation into memory-like KLRG1 + iNKT cells in the periphery., Competing Interests: The authors declare no competing interests.

The CD1d-restricted Vα14 invariant NKT (iNKT) cell lineage in mice (Vα24 in humans) represents an evolutionary conserved innate-like immune cell type that recognizes glycolipid antigens. Because of their unique ability to promptly secrete copious amounts of both pro-inflammatory and anti-inflammatory cytokines, typically produced by different T helper cell types, iNKT cells are implicated in the regulation of various pathologic conditions such as infection, allergy, autoimmune disease, maintenance of transplantation tolerance, and cancer. This striking multifaceted role in immune regulation is correlated with the presence of multiple functionally distinct iNKT cell subsets that can be distinguished based on the expression of characteristic surface markers and transcription factors. However, to date it, remains largely unresolved how this puzzling diversity of iNKT cell functional subsets emerges and what factors dictate the type of effector cell differentiation during the thymic differentiation considering the mono-specific nature of their T cell receptor (TCR) and their selecting molecule CD1d. Here, we summarize recent findings focusing on the role of TCR-mediated signaling and discuss possible mechanisms that may influence the sub-lineage choice of iNKT cells.

Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert to regulate the levels of reactive oxygen species (ROS). The Keap1-Nrf2 antioxidant system also participates in T cell differentiation and inflammation, but its role in innate T cell development and functions remains unclear. We report that T cell-specific deletion of Keap1 results in defective development and reduced numbers of invariant natural killer T (NKT) cells in the thymus and the peripheral organs in a cell-intrinsic manner. The frequency of NKT2 and NKT17 cells increases while NKT1 decreases in these mice. Keap1-deficient NKT cells show increased rates of proliferation and apoptosis, as well as increased glucose uptake and mitochondrial function, but reduced ROS, CD122, and Bcl2 expression. In NKT cells deficient in Nrf2 and Keap1, all these phenotypic and metabolic defects are corrected. Thus, the Keap1-Nrf2 system contributes to NKT cell development and homeostasis by regulating cell metabolism., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Invariant NKT (iNKT) cells are innate lipid-reactive T cells that develop and differentiate in the thymus into iNKT1/2/17 subsets, akin to T H 1/2/17 conventional CD4 T cell subsets. The factors driving the central priming of iNKT cells remain obscure, although strong/prolonged TCR signals appear to favor iNKT2 cell development. The Src homology 2 domain-containing phosphatase 1 (Shp1) is a protein tyrosine phosphatase that has been identified as a negative regulator of TCR signaling. In this study, we found that mice with a T cell-specific deletion of Shp1 had normal iNKT cell numbers and peripheral distribution. However, iNKT cell differentiation was biased toward the iNKT2/17 subsets in the thymus but not in peripheral tissues. Shp1-deficient iNKT cells were also functionally biased toward the production of T H 2 cytokines, such as IL-4 and IL-13. Surprisingly, we found no evidence that Shp1 regulates the TCR and Slamf6 signaling cascades, which have been suggested to promote iNKT2 differentiation. Rather, Shp1 dampened iNKT cell proliferation in response to IL-2, IL-7, and IL-15 but not following TCR engagement. Our findings suggest that Shp1 controls iNKT cell effector differentiation independently of positive selection through the modulation of cytokine responsiveness., (Copyright © 2019 by The American Association of Immunologists, Inc.)

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells' glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function., Competing Interests: The authors declare no conflict of interest.

Peripheral invariant NKT cells (iNKT) and CD8 + tissue-resident memory T cells (T RM ) express high levels of the extracellular ATP receptor P2RX7 in mice. High extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8 + T RM Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and T RM The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and T RM is unclear, however, as is the impact of recovery from other nonlymphoid sites. In this study, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8 + T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8 + T RM express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17, and CD8 + circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and T RM recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of T RM , although to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and T RM from nonlymphoid tissues, and it represents a crucial step for functional studies of these populations., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor (TCR), which recognizes glycolipid antigens presented on CD1d molecules. These cells are phenotypically and functionally distinct from conventional T cells. When we characterized the metabolic activity of iNKT cells, consistent with their activated phenotype, we found that they had much less mitochondrial respiratory capacity but increased glycolytic activity in comparison to naïve conventional CD4 + T cells. After TCR engagement, iNKT cells further increased aerobic glycolysis, which was important for the expression of interferon-γ (IFN-γ). Glycolytic metabolism promoted the translocation of hexokinase-II to mitochondria and the activation of mammalian target of rapamycin complex 2 (mTORC2). Inhibiting glycolysis reduced the activity of Akt and PKCθ, which inhibited TCR recycling and accumulation within the immune synapse. Diminished TCR accumulation in the immune synapse reduced the activation of proximal and distal TCR signaling pathways and IFN-γ production in activated iNKT cells. Our studies demonstrate that glycolytic metabolism augments TCR signaling duration and IFN-γ production in iNKT cells by increasing TCR recycling., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Although natural killer T cells (NKT cells) are thought to be generated from CD4 + CD8 + (DP) thymocytes, the developmental origin of CD4 - CD8 - (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44 + CD25 - (DN 1 ) cells, after that CD44 + CD25 + (DN 2 ), finally, CD44 - CD25 - (DN 3 ) and CD44 - CD25 + (DN 4 ) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN 1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3 + surface CD3 - and surface CD3 + TCR-β - cells in DN 1 -NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.

Background/aim: Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo., Materials and Methods: Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging., Results: Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5., Conclusion: CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Invariant natural killer T cells (iNKTs) are distinct from conventional T cells. iNKT cells express a semi-invariant T cell receptor (TCR) that can specifically recognize lipid antigens presented by CD1d, an MHC class I-like antigen-presenting molecule. Currently, iNKT cells are distinguished in three functionally distinct subsets. Each subset is defined by lineage-specifying factors: T-bet shapes the fate of NKT1 subset that mainly secretes IFNγ, Gata3 specifies the NKT2 subset that produces robustly IL-4 whereas RORγt seals the differentiation of NKT17 subset that secretes IL-17. In the present review, the focus is placed on the regulation of NKT17 specification and their function., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background & Aims: GPBAR1, also known as TGR5, is a G protein-coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis., Material and Methods: Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s., Results: In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1 -/- mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model., Conclusion: Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: The development of atherosclerosis is tightly regulated by the innate and adaptive immune system. Communication between these two compartments occurs, among others, upon presentation of lipid antigens to the NKT cell population by CD1d-expressing antigen-presenting cells. Recent evidence states that also mast cells express CD1d and can directly communicate with NKT cells. However, no such relationship has been reported in atherosclerosis. Here, we aimed to elucidate in vivo the CD1d-mediated interaction between mast cells and NKT cells upon atherosclerosis progression., Methods: We adoptively transferred CD1d -/- or control mast cells to mast cell-deficient apoE -/- Kit W-sh/W-sh mice and subsequently placed the animals on a Western-type diet for 10 weeks., Results: At the end of the Western-type diet period, the aortic root of CD1d -/- mast cell-reconstituted mice displayed increased plaque size, with less collagen deposition and higher intraplaque CD4 + T cells, as compared to control mice. In addition, T cells inside the aortic arch showed higher pro-inflammatory cytokine production in the form of IFNγ, TNFα and IL-17. Finally, T-bet expression was found elevated in both CD4 + and CD8 + circulating T cells., Conclusions: This study is the first to illustrate that disruption of the CD1d communication pathway between mast cells and NKT cells aggravates atherosclerosis, through a shift towards pro-inflammatory T cell responses. This ability of mast cell action during plaque progression sheds new light on their role in atherosclerosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Natural killer T (NKT) cells are innate-like lymphocytes that bridge the gap between the innate and adaptive immune responses. Like innate immune cells, they have a mature, effector phenotype that allows them to rapidly respond to threats, compared to adaptive cells. NKT cells express T cell receptors (TCRs) like conventional T cells, but instead of responding to peptide antigen presented by MHC class I or II, NKT cell TCRs recognize glycolipid antigen in the context of CD1d. NKT cells are subdivided into classes based on their TCR and antigen reactivity. This review will focus on type I iNKT cells that express a semi invariant Vα14Jα18 TCR and respond to the canonical glycolipid antigen, α-galactosylceramide. The innate-like effector functions of these cells combined with their T cell identity make their developmental path quite unique. In addition to the extrinsic factors that affect iNKT cell development such as lipid:CD1d complexes, co-stimulation, and cytokines, this review will provide a comprehensive delineation of the cell intrinsic factors that impact iNKT cell development, differentiation, and effector functions - including TCR rearrangement, survival and metabolism signaling, transcription factor expression, and gene regulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Salivary gland cancers are not sensitive to conventional radiotherapy or chemotherapy regimens. Therefore, the development of a new treatment strategy is of critical importance for improving the prognosis. We examined the expression of mesothelin molecules in salivary gland cancers and the efficacy of adoptive cell therapy based on mesothelin-specific chimeric antigen receptor transduced T cells., Methods: The expression of mesothelin molecule was studied in salivary gland cancer samples obtained from 16 patients as well as a salivary gland cancer cell line (A-253) and five other cell lines. The activation of mesothelin-specific chimeric antigen receptor-expressing CD8 T cells after stimulation with mesothelin and the effects of invariant natural killer T cells on this activation were evaluated., Results: Mesothelin was detected in the A-253 cells and the surgical specimens except for the case of squamous cell carcinoma to various degrees. Following stimulation with mesothelin expressing cancer cells, chimeric antigen receptor T cells were dose-dependently activated; this activation was enhanced by co-culture with invariant natural killer T cells and subsequently abrogated by treatment with anti-interferon-γ antibodies. Furthermore, the cytotoxicity of chimeric antigen receptor T cells against various cancer cells was further augmented by invariant natural killer T cells., Conclusions: The use of adoptive transfer with mesothelin-specific chimeric antigen receptor-expressing CD8 T cells against salivary gland cancers is an effective therapy and invariant natural killer T cells are expected to be used in adjuvant treatment for T cell-based immunotherapy.

Low levels of peripheral blood natural killer T (NKT) cells in cancer patients and a favorable outcome associated with a high number of tumor-infiltrating NKT cells demonstrated in several studies indicated the important role of these immune cells in the antitumor response. With effective antitumor immunity via direct tumor lysis, cytokine modulation of effector cells and regulation of immunosuppressive cells, type I NKT cells display interesting features/properties for the rapidly developing chimeric antigen receptor (CAR) technology. Due to their restriction to the monomorphic HLA-like molecule CD1d, but not to the polymorphic human leukocyte antigen (HLA), NKT CAR cells show potential for enabling autologous and allogeneic/off-the-shelf cancer immunotherapy. Promising results were obtained in preclinical NKT CAR cell studies, but clinical trials have not yet been conducted. In this review, we summarize the biological features of NKT cells, their role in antitumor immunity and recent advances in the development of NKT CAR cells., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)