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.

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, Cd1ddeficient 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.
Numerous studies investigating the role of iNKT cells have utilized mouse models of iNKT cell deficiency. One such model directly targets Jα18 (Traj18 −/− ) (10), which is required for iNKT-TCR formation. However, the overall TCR repertoire diversity is impaired in Traj18 −/− mice, in which Traj18 was replaced with a PGK-Neo r cassette, which had inadvertent but substantial effects on transcription and TCRα gene rearrangements (11). Another model makes use of mice deficient in CD1d (Cd1d1 −/− ) (12), which prevents the development of any CD1d-restricted T cells including iNKT cells. However, in mice, although not in humans, there has been a gene duplication event and so two homologous genes, Cd1d1 and Cd1d2, encode CD1d proteins. Even though Cd1d1 −/− Cd1d2 −/− mice have also been developed (13,14), the role of CD1d2 in iNKT cell development and function is still unclear. Consequently, any changes in immunological activity attributed to iNKT cells that are based on studies of Traj18 −/− or Cd1d −/− mice need to be reassessed.
On the other hand, mice that have been genetically manipulated to have elevated numbers of iNKT cells are also useful tools for iNKT cell study. Therefore, rearranged Vα14-Jα18 and Vβ8 genes were introduced into recombination-activating gene-deficient mice, and there was preferential generation of iNKT cells but no mature B and T lymphocytes (15). iNKT-TCRα transgenic mice that overexpressed iNKT-TCRα (mVα14-Jα18) were firstly generated by Bendelac et al. (16) resulting in preponderance of iNKT cells, while abnormal development of other immune cells were also observed. A human iNKT-TCRα (hVα24-Jα18) transgenic mouse has also been developed by similar approach (17). Moreover, iPS cell lines obtained by reprogramming of mature iNKT cells (iNKT-iPSC) from C57BL/6 (B6) mice preferentially generate iNKT cells but no conventional αβ T or γδ T cells, NK cells, DCs or B cells in vitro (18). Furthermore, mice generated from the iNKT-iPSC had a much larger number of iNKT-like cells (19) compared to mice with a rearranged Vα14-Jα18 transgene (16). It is therefore important to compare the development and function of iNKT cells and their subtypes that differentiate in vivo in these iNKT cell overexpressed mice.

Traj18-DeFiCienT AnD Cd1d-DeFiCienT MiCe
Because iNKT cells are highly conserved among species inclu ding mice and humans, mouse models of iNKT cell deficiency represent useful tools for the analysis of iNKT cell biology. However, as described above, the originally generated Traj18 −/− mice (10) lack transcripts not only of Traj18 but also of genes encoding Jα regions upstream of Traj18, resulting in an almost 60% reduction in the diversity of the TCRα repertoire (11). It is possible that the lower overall αβ TCR diversity of the original Traj18 −/− mice contributed to the divergent results that have been reported by some of the studies that used the mice. Recently, four new Traj18 −/− mouse lines have been established by different research groups including ours. Two lines were generated by classical P1 bacteriophage cyclization recombination/locus of crossover in P1 (Cre/ loxP) technology (20,21), a third was generated by transcription activator-like effector nuclease (TALEN) methodology (TALEN-Traj18 −/− ) (22), and a fourth by using the clustered regularly interspaced short palindromic repeat (CRISPR)/ Cas9 technology (CRISPR-Traj18 −/− ) (23). All four groups analyzed TCRα diversity in CD4 + CD8 + double-positive (DP) thymocytes by next-generation sequencing and found that the usage frequency of Jα gene segments in these new Traj18 −/− mouse lines was comparable with WT B6 mice (20)(21)(22)(23).
In addition to canonical Vα14-Jα18 iNKT cells, another minor α-GalCer/CD1d reactive subset of T cells harboring Trav10-Traj50 was recently described as type Ib NKT cells (24). However, type Ib NKT cells were discovered in mice that lack expression of Traj gene segments upstream of Traj18 (10). We (23) and Chandra et al. (20) could not detect any type Ib NKT cells in the new mouse strains lacking iNKT cells. By contrast, Zhang et al. (22) did find type Ib NKT cells in their TALEN-Traj18 −/− mice. However, these mice express Trav11-Traj18 mRNA with a partial deletion, indicating that a mutant iNKT-TCRα has the unexpected ability to recognize α-GalCer/CD1d. Based on these results, we should rethink the existence of type Ib NKT cells.
It is known that iNKT cells are restricted by CD1d molecules, but that two CD1d isoforms, CD1d1 and CD1d2, are present in mice. Two gene manipulated lines has been developed, Cd1d1 −/− (12) and Cd1d1 −/− Cd1d2 −/− (13,14). iNKT cells are severely impaired in both lines, indicating that CD1d2 cannot substitute for CD1d1 to support iNKT cell development. CD1d1 and CD1d2 in 129/Sv mice share 93% amino acid identity. Although CD1d2 on thymocytes cannot substitute for the development of iNKT cells (25), we cannot eliminate the potential role of CD1d2 in the development or function of iNKT cells. Sundararaj et al. (26) recently reported that the structure of the CD1d2 A′-pocket was restricted in size compared with CD1d1 in complex with endogenous lipids or a truncated acyl-chain analog of α-GalCer. They found that the majority of iNKT cells in the Cd1d1 −/− mice showed an increase in the iNKT2 and iNKT17 populations and a concomitant decrease in iNKT1 compared with WT mice (26). A small but consistent increase in the proportion of cells using the Vβ8 gene segment, concomitant to a reduction in Vβ7 gene usage, was also observed for CD1d2-selected iNKT cells compared with CD1d1-selected iNKT cells (26). B6 mice, but not BALB/c or 129/Sv mice, harbor a two-nucleotide insertion in exon 4 of Cd1d2, which encodes the α3 domain (27). This frameshift mutation introduces a stop codon, abolishing surface expression but possibly still allowing expression of a soluble CD1d2 molecule ( Figure 1A). These results indicate that the CD1d2 molecule can present different sets of self-antigen(s) in the thymus of different mouse strains, thereby potentially impacting the development of iNKT cells. Even though Cd1d2 −/− mice have not yet been established, they should provide an answer to this controversy.
CD1d is also an Ag presenting molecule for cells other than iNKT cells. Another type of CD1d-restricted cell is the type II or variant NKT cell, which has a more diverse TCR repertoire and appears to recognize various lipid Ags including sulfatides, also known as 3-O-sulfogalactosylceramide, which are a class of glycolipids that contain a sulfate group (28). Unfortunately, no tools are yet available that can be used to analyze the entire population of type II NKT cells. Thus, when we discuss type II NKT cells, it is important to understand the advantages and limitations of each experimental tool, as well as the precise definition of type II NKT cells being analyzed (29). There is also a report that the homeostasis of liver-resident IL-17A-producing γδT (γδT-17) cell depends on hepatocyte-expressed CD1d that presents lipid Ag (30); however, it is unclear whether CD1d is required for the development of γδT-17 cells in the thymus (Figure 1B).
Despite a high degree of conservation, subtle but important differences exist between the CD1d Ag presentation pathways of humans and mice. Wen et al. (31) have generated a human CD1d knock-in mouse (hCD1d-KI) which substitute mouse Cd1d1 to human CD1D locus. Reduced numbers of iNKT cells were observed, but at an abundance comparable to that in most humans. They further generated human iNKT-TCRα chain knock-into the hCD1d-KI (32). Similar to humans, the mice developed a subset of CD8αβ + iNKT cells among other human-like iNKT subsets. The results support human CD1D is functionally and phenotypically ortholog of mouse Cd1d1. These hCD1d-KI mice will allow more accurate in vivo modeling of human iNKT cell responses as some human pathogens specifically target human CD1D for pathogenicity and will facilitate the preclinical optimization of iNKT cell-targeted immunotherapies.

inKT CeLLS AnD OBeSiTY
Obesity research is an illustrative example of how the different genetically engineered animals have been employed to study the role of iNKT cells in a complex disease. Both the original Traj18 −/− (10) and the Cd1d −/− (12-14) mice have been used by many different research groups to study the role of iNKT cells and/or type II NKT cells in obesity-related pathologies in high fat diet (HFD)-induced obesity models. However, these studies have reported very conflicting results, with some groups finding no effect (33,34), some protection (35)(36)(37) and others finding promotion (38, 39) of obesity-associated disease.
Among these studies, only one paper, that published by Lynch et al. (33), has focused on the protective role of iNKT cells in obesity by studying HFD-induced obese Traj18 −/− mice on a B6 background. In this study, they found that when mice lacking iNKT cells were placed on an HFD they showed enhanced weight gain, larger adipocytes, fatty livers, and insulin resistance. By contrast, many other research groups suggested a pathogenic role of iNKT cells in obesity by showing an ameliorated metabolic phenotype in HFD-induced obese Traj18 −/− or Cd1d −/− mice. In the studies that used B6 background Traj18 −/− mice, Wu et al. (39) reported ameliorated hepatic steatosis, glucose tolerance, and insulin sensitivity, as well as reduced tissue inflammation in Traj18 −/− mice on an HFD. Pathological roles of NKT cells in obesity were also reported by Satoh et al. (38) in Cd1d1 −/− mice; however, no difference in the metabolic parameters between Traj18 −/− and WT B6 mice on an HFD was observed in their study, arguing for a pathogenic role of type II NKT rather than iNKT cells in these pathologies. Similarly, Kotas et al. (40) and Lee et al. (41) have also reported a minor role of iNKT cells in the development of obesity, by comparing Traj18 −/− and Cd1d1 −/− C d1d2 −/− mice with WT B6 mice on an HFD.
Many reasons for these divergent results have been proposed and discussed, including the age, gender, and background of the mice, HFD type and duration, and the gut flora or environmental microbial distribution among the animals employed by the different research groups. Nevertheless, if we focus only on the results obtained from B6 background Traj18 −/− mice, it is interesting to note that most of the studies have used an HFD of 60% fat calories, and none of them have reported a decreased level of weight gain in Traj18 −/− mice as compared with WT B6 mice (36)(37)(38)(39)(40). It seems that a consensus has been reached that iNKT cells do not participate in promoting the development of obesity, at least as measured by gain in bodyweight.
To exclude the possible effect of impaired TCR repertoire diversity on diet-induced obesity observed in the original Traj18 −/− mice (10, 11), we re-investigated the contribution of iNKT cells to the development of obesity using our CRISPR-Traj18 −/− mice (23) with an unbiased TCR repertoire. The results were clear cut, obese CRISPR-Traj18 −/− mice gained less body weight and had smaller visceral fat-pads and adipocytes, less fat deposits in the liver, and ameliorated glucose tolerance and insulin resistance (23). The ameliorated levels were almost equivalent to those seen in obese Cd1d1 −/− mice, indicating that iNKT cells play a pathogenic role in diet-induced obesity and that the impact of CD1d deficiency on metabolism is iNKT cell dependent.
It is notable that one of the T cell populations affected by impaired TCR repertoire diversity in the original Traj18 −/− mice, the mucosal-associated invariant T (MAIT) cells that use the invariant Vα19-Jα33 (Trav1-Traj33) chain in mice (42), were recently reported to have an altered distribution and cytokine productions in obese patients, and were found to be positively associated with insulin resistance (43,44). Considering the potential role of MAIT cells and other T cell subsets in obesity, results obtained with the original Traj18 −/− mouse model should be interpreted with caution.

inKT-TCRα TRAnSGeniC AnD inKT CeLL CLOneD MiCe
Mice that have been genetically manipulated to have elevated numbers of iNKT cells were first attempted to generated by overexpressed Vα14-Jα18 iNKT TCRα (mVα14-Jα18) as a transgene (16). Consistent with the results from Cd1d-deficient mice (12)(13)(14), the mVα14-Jα18 transgenic mice exhibited increased IL-4 and immunoglobulin (Ig) E in serum, indicating that mouse iNKT cells are one of the important sources of IL-4 and IgE. Because both human and mouse iNKT cells are restricted to α-GalCer/CD1d, a human iNKT TCRα (hVα24-Jα18) transgenic mouse has also been developed (17). Interestingly, analysis of the mice and derived hVα24-Jα18 + T cells revealed that type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] is associated with an extreme T helper (Th) 1 phenotype of hVα24-Jα18 + T cells, suggesting that there is a strong link between hVα24-Jα18 + T cells and human type 1 diabetes. On the other hand, there is evidence that IL-4 exerts a dominant-negative effect on the progression to IDDM in non-obese diabetic (NOD) mice (45)(46)(47), and NOD mice with the mVα14-Jα18 transgene were protected from diabetes (48), indicating that not only the number but also the phenotype of iNKT cells influences the incidence of diabetes both in humans and mice. The fact that the gut microbiota can impact iNKT cell development and functions (49)(50)(51) and is associated with diabetes onset, regulatory imbalance, and IFN-γ levels in NOD mice should be also considered (52). Another iNKT cell-overexpressing mouse line was derived from iPSCs. iPSCs hold tremendous potential for applications not only in drug discovery, regenerative medicine, and cell replacement therapy (53)(54)(55), but also in basic biology. We have succeeded in reprogramming splenic iNKT cells from WT B6 mice (18). These iPSC-iNKT cells could be differentiated into iNKT cells in vitro and secreted large amounts of IFN-γ. Importantly, iPSC-iNKT cells recapitulated the known adjuvant effects of natural iNKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent iNKT cells via an iPSC phase, an approach that may be adapted for iNKT cell-targeted therapy in humans (56,57). We further succeeded in generating iNKT cell cloned Trav11-Traj18 +/+ mice from iPSC-iNKT cells through germline transmission and breeding with WT B6 mice (19). The absolute numbers and percentages of α-GalCer/CD1d dimer + TCRβ + cells in the thymus and periphery of Trav11-Traj18 +/+ mice were elevated by 10-20fold compared to B6 mice and 10-20-fold compared to B6 mice and by 3-10-fold compared to iNKT-TCRα transgenic mice due to the bypass of TCRα rearrangement at the double-negative (DN) stage. They lacked γδ T cells due to the deletion of the δ locus and had reduced numbers of αβ T cells while NK, B, and DC numbers were normal. However, the surface phenotype of α-GalCer/CD1d dimer + TCRβ + cells in Trav11-Traj18 +/+ mice was different from that in WT B6 mice; there was a partial reduction of CD44 + cells and changes in the CD4 + :NK1.1 + ratio (19). We think this is due to the shortage of CD1d molecules in the face of an excess number of α-GalCer/CD1d dimer + TCRβ + cells because the surface phenotype of the iNKT cells changed into the mature phenotype as seen in WT B6 when these cells were sorted and transferred into Traj18 −/− mice (58). Generation of Trav11-Traj18 +/+ mice carrying a Cd1d1 transgene should clarify this point.

inKT CeLL DeveLOPMenT in THe THYMUS
Until recently, the iNKT cell field had embraced a sequential lineage developmental model (60) in which "developmental intermediates" produce Th2-type cytokines and "mature" NK1.1 + iNKT cells produce Th1 cytokines. However, based on the finding of the expression of distinct transcription factors, T-bet (Tbx21), PLZF (Zbtb16), and RORγt (Rorc) (61) and surface markers, CD4, NK1.1, and IL-17RB (7) in iNKT cell subsets, we considered an alternative "lineage diversification" model for iNKT cells (62), analogous to the differentiation of effector Th cells (63) and innate lymphoid cells (64,65). Three major subsets of iNKT cells (iNKT1, iNKT2, and iNKT17) that produce distinct cytokines have been defined (7,61,66,67) (Figure 2B), and these represent diverse lineages and not developmental stages, as previously thought. In fact, it was recently reported that some iNKT1 cells developed through an alternative DN pathway that bypasses the DP pathway (68), supporting the above findings that iNKT subtypes possibly arise from different precursors in the thymus. Of note, thymic α-GalCer/CD1d dimer + TCRβ + cells from Cd1d1 −/− Trav11-Traj18 +/+ mice described above exhibit the precursors of all iNKT cell subtypes. The poised effector state is acquired during development in the thymus, where iNKT precursors differentiate into one of three distinct subsets, while it has still been unclear which signals control the divergence of iNKT1, iNKT2, and iNKT17. The precise molecular mechanisms should be clarified that are important for iNKT lineage diversification but are dispensable for conventional αβ T cell development. Taken collectively, it can be proposed that the acquisition of diverse functional characteristics by iNKT subtypes might be dependent on the environment providing an appropriate cytokine milieu, as well as on the cytokine receptor signaling in precursor cells undergoing CD1d selection.

COnCLUSiOn
The gene manipulated mice described here will reveal more insights into mouse iNKT cell development and function, and these insights should also be applicable to human iNKT cell studies. Overall, some reported differences between Cd1d −/− and Traj18 −/− mice are likely due to the loss of some T cell populations including MAIT cells in the original Traj18 −/− mice. Traj18 −/− mice with unbiased TCR diversity will inform us of the actual role of iNKT cells and type II NKT cells. Cd1d1 −/− Trav11-Traj18 +/+ mice may further reveal differences in iNKT cell thymic development and may account for the observed mouse strain specific differences. iNKT cells hold great promise for treatment of a myriad of diseases, and these gene manipulated mice will be invaluable in deciphering the role of iNKT cells in health and disease.

AUTHOR COnTRiBUTiOnS
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

ACKnOwLeDGMenTS
This work was supported mainly by a Grant-in-Aid for challenging Exploratory Research (16K14591) by the Japan Society for the Promotion of Science to HW and in part by the Foundation for Young Scholars of Jiangxi Province, China (20161BAB215259) and Key Research and Development Project of Jiangxi Province, China (20161ACG70018) to YR. We thank Ms. Kazuko Okunuki for secretarial assistance.