New Genetically Manipulated Mice Provide Insights Into the Development and Physiological Functions of Invariant Natural Killer T Cells.

Invariant natural killer T (iNKT) cells are a unique T cell subset that exhibits characteristics of both innate immune cells and T cells. They express Vα14-Jα18 (Trav11-Traj18) as an invariant chain of the T cell receptor (TCR) and are restricted to the MHC class I-like monomorphic antigen presenting molecule CD1d. iNKT cells are known as immune regulators that bridge the innate and acquired immune systems by rapid and massive production of a wide range of cytokines, which could enable them to participate in immune responses during various disease states. Thus, Traj18-deficient mice, Cd1d-deficient mice, or iNKT cell-overexpressing mice such as iNKT TCRα transgenic mice and iNKT cell cloned mice which contain a Vα14-Jα18 rearrangement in the TCRα locus are useful experimental models for the analysis of iNKT cells in vivo and in vitro. In this review, we describe the pros and cons of the various available genetically manipulated mice and summarize the insights gained from their study, including the possible roles of iNKT cells in obesity and diabetes.

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.

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines.

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T(H)2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺iNKT cells are present in the thymic CD44⁺/⁻ NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺iNKT cells produce T(H)2 (IL-13), T(H)9 (IL-9 and IL-10), and T(H)17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T(H)17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.

Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells.

NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with alpha-galactosylceramide, the prototypic NKT cell-activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell-specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-gamma. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell-targeted therapy in humans.

Induction of Th1-biased cytokine production by alpha-carba-GalCer, a neoglycolipid ligand for NKT cells.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice.

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.