A Novel Mouse Model for Invariant NKT Cell Study1

We have generated a novel mouse model harboring the in-frame rearranged TCRVα specific for invariant NKT (iNKT) cells (Vα14-Jα18) on one allele by crossing the mouse cloned from NKT cells with wild-type mice. This genomic configuration would ensure further rearrangement and expression of TCRVα14-Jα18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRVα (Vα14-Jα18 mouse) possessed an increase in iNKT cells in the thymus, liver, spleen, and bone marrow. Intriguingly, both Th1- and Th2-type cytokines were produced upon stimulation with αGalactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the Vα14-Jα18 mouse. These features markedly distinguish the nature of iNKT cells present in the Vα14-Jα18 mouse from that of iNKT cells found in the Vα14-Jα18 transgenic mouse. Besides these, the expression of TCRVγδ cells remained intact, and the use of the TCRVβ repertoire in iNKT cells was highly biased to TCRVβ8 in the Vα14-Jα18 mouse. Furthermore, αGalactosylceramide-CD1d dimer-reactive immature iNKT cells expressed less Rag2 as compared with the conventional immature T cells at the positive selection stage. Cell cycle analysis on the thymocytes revealed that no particular subset proliferated more vigorously than the others. Crossing the Vα14-Jα18 mouse with the CD1d knockout mouse revealed a novel population of iNKT cells whose coreceptor expression profile was similar to that assigned to iNKT precursor cells. These mice will be useful for the study on the development of iNKT cells as well as on their functions in the immune system.

I nvariant NKT (iNKT) 3 cells are a subset of TCR␣␤ T cells possessing a set of markers for the NK cell lineage, such as NK1.1 and a member of Ly-49 family, as well as the invariant V␣14-J␣18 TCR (1)(2)(3). They may function as regulatory cells because they produce a copious amount of cytokines. Defective development of iNKT cells has been linked to autoimmunity (4,5). Intriguingly, the development of iNKT cells is not dependent on the classical MHCs, but is conditional upon CD1d, a MHC-like molecule, mainly expressed on double-positive (DP) thymocytes (6,7). Furthermore, they occupy up to 0.7% of the thymocytes as revealed by CD1d tetramer or dimer loaded with a synthetic ligand, ␣-galactosylceramide (␣GalCer) (8,9). Such an unusual abundance in the invariant TCRV␣ is intriguing in that their development may be somehow unique as compared with that of the mainstream ␣␤T cells. Many efforts have been devoted to elucidate the development of such cells and suggested that their commitment is "selected" during thymocyte development (10). Ac-cording to this, iNKT cells diverge from CD4 ϩ CD8 ϩ DP in which cells undertake random TCRV␣ rearrangement, and those succeeding in rearranging V␣14 with J␣18 are "selected" to follow the fate to become iNKT cells. The fact that there are no iNKT cells present in CD1d knockout mice argues that CD1d is indispensable for the development of iNKT cells as well as of non-iNKT cells (8,(11)(12)(13).
The study on the V␣14-J␣18 transgenic (Tg) mice demonstrates 1) a preponderance of iNKT cells in the thymocytes and 2) a biased decrease of TCRV␤7 and V␤8 in CD8 single-positive (SP) cells that gives ground for a favorable selection of V␣14-J␣18 (8,14). Such a bias in TCRV␤ repertoire, however, does not necessarily insure the lineage commitment of iNKT cells upon assemblage of V␣14-J␣18 and TCRV␤7 or TCRV␤8, as it is conceivable that such a combination may result in an inhibition of the positive selection imposed by TCRV␤8 ϩ /CD8 SP cells. Similarly, V␣24-J␣Q Tg/C␣ Ϫ/Ϫ mice show a slight increase in iNKT cell number and an enhanced V␤7 usage concomitant with a decrease in V␤8.2 usage (15). Furthermore, the V␣14-J␣18 Tg mouse produced only IL-4, but not IFN-␥, upon the Ag stimulation, and the mice possessed Th-2 biased Ig production in the serum (14). Although V␣24-J␣Q Tg/C␣ Ϫ/Ϫ mice produced both cytokines, the amount of the cytokines did not reflect the increase of iNKT cells (15). Because the production of both Th1-and Th2-type cytokines is a hallmark of iNKT cells, the V␣14-J␣18 or V␣24-J␣Q Tg mouse may not be suitable for certain experiments. These Tg mice have other disadvantages in that the spatiotemporal expression of the V␣14-J␣18 or V␣24-J␣Q is not ensured, and that cells are not allowed to delete such a transgene by further rearrangement. It is, therefore, imperative to develop a novel mouse model in which the development and the functions of iNKT cells mimic those found in the control as faithfully as possible.
Our recent work has provided evidence that nuclei from iNKT cells are competent for reprogramming a genome in such a way that they exert the totipotency when transferred into enucleated oocytes (16). Because such cloned mice possess a rearranged set of the in-frame rearranged V␣14-J␣18 and V␤8-D-J, some progenies from the clone will inherit the in-frame rearranged V␣14-J␣18 in one allele (hereafter referred as the V␣14-J␣18 mouse). In such circumstances, expression of the V␣14-J␣18 TCR would be under the control of the endogenous promoter(s) and enhancers that ensure their proper expression, and the V␣14-J␣18 TCR is readily subject to further rearrangement.
We found that V␣14-J␣18 mice harbored an increased population of iNKT cells in the thymus as well as liver, spleen, and bone marrow. Concomitantly, these mice produced a copious amount of Th1-and Th2-type cytokines upon an appropriate stimulus both in vitro and in vivo relative to the control. These data indicate that the V␣14-J␣18 mouse would be a novel model suitable for studying the development and the functions of iNKT cells.

Materials and Methods
Generation of the in-frame rearranged V␣14-J␣18 mice The cloned mouse harboring the in-frame rearranged V␣14-J␣18 locus derived from C57BL/6 was mated with female C57BL/6 (Charles River Laboratories) and resulting progenies were analyzed by Southern blot for the inheritance of the V␣14-J␣18 locus (16). Mice harboring the in-frame rearranged V␣14-J␣18 on one allele (V␣14-J␣18 mice) were used throughout the study unless specified. V␣14-J␣18 mice were mated with CD1d knockout mice to generate mice possessing the in-frame rearranged V␣14-J␣18 on the CD1d Ϫ/Ϫ background in F 2 . Mouse colonies were maintained in a specific pathogen-free facility at the Research Center for Allergy and Immunology and all experiments were conducted in compliance with the protocol approved by the RIKEN Animal Care and Use committee.

Cell cycle analysis
Thymocytes from C57BL/6 and V␣14-J␣18 mice were stained with anti-TCRV␤ and -TCRV␤8 mAbs, and sorted into four populations as described in the legend for Fig. 5b. Cell cycle analysis on these thymocytes was performed with propidium iodide according to the protocol provided by BD Biosciences after RNase treatment.

Inheritance of the rearranged V␣14-J␣18 locus in the progeny
We have established the cloned mice from the peripheral NKT cells by direct nuclear transfer (16). In such mice, both TCRV␣ and TCRV␤ loci are rearranged in-frame in the germline. As for the TCRV␣␤ combination, clone 1 had V␣14-J␣18 and V␤8S2-D␤1-J␤2S5, while clone 2 possessed V␣14-J␣18 and V␤8S3-D␤1-J␤1S4. Because the genomic sequence for V␣14-J␣18 in clone 1 is from C57BL/6 (16), we have focused our study on the progenies using clone 1 as a founder. We first determined how these loci are segregated in F 1 progeny. Mating with C57BL/6 resulted in the pups possessing the rearranged V␣14-J␣18, V␤8S2-D␤1-V␤2S5, or both (Fig. 1a). Mating these F 1 with C57BL/6 gave rise to mice harboring the in-frame rearranged V␣14-J␣18 in one allele (Fig.  1b, lower panel). The data also demonstrate that the rearranged V␣14-J␣18 locus is inherited by the progenies without affecting the fertility.

Increase of iNKT cells in the thymocytes and peripheral lymphoid organs in V␣14-J␣18 mice
We examined the presence of iNKT cells in V␣14-J␣18 mice and found that 16% of the total thymocytes were iNKT cells (Fig. 2a). The size of thymi from V␣14-J␣18 mice was, however, reduced compared with the control mice. Indeed, the total cell number of the thymocytes from such mice represented ϳ15% of that from the control mice (Table I). Although the absolute number of the thymocytes decreased, the net iNKT cell number was augmented six times over the control (Table I and Fig. 2a). In the spleen, the number of iNKT cells increased Ͼ20 times as compared with the control, and iNKT cells occupied Ͼ50% of the total T lymphocytes (Fig. 2a). In the liver mononuclear cells, more than half were iNKT cells (Fig. 2a). A similar increase of iNKT cells was also observed in the bone marrow where the number of such cells exhibited almost 20 times that of the control (Fig. 2a). We then examined the CD4/CD8 expression profile in the thymocytes. CD4 ϩ /CD8 ϩ DP cells showed a decrease (47%) accompanying an increase in CD4 ϩ (20%) or CD8 ϩ (13%) SP cells (Fig. 2b). Concomitantly, double-negative (DN) cells represented ϳ20% of the total thymocytes (Fig. 2b). Because most of the iNKT cells reported to date are either CD4 ϩ /CD8 Ϫ or CD4 Ϫ /CD8 Ϫ , we evaluated the percentage of iNKT cells within the V␣14-J␣18 mice thymocytes. More than 40% of CD4 ϩ /CD8 Ϫ cells were iNKT cells, while almost half of CD4 Ϫ /CD8 Ϫ cells represented iNKT cells. Among CD4 ϩ / CD8 ϩ and CD4 Ϫ /CD8 ϩ cells, few iNKT cells were present (Fig. 2b). In the periphery, a similar tendency was observed except that some iNKT cells were CD4 Ϫ /CD8 ϩ in the liver (data not shown). The results demonstrated that iNKT cells in V␣14-J␣18 mice are essentially either CD4 ϩ /CD8 Ϫ or CD4 Ϫ /CD8 Ϫ .
We analyzed the expression of the other markers on iNKT cells in the thymus. The profile for the NKRs such as Ly49A, Ly49D, and CD25 in V␣14-J␣18 mice was almost equivalent to that found in the control thymocytes (Fig. 2c, data not shown). In contrast, CD69 ϩ and NK1.1 ϩ cells decreased concomitant with an increase of CD44 dull cells, while CD24 ϩ cells increased in the V␣14-J␣18 mouse thymocytes (Fig. 2c).

Production of a copious amount of the cytokines upon ␣GalCer stimulation in V␣14-J␣18 mice
We next evaluated the potential of iNKT cells to respond to ␣GalCer and the resulting production of the cytokines in the serum. Without any stimulation, no cytokine was produced in both control and V␣14-J␣18 mice. Upon ␣GalCer stimulation, however, both types of mice simultaneously produced Th1-type cytokines such as IL-2 and IFN-␥, Th2-type cytokines, IL-4, IL-5, and IL-10, and inflammatory cytokines such as IL-1␤ and TNF-␣ (Fig. 3a). Cytokine production peaked at 4 h poststimulation for all the cytokines. V␣14-J␣18 mice produced Ͼ10 times cytokines such as IL-2, IL-4, IL-10, GM-CSF, and IFN-␥ relative to the control at 4 h. In contrast, no significant increment in the amount of IL-1␤ and IL-5 was detected in V␣14-J␣18 mice under the same conditions (Fig. 3a).
We also examined whether stimulation with ␣GalCer-pulsed DCs resulted in enhanced cytokine production in vitro. DCs prepared from the spleen of V␣14-J␣18 or control mice were cocul-tured with the whole spleen cells from these mice. Coculture of the control spleen cells with DCs from either control or V␣14-J␣18 mice led to a modest production of IFN-␥ and IL-4 (Fig. 3b). In contrast, irrespective of the origin of DCs, use of spleen cells from V␣14-J␣18 mice resulted in a 5-and 10-fold increase for IL-4 and IFN-␥, respectively (Fig. 3b). This increment mirrored well the abundance of iNKT cells in the V␣14-J␣18 mouse spleen. The  1-9) was digested with EcoRI (for TCRV␣14) or BamHI (for TCRV␤) and subjected to Southern blot with the specific probes (16). Arrowheads show the bands indicating the TCR rearrangement. C: control DNA from C57BL/6. b, Schematic representation of the TCRV␣14 locus in the C57BL/6 and V␣14-J␣18 mouse. The TCRV␣14 locus in nonrearranged and rearranged germline configuration is shown with EcoRI sites for C57BL/6 (upper panel) and V␣14-J␣18 mice (lower panel), respectively. Ⅺ, V␣14 exon; u, J␣18 exon; f, probe used for Southern blot. production of IL-4 and IFN-␥ from the splenic iNKT cells was confirmed by intracellular staining (Fig. 3c).
Because V␣14-J␣18 Tg mice harbor a Th2-biased Ig isotype in serum, we examined whether this was the case for V␣14-J␣18 mice (14). A modest increase in Th2-type IgG1 (5-fold above controls on averages) concomitant with a decrease in Th1-type IgG2a production (one-tenth relative to the controls on average) was detected. Nevertheless, there was little bias in the amount of IgE between V␣14-J␣18 mice and the littermates (Fig. 3d).

␥␦T cells in V␣14-J␣18 mice
To exclude the possibility that the increase of iNKT cells in the thymus was due to the results of ␣␤ vs ␥␦ lineage commitment competition (17) or to exclusive transcription of the allele harboring the rearranged V␣14-J␣18 whatever be the reason, ␥␦T cells in the intestinal intraepithelial lymphocytes (iIEL) were analyzed. It turned out that the cell number of the each subset and the ratio of ␣␤:␥␦ in V␣14-J␣18 mice were quasiequivalent to those of the control mice (Fig. 3e), and there were ␥␦T cells in the thymocytes (data not shown). These experiments also demonstrated that ␥␦ T cells stemmed from the other nonrearranged allele (Fig. 1b). When B and NK cells from V␣14-J␣18 mice were examined, little difference in the cell number and in the receptor expression profile was noticed relative to the controls (data not shown). Together, these data suggested that the in-frame rearranged V␣14-J␣18 locus mainly affects T lymphocyte development.

Decreased further rearrangement in immature iNKT cells relative to the conventional T cells at the DP stage
We next examined whether there was a difference in further rearrangement between immature iNKT and the immature conventional T cells. Rag2 expression was examined with RT-PCR using TCRV␤ med /TCRV␤8 med and TCRV␤ med /TCRV␤8 Ϫ populations, both of which represent DP cells. A population stained with ␣Gal-Cer-CD1d dimers in DP is considered as immature iNKT cells under maturation, while cells not stained with this reagent are considered as the conventional T cells under positive selection. RT-PCR analysis revealed that immature iNKT cells expressed less Rag2 than the conventional T cells in both TCRV␤ med / TCRV␤8 med and TCRV␤ med /TCRV␤8 Ϫ populations (Fig. 5a). As an internal control, there was no difference in the expression of Myd118 and Hprt between ␣GalCer-CD1d dimer-reactive and nonreactive cells (Fig. 5a).   (2) thymocytes derived from V␣14-J␣18 mice were subdivided into ␣GalCer-CD1d dimer-reactive (dim ϩ ) and nonreactive (dim Ϫ ) cells, and subjected to semiquantitative RT-PCR for the expression of Rag2, Myd118, and Hprt. Each sample was analyzed with a 3-fold serial dilution. b, No massive apoptosis and vigorous expansion in the V␣14-J␣18 mice thymocytes. Thymocytes from C57BL/6 (control) and V␣14-J␣18 mice were stained with anti-TCRV␤ and -TCRV␤8 Abs. Four subsets representing TCRV␤ high /TCRV␤8 Ϫ (1), TCRV␤ med /TCRV␤8 Ϫ (2), TCRV␤ Ϫ /TCRV␤8 Ϫ (3), and TCRV␤ ϩ /TCRV␤8 ϩ (4) were analyzed for the cell cycle status and apoptosis with propidium iodide staining. The number indicates the percentage of cells in G 2 -M phase. One representative data from three experiments is shown.

No vigorous proliferation and no massive apoptosis in the V␣14-J␣18 mouse thymocytes
Because the above data indicated that immature iNKT cells less frequently perform further rearrangement relative to the conventional immature T cells, it was necessary to examine whether a particular subset of the thymocytes proliferated more vigorously than the others, and/or whether there was massive apoptosis to give grounds for the preponderance of iNKT cells in the thymus. We, therefore, have examined the cell cycle status of the thymocytes. When TCRV␤ high /TCRV␤8 Ϫ cells (mature T cells) from the control mouse thymocytes were stained with propidium iodide, Ͼ98% of cells were in the G 0 /G 1 phase (Fig. 5b, 1, control). This was also true for TCRV␤ high / TCRV␤8 ϩ cells (Fig. 5b, 4). In TCRV␤ med /TCRV␤8 Ϫ cells that represent the immature T cells, ϳ3% of cells were actively cycling (Fig. 5b, 2). On the contrary, Ͼ9% of TCRV␤ Ϫ / TCRV␤8 Ϫ cells that mostly represent DN cells were in the cycling status (Fig. 5b, 3). In V␣14-J␣18 mice, the percentage of the cycling cells was almost equivalent to that of the control for each subset except TCRV␤ Ϫ /TCRV␤8 Ϫ cells in which Ͼ15% were actively cycling (Fig. 5b, 3, V␣14-J␣18). This may reflect the fact that the absolute number of DN cells increased ϳ2-fold in V␣14-J␣18 mice ( Fig. 2b and Table I). The analyses revealed that no subset of the thymocytes proliferated more vigorously than the others within the V␣14-J␣18 mouse thymocytes concomitant with no extensive apoptosis (Fig. 5b,  V␣14-J␣18).

Discussion
The V␣14-J␣18 mouse as a novel model to study the function of iNKT cells Our present data showed that V␣14-J␣18 locus has a large impact on the development of T lymphocytes in the thymus accompanying a significant increase in iNKT cells (Fig. 2a). Cytokine production experiments suggested that the function of DC remained intact in V␣14-J␣18 mice. Analysis of DC markers such as CD40, CD80, CD86, CD1d, and I-A b on DC revealed no significant difference in their expression profiles relative to the control mice (data not shown). From these data, we could conclude that the functions of iNKT cells in V␣14-J␣18 mice mimic those found in the control mouse. Thus, V␣14-J␣18 mice could represent a novel model to study further the roles of iNKT cells in the immune system.
The V␣14-J␣18 mouse to study the development mode of iNKT cells In line with the assumption that iNKT cells pass through DP thymocytes, the recent fate-mapping study has demonstrated that indeed these cells derive from DP (21). Nonetheless, there remains a fundamental question as to how iNKT cells develop in the thymus. The identification of immature iNKT cells in V␣14-J␣18/CD1d Ϫ/Ϫ mice underpinned the phenotype described in the other studies (Fig. 4b) (18,19). CD1d may be responsible for the expression of NK1.1, CD44, and CD69, and for down-regulation of CD24 (compare Figs. 2c and 4b). The fact that iNKT precursor cells undergo extensive proliferation during their maturation suggests that CD1d is required for iNKT cell survival/proliferation (10,18). Alternatively, it may serve as an extrinsic stimulus that instructs the development of iNKT cells. Extrinsic factors can impact on the consequence of fate decision by multipotent progenitors through two distinct mechanisms. In a permissive mechanism, the cell fate decision of progenitors to differentiate into a particular lineage is made independent of extrinsic factors, and their raison-d'être is to support the survival and proliferation of the committed cells. On the contrary, in an instructive mechanism, extrinsic factors impose upon the progenitors to select one lineage at the expense of others (22). The intensive scrutiny in the neural crest stem cells has demonstrated that neural cell fate is instructively determined by the extrinsic factors such as bone marrow protein 2, neuregulin, TGF␤, and cilial neurotrophic factor (23)(24)(25).
In lymphopoiesis, AgR signals play a pivotal role in the development of lymphocytes. However, the relative contribution of the above two mechanisms in determining the fate of progenitors has been a matter of debate. It is proposed that formation of the pre-TCR complex composed of TCRV␤ together with pT␣ and CD3 instructively induces ␣␤T cells over ␥␦T cells, based on the fact that pT␣ knockout mice contain less in-frame rearranged TCRV␤-harboring cells relative to the control (17). Nonetheless, it can be interpreted that such a pre-TCR complex formation merely supports the survival and/or proliferation of the committed ␣␤T cells, indicating the permissive role for the above pre-TCR complex. Another report has suggested that the ␣␤-␥␦T cell lineage diversification occurs well before the TCR rearrangement, rather supporting the permissive role for TCR signals in the ␣␤T cell lineage commitment (26).
Similarly, the mode of the cell fate decision to be helper (CD4) or cytotoxic (CD8) T cells has also been a long-standing dispute. Transgenic expression of CD8 substantially improves MHC class I-specific CD8 selection while affecting little on class II-specific CD4 selection, arguing that the CD8 cell lineage commitment is instructively determined (27). Subsequent study on MHC class I knockout mice revealed that indeed there are no CD8 cells. However, reintroduction of CD4 using the transgene into such mice restored CD8 cells and vice versa (28,29). These studies simply denote that the lineage choice to be CD4 or CD8 cells is not dictated by CD4 or CD8 per se; it conferred room for an interpretation that signals elicited from these molecules permissively contribute to the survival and/or proliferation of the committed CD4 or CD8 cells. These seemingly conflicting interpretations stem from the fact that transgenic expression or ablation of the signaling molecules tend to support their permissive action on the cell fate decision through providing or depriving of survival signals.
Another caveat to address the above issue is that the lymphocyte progenitors proliferate more vigorously than the neural progenitors. Because the presence of precommitted and committed cells is a prerequisite for addressing the issue, such a propensity often blunts the interpretation. It is thus imperative to examine whether the lineage precommitted cells already predominated the lymphocytes early in development. Hitherto, due to lack of appropriate assays, it has not been possible to address properly whether the AgR signals instructively or permissively affect the destiny of the progenitor cells.
The analysis on the V␣14-J␣18 mouse thymocytes revealed that iNKT cells comprise CD1-interacted and CD1d-noninteracted cells as evidenced by the presence of CD24 low /NK1.1 ϩ / CD44 high and CD24 high /NK1.1 Ϫ /CD44 low cells, respectively (Figs. 2c, 4b, and 6, a and b). This antecedent allows us to investigate further whether the development of iNKT cell is determined instructively or permissively by CD1d through the interaction with the V␣14-J␣18TCR. Our present data rather support the former possibility based on the following observations. 1) The preponderance of iNKT cells in the thymus (Fig.  2a).
2) The CD1d-dependent preferential association of V␣14-J␣18TCR with V␤8 (Fig. 4c). 3) Immature iNKT cells found in DP express less Rag2 relative to the conventional T cells under positive selection (Fig. 5a). 4) Few subsets of the thymocytes proliferate more vigorously than the others, and show a massive apoptosis (Fig. 5b). These data are in line with the instructive mode of iNKT cell development in that upon interaction with CD1d, immature iNKT cells obey their fate to be iNKT cells without striving to change the TCR␣ repertoire by operating the recombination machinery, and without dying massively by apoptosis (Fig. 6c). Although further study has yet to be performed to rigorously prove the above hypothesis, the V␣14-J␣18 mouse is an invaluable model to shed light on the mode of iNKT cell development. FIGURE 6. a, iNKT cells before/after CD1d interaction. The status of iNKT cells before and after interaction with CD1d is schematically depicted. Half-tone text shows that the commitment has not yet completed, thereby the nascent V␣14-J␣18 TCR may be subject to further rearrangement (left panel, CD1d-nonreacted). Plain text indicates that the commitment is over upon CD1dϪ (V␣14-J␣18 TCR) interaction and further rearrangement of the V␣14-J␣18 TCR that culminates in conversion to Th or cytotoxic T cell (Th/Tc) lineage is blocked (right panel, CD1d-reacted). b, Schematic representation of iNKT cells in the thymocytes. CD1d-nonreacted (half tone text and open circle) and CD1d-reacted (plain text and shaded circle) iNKT cells in the thymocytes are shown. However, the relative abundance of the CD1d-nonreacted cells over the CD1d-reacted cells is different in each thymocyte. In the control thymocytes, a few CD1d-nonreacted immature iNKT are present (left panel), while the V␣14-J␣18 thymocytes consist of CD1dnonreacted and CD1d-reacted iNKT cells (middle panel). In contrast, no CD1d-reacted iNKT cells are present in the V␣14-J␣18/CD1d Ϫ/Ϫ mouse thymocytes (right panel). Note that the number of CD1d-nonreacted and of CD1d-reacted iNKT cells depicted in the figure does not necessarily reflect the real number of the each subset. c, Instructive vs permissive cell commitment. Instructive model (left): The (V␣14-J␣18TCR)-CD1d signal instructs the multipotent progenitors (NKT/Th/ Tc) to commit to be iNKT cells at the expense of the Th and CTLs (half tone). Permissive model (right): In contrast, the (V␣14-J␣18TCR)-CD1d signal solely supports the survival and proliferation of the committed iNKT cells. Note that the commitment of iNKT cells from the multipotent progenitors is determined independent of the (V␣14-J␣18TCR)-CD1d signal. In this case, simultaneously generated Th and CTLs that happened to express the V␣14-J␣18 TCR should be deleted by apoptosis, or they should alter V␣14-J␣18 TCR to change their fate by operating recombination machinery to give ground for the predominance of the thymic iNKT cells upon (V␣14-J␣18 TCR)-CD1d signal.