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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.)

Invariant natural killer T (iNKT) cells develop into three subsets (NKT1, NKT2, and NKT17) expressing a distinct transcription factor profile, which regulates cytokine secretion upon activation. iNKT cell development in the thymus is modulated by signaling lymphocytic activation molecule family (SLAMF) receptors. In contrast to other SLAMF members, Ly9 (SLAMF3) is a non-redundant negative regulator of iNKT cell development. Here, we show that Ly9 influences iNKT cell lineage differentiation. Ly9-deficient mice on a BALB/c background contained a significantly expanded population of thymic NKT2 cells, while NKT1 cells were nearly absent in BALB/c.Ly9 -/- thymus. Conversely, the number of peripheral NKT1 cells in BALB/c.Ly9 -/- mice was comparable to that in wild-type mice, indicating that the homeostasis of the different iNKT cell subsets may have distinct requirements depending on their tissue localization. Importantly, Ly9 absence also promoted NKT2 cell differentiation in the NKT1-skewed C57BL/6 background. Furthermore, treatment of wild-type mice with an agonistic monoclonal antibody directed against Ly9 impaired IL-4 and IFN-γ production and reduced by half the number of spleen iNKT cells, with a significant decrease in the proportion of NKT2 cells. Thus, anti-Ly9 targeting could represent a novel therapeutic approach to modulate iNKT cell numbers and activation., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mammalian Target of Rapamycin (mTOR) protein controls the machinery necessary for T-cell activation, differentiation, and memory formation, as a component of mTOR complex 1 (mTORC1) and mTORC2, which function both downstream and upstream of AKT. Invariant natural killer T (iNKT) cells are a unique T-cell subset that exist in a primed state, capable of rapid activation, and produce large quantities of cytokines. iNKT-cell effector differentiation is dependent on the mTORC1 complex; however, the requirements for mTORC2 in iNKT cells have been controversial. In this issue, Sklarz et al. [Eur. J. Immunol. 2017. 47: 516-526] provide a careful analysis of the requirements for the mTORC2 component Rictor in iNKT cells, providing a new twist in this unfolding tale. The authors demonstrate that Rictor is required for iNKT-cell proliferation and survival during the key stage of intrathymic expansion and that Rictor supports the development of NKT17 cells, an effector subset which depends on the transcription factor RORγt and produces interleukin (IL)-17, in both the thymus and the lung. IL-4-producing NKT2 cells develop in the absence of Rictor but the cytotoxic potential of iNKT cells is Rictor-dependent., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invariant NKT (iNKT) cells differentiate in the thymus into three distinct lineages defined by their cytokine and transcription factor expression. Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is essential for early stages of iNKT cell development, but its role during terminal differentiation of iNKT1, iNKT2, or iNKT17 cells remains unclear. Taking advantage of SAP-deficient mice expressing a Vα14-Jα18 TCRα transgene, we found that SAP is critical not only for IL-4 production but also for the terminal differentiation of IL-4-producing iNKT2 cells. Furthermore, without SAP, the IL-17 producing subset is expanded, while IFN-γ-producing iNKT1 differentiation is only moderately compromised. Lack of SAP reduced the expression of the transcription factors GATA-3 and promyelocytic leukemia zinc finger, but enhanced the levels of retinoic acid receptor-related orphan receptor γt. In the absence of SAP, lineage commitment was actually shifted toward the emergence of iNKT17 over iNKT2 cells. Collectively, our data unveil a new critical regulatory function for SAP in thymic iNKT cell fate decisions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cell surface receptor CD155 influences a variety of immune processes by binding to its ligands CD226, CD96, or TIGIT. Here, we report that the interaction of CD155 with CD226 in the thymus of BALB/c mice has a dual function. It directly influences the dwell time of memory-like CD8(+) T cells, while it is indirectly involved in generating these cells. It was shown earlier that a massive emergence of memory-like CD8 T cells in thymus crucially depends on abundant IL-4, secreted in steady state by iNKT2 (where iNKT is invariant NKT) cells, a subclass of iNKT cells. Here, we show that absence of either CD155 or CD226 in BALB/c mice causes a profound shift in the iNKT subtype composition in thymus, expanding the frequency and numbers of iNKT1 cells at the expense of iNKT2 cells, as well as iNKT17 cells. This shift results in a drop of available IL-4 and creates a scenario similar to that observed in C57BL/6 mice, where iNKT1 cells predominate and iNKT2 cells are much less frequent when compared with BALB/c mice. Yet also in C57BL/6 mice, lack of CD155 or CD226 provokes a further decline in iNKT2 cells, suggesting that the observed effects are not restricted to a particular inbred strain., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Itk(-/-) mice exhibit defects in the activation, development, and function of CD4(+) and CD8(+) T cells and iNKT cells. These and other defects in these mice make it difficult to uncouple the developmental versus functional requirement of Itk signaling. Here, we report an allele-sensitive mutant of Itk (Itkas) whose catalytic activity can be selectively inhibited by analogs of the PP1 kinase inhibitor. We show that Itkas behaves like WT Itk in the absence of the inhibitor and can rescue the development of Itk(-/-) T cells in mice. Using mice carrying Itkas, we show using its inhibitor that Itk activity is required not only for Th2, Th17, and iNKT-cell cytokine production, but also surprisingly, for Th1 cytokine production. This work has important implications for understanding the role of Itk signaling in the development versus function of iNKT cells, Th1, Th2, and Th17 cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The organization of the thymus into distinct cortical and medullary regions enables it to control the step-wise migration and development of immature T-cell precursors. Such a process provides access to specialized cortical and medullary thymic epithelial cells at defined stages of maturation, ensuring the generation of self-tolerant and MHC-restricted conventional CD4(+) and CD8(+) αβ T cells. The migratory cues and stromal cell requirements that regulate the development of conventional αβ T cells have been well studied. However, the thymus also fosters the generation of several immunoregulatory T-cell populations that form key components of both innate and adaptive immune responses. These include Foxp3(+) natural regulatory T cells, invariant γδ T cells, and CD1d-restricted invariant natural killer T cells (iNKT cells). While less is known about the intrathymic requirements of these nonconventional T cells, recent studies have highlighted the importance of the thymus medulla in their development. Here, we review recent findings on the mechanisms controlling the intrathymic migration of distinct T-cell subsets, and relate this to knowledge of the microenvironmental requirements of these cells., (© 2015 The Authors. European Journal of Immunology published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Atopic eczema (AE) is a chronic relapsing inflammatory skin disease where the commensal yeast Malassezia can act as a microbial trigger factor. Malassezia activates human DC to produce IL-18, an innate cytokine that is elevated in serum of AE patients; however, the precise role of IL-18 in human AE etiology is unknown. Herein, we investigated the effect of IL-18 on the human invariant NKT (iNKT) cell compartment in AE. We found that IL-18 was a potent activator of human iNKT-cells and promoted a pro-inflammatory CD1d-dependent response, even in the absence of exogenous ligands. Chronic activation via IL-18 on the other hand was inhibitory and skewed the iNKT-cell pool by selectively suppressing CD4(+) iNKT-cells. This was mimicked in AE patients where the proportion of CD4(+) iNKT-cells was reduced in peripheral blood and coincided with elevated plasma levels of IL-18. Furthermore, a reduced CD4(+) iNKT-cell pool was associated with elevated IgE levels in plasma, and the plasma levels of IL-18 correlated with both total IgE and disease severity in the AE patients. Based on these findings, we propose that IL-18-mediated activation and subsequent dysregulation of the CD1d-restricted iNKT-cells plays a role in the pathogenesis of human AE.

The glycosphingolipid sulfatide (SO(3)-3Galbeta1Cer) is a demonstrated ligand for a subset of CD1d-restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic beta-cells, is one of several natural ligands for type II CD1d-restricted NKT cells.

The pathogenesis of immune-mediated drug-induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto-antibodies in anesthetic DILI patients suggests a role for IL-4 in this hapten-mediated process. We investigated IL-4-mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL-4(-/-) (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T-cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4(+) T cells adoptively transferred hepatitis to naïve Rag(-/-) mice (p<0.01). Pro-inflammatory cytokines were expectedly decreased in TFA hapten-stimulated KO splenocyte supernatants (p<0.001); however, IL-2 and IFN-gamma (p<0.05), as well as IL-6 and IL-10 (p<0.001) levels were elevated in CYP2E1-stimulated KO splenocyte supernatants, suggesting dual IL-4-mediated pro-inflammatory and regulatory responses. Anti-IL-10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL-4. This is the first demonstration of dual roles for IL-4 in the pathogenesis of immune-mediated DILI by suppressing auto-antigen-induced regulatory responses while promoting hapten-induced pro-inflammatory responses., Competing Interests: None

Development of NKT cells was shown to depend on lymphotoxin (LT) and IL-15 signaling pathways as well as on cytokine receptor common gamma chain. After positive selection, NKT-cell precursors transit through progressive maturation stages including proliferative expansion within the NK1.1(-) window. The transcription factors that integrate different signaling pathways into different stages of NKT-cell development are not well characterized. Here, we show that the Rel/NF-kappaB family member RelA regulates the NK1.1(-) to NK1.1(+) transition during NKT-cell development. RelA is also required for both IL-15- and IL-7-induced proliferation of CD44(hi)NK1.1(-) NKT-cell precursors. Activation of the invariant NKT-cell receptor induces both IL-15 receptor alpha and gamma chains' expression in an NF-kappaB-dependent manner, suggesting a molecular mechanism by which NF-kappaB regulates NKT-cell development. NF-kappaB also regulates TCR-induced expression of LT-alpha and LT-beta within NKT cells. In contrast to previous reports, however, we show that LT signaling is dispensable for thymic NKT-cell development but is essential for their colonization of peripheral organs such as liver.