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* Environmental Biology, School of Medicine, Hokkaido University, Sapporo, Japan;
Laboratory for Immune Regulation,
Laboratory for Lymphocyte Development, and
Developmental Genetics Group, RIKEN, Research Center for Allergy and Immunology, Yokohama-shi, Japan; and
¶ Bioresource Engineering Division, RIKEN, Bioresource Center, Tsukuba, Japan
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Abstract |
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specific for invariant NKT (iNKT) cells (V14-J18) 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 TCRV14-J18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRV (V14-J18 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 Galactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the V14-J18 mouse. These features markedly distinguish the nature of iNKT cells present in the V14-J18 mouse from that of iNKT cells found in the V14-J18 transgenic mouse. Besides these, the expression of TCRV
cells remained intact, and the use of the TCRV
repertoire in iNKT cells was highly biased to TCRV8 in the V14-J18 mouse. Furthermore, Galactosylceramide-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 V14-J18 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. |
Introduction |
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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 V14-J18 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). According to this, iNKT cells diverge from CD4+CD8+ DP in which cells undertake random TCRV rearrangement, and those succeeding in rearranging V14 with J18 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 V14-J18 transgenic (Tg) mice demonstrates 1) a preponderance of iNKT cells in the thymocytes and 2) a biased decrease of TCRV7 and V8 in CD8 single-positive (SP) cells that gives ground for a favorable selection of V14-J18 (8, 14). Such a bias in TCRV repertoire, however, does not necessarily insure the lineage commitment of iNKT cells upon assemblage of V14-J18 and TCRV7 or TCRV8, as it is conceivable that such a combination may result in an inhibition of the positive selection imposed by TCRV8+/CD8 SP cells. Similarly, V24-JQ Tg/C–/– mice show a slight increase in iNKT cell number and an enhanced V7 usage concomitant with a decrease in V8.2 usage (15). Furthermore, the V14-J18 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 V24-JQ Tg/C–/– 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 V14-J18 or V24-JQ Tg mouse may not be suitable for certain experiments. These Tg mice have other disadvantages in that the spatiotemporal expression of the V14-J18 or V24-JQ 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 V14-J18 and V8-D-J, some progenies from the clone will inherit the in-frame rearranged V14-J18 in one allele (hereafter referred as the V14-J18 mouse). In such circumstances, expression of the V14-J18 TCR would be under the control of the endogenous promoter(s) and enhancers that ensure their proper expression, and the V14-J18 TCR is readily subject to further rearrangement.
We found that V14-J18 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 V14-J18 mouse would be a novel model suitable for studying the development and the functions of iNKT cells.
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Materials and Methods |
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14-J18 mice
The cloned mouse harboring the in-frame rearranged V14-J18 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 V14-J18 locus (16). Mice harboring the in-frame rearranged V14-J18 on one allele (V14-J18 mice) were used throughout the study unless specified. V14-J18 mice were mated with CD1d knockout mice to generate mice possessing the in-frame rearranged V14-J18 on the CD1d–/– background in F2. 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.
Flow cytometry
Liver lymphocytes were prepared with the Percoll gradient method by centrifugation. Cells from 8- to 10-wk-old mice were stained with mAbs, anti-CD4 (L3T4 RM4-5), anti-CD8 (LY-2 53-6.7), anti-TCRV (H57-597), anti-TCRV8 (F23.1), anti-TCR (GL3), anti-NK1.1 (PK136), anti-CD24 (M1/69), anti-CD44 (Im7), anti-CD69 (H1.2F3), anti-CD1d (1B1) (BD Pharmingen). GalCer was obtained from Kirin Brewery and CD1d:Ig dimers were obtained from BD Biosciences. GalCer-CD1d dimers were prepared according to the manufacturer’s protocol (BD Biosciences). For GalCer-CD1d dimer staining, cells were blocked with anti-CD16/32 (2.4G2; BD Biosciences) for 5 min on ice. After brief centrifugation, the supernatant was removed and mixed with fluorescently labeled GalCer-CD1d dimers followed with the appropriate mAbs for 30 min on ice. Stained cells were analyzed with a FACSCalibur or FACSVantage Diva (BD Immunocytometry Systems). Data were analyzed with FlowJo software (Tree Star).
In vitro and in vivo stimulation with GalCer
Dendritic cells (DCs) (5 x 104 cells) were enriched with CD11c Microbeads (Miltenyi Biotec) from the control and V14-J18 mice spleens and pulsed with GalCer (200 ng/ml) for 6 h before mixture with the whole spleen cells derived from both types of mice (1 x 106 cells). After 72-h coculture, the concentration of IFN- and IL-4 was measured with an ELISA kit (Genzyme Techne). For in vivo stimulation, mice received i.p. GalCer (2 µg/mouse) and the serum was sampled at the indicated time. Concentration of the resultant cytokines was measured with the Bio-Plex Suspension Array system (Bio-Rad). The relative amounts of IgG1, IgE, IgG2a, IgG2b, IgG3, IgM, and IgA were measured with the Mouse Ig ELISA-Quantification kit (Bethyl Laboratories).
Intracellular staining
Three hours after i.p. GalCer injection (2 µg/mouse), TCRV8+ cells from the V14-J18 mouse spleen were subject to intracellular staining with anti-IL-4 or anti-IFN- Abs in parallel with the isotype control (Cytofix/Cytoperm kit; BD Biosciences).
RT-PCR
Thymocytes from V14-J18 mice were stained with anti-TCRV, anti-TCRV8 mAbs, and GalCer-CD1d dimers, and sorted into four populations as described in the legend for Fig. 5a. Typically, starting from 3.0 x 108 V14-J18 mouse thymocytes, 2.5 x 105 GalCer-reactive TCRVmed/TCRV8– cells and 1.0 x 106 GalCer-nonreactive TCRVmed/TCRV8– cells were obtained after cell sorting. Similarly, the number of GalCer-reactive TCRVmed/TCRV8med cells and that of GalCer-nonreactive TCRVmed/TCRV8med cells were 2.1 x 105 and 2.3 x 105, respectively. RNA was prepared with the RNeasy Micro kit (Qiagen) and the first strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer’s instruction. The sequences of primers were: 5'-CACAggACTAgAACACCTgC-3' and 5'-gCTggTgAAAAggACCTCT-3' for hypoxanthine phosphoribosyltransferase (HPRT); 5'-CTCCTggTCACgAACTgTCA-3' and 5'-gggTAgggTAgCCTTTgAgg-3' for MyD118, and 5'-CCCAgAgAACCACAgAAAAAT-3' and 5'-TAACCACCCACAATAACAAAT-3' for RAG2.
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Thymocytes from C57BL/6 and V14-J18 mice were stained with anti-TCRV and -TCRV8 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.
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Results |
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14-J18 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 V14-J18 and V8S2-D1-J2S5, while clone 2 possessed V14-J18 and V8S3-D1-J1S4. Because the genomic sequence for V14-J18 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 F1 progeny. Mating with C57BL/6 resulted in the pups possessing the rearranged V14-J18, V8S2-D1-V2S5, or both (Fig. 1a). Mating these F1 with C57BL/6 gave rise to mice harboring the in-frame rearranged V14-J18 in one allele (Fig. 1b, lower panel). The data also demonstrate that the rearranged V14-J18 locus is inherited by the progenies without affecting the fertility.
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14-J18 mice
We examined the presence of iNKT cells in V14-J18 mice and found that 16% of the total thymocytes were iNKT cells (Fig. 2a). The size of thymi from V14-J18 mice was, however, reduced compared with the control mice. Indeed, the total cell number of the thymocytes from such mice represented 
15% 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 >20 times as compared with the control, and iNKT cells occupied >50% 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+/CD8+ DP cells showed a decrease (47%) accompanying an increase in CD4+ (20%) or CD8+ (13%) SP cells (Fig. 2b). Concomitantly, double-negative (DN) cells represented 20% of the total thymocytes (Fig. 2b). Because most of the iNKT cells reported to date are either CD4+/CD8– or CD4–/CD8–, we evaluated the percentage of iNKT cells within the V14-J18 mice thymocytes. More than 40% of CD4+/CD8– cells were iNKT cells, while almost half of CD4–/CD8– cells represented iNKT cells. Among CD4+/CD8+ and CD4–/CD8+ cells, few iNKT cells were present (Fig. 2b). In the periphery, a similar tendency was observed except that some iNKT cells were CD4–/CD8+ in the liver (data not shown). The results demonstrated that iNKT cells in V14-J18 mice are essentially either CD4+/CD8– or CD4–/CD8–.
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14-J18 mice was almost equivalent to that found in the control thymocytes (Fig. 2c, data not shown). In contrast, CD69+ and NK1.1+ cells decreased concomitant with an increase of CD44dull cells, while CD24+ cells increased in the V14-J18 mouse thymocytes (Fig. 2c).
Production of a copious amount of the cytokines upon GalCer stimulation in V14-J18 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 V14-J18 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. V14-J18 mice produced >10 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 V14-J18 mice under the same conditions (Fig. 3a).
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GalCer-pulsed DCs resulted in enhanced cytokine production in vitro. DCs prepared from the spleen of V14-J18 or control mice were cocultured with the whole spleen cells from these mice. Coculture of the control spleen cells with DCs from either control or V14-J18 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 V14-J18 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 V14-J18 mouse spleen. The production of IL-4 and IFN- from the splenic iNKT cells was confirmed by intracellular staining (Fig. 3c).
Because V14-J18 Tg mice harbor a Th2-biased Ig isotype in serum, we examined whether this was the case for V14-J18 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 V14-J18 mice and the littermates (Fig. 3d).
T cells in V14-J18 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 V14-J18 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 V14-J18 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 V14-J18 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 V14-J18 locus mainly affects T lymphocyte development.
CD1d may be responsible for the up-regulation of CD44, CD69, and NK1.1 and down-regulation of CD24
Mice deficient for CD1d are devoid of iNKT cells, as CD1d is responsible for the development of iNKT and non-iNKT cells (11, 12, 13). iNKT cells present in the periphery express the coreceptor such as CD44, CD69, and NK1.1. Studies on the ontogeny of iNKT cells have indicated that iNKT cells emerge from CD24high/NK1.1–/CD44low cells in the thymus, and they acquire the CD24low/NK1.1+/CD44high phenotype upon emigration from the thymus (18, 19). We have mated V14-J18 mice with CD1d–/– mice to obtain mice harboring the hemizygous V14-J18 and no CD1d (V14-J18/CD1d–/– mouse). If a V14-J18 TCR signal is critical for iNKT cell development, an immature iNKT cell population would be tangible on the CD1d–/– background. Analyses of the V14-J18/CD1d–/– mouse revealed that 1% of the thymocytes were stained with GalCer-CD1d dimers, harboring a little expression of CD69, but little NK1.1, denoting that NK1.1 expression is most likely dependent on CD1d. In contrast, CD24- and CD44low-expressing cells were abundant in the population (Fig. 4a, compare Figs. 2c and 4b). Thus, immature iNKT cells that did not react with CD1d were CD24+CD44dullCD69+/–NK1.1–. It would be intriguing to compare these cells with V14-J18 Tg DN cells in CD1d–/– mice (20).
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usage in iNKT cells in the V14-J18 mouse
Previous studies showed that the usage of TCRV in iNKT cells is highly biased to TCRV8, 7, and 5 (1, 2, 3). The analysis of TCRV+/TCRV8+ cells revealed that >80% of this population comprised iNKT cells (Fig. 4c, upper panel). A similar tendency was observed in TCRV+/TCRV7+ cells but not in TCRV+/TCRV5+ cells (data now shown). These data indicated that TCRV8 was the preferred repertoire of iNKT cells in V14-J18 mice. In contrast, only 20% of TCRV+/TCRV8+ cells were iNKT cells in the V14-J18/CD1d–/– thymocytes (Fig. 4c, lower panel).
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 TCRVmed/TCRV8med and TCRVmed/TCRV8– populations, both of which represent DP cells. A population stained with GalCer-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 TCRVmed/TCRV8med and TCRVmed/TCRV8– 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).
No vigorous proliferation and no massive apoptosis in the V14-J18 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 TCRVhigh/TCRV8– cells (mature T cells) from the control mouse thymocytes were stained with propidium iodide, >98% of cells were in the G0/G1 phase (Fig. 5b, 1, control). This was also true for TCRVhigh/TCRV8+ cells (Fig. 5b, 4). In TCRVmed/TCRV8– cells that represent the immature T cells, 3% of cells were actively cycling (Fig. 5b, 2). On the contrary, >9% of TCRV–/TCRV8– cells that mostly represent DN cells were in the cycling status (Fig. 5b, 3). In V14-J18 mice, the percentage of the cycling cells was almost equivalent to that of the control for each subset except TCRV–/TCRV8– cells in which >15% were actively cycling (Fig. 5b, 3, V14-J18). This may reflect the fact that the absolute number of DN cells increased 2-fold in V14-J18 mice (Fig. 2b and Table I). The analyses revealed that no subset of the thymocytes proliferated more vigorously than the others within the V14-J18 mouse thymocytes concomitant with no extensive apoptosis (Fig. 5b, V14-J18).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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14-J18 mouse as a novel model to study the function of iNKT cells
Our present data showed that V14-J18 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 V14-J18 mice. Analysis of DC markers such as CD40, CD80, CD86, CD1d, and I-Ab 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 V14-J18 mice mimic those found in the control mouse. Thus, V14-J18 mice could represent a novel model to study further the roles of iNKT cells in the immune system.
The V14-J18 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 V14-J18/CD1d–/– 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 V14-J18 mouse thymocytes revealed that iNKT cells comprise CD1-interacted and CD1d-noninteracted cells as evidenced by the presence of CD24low/NK1.1+/CD44high and CD24high/NK1.1–/CD44low 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 V14-J18TCR. 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 V14-J18TCR with V8 (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 V14-J18 mouse is an invaluable model to shed light on the mode of iNKT cell development.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by RIKEN and in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A. Ogura). 
2 Address correspondence and reprint requests to Dr. Hiroshi Wakao, Environmental Biology, School of Medicine, Hokkaido University N15W7 Sapporo 060-8638 Japan. E-mail address: hwakao{at}med.hokudai.ac.jp
3 Abbreviations used in this paper: iNKT, invariant NKT; DP, double positive; GalCer, Galactosylceramide; Tg, transgenic; SP, single positive; DC, dendritic cell; HPRT, hypoxanthine phosphoribosyltransferase; DN, double negative; iIEL, intestinal intraepithelial lymphocyte.
Received for publication April 18, 2007. Accepted for publication July 6, 2007.
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References |
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14 NKT cells in innate and acquired immune response. Annu. Rev. Immunol. 21: 483-513. [Medline]14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188: 1831-1839. 24-JQ TCR supports the development of CD1d-dependent NK1.1+ and NK1.1– T cells in transgenic mice. J. Immunol. 170: 2390-2398. versus T lineage commitment. Immunity 9: 649-655. [Medline] bias of V14 natural killer T cells and consequences for microbial glycolipid recognition. J. Exp. Med. 203: 1197-1207. 14i NKT cell lineage from double-positive thymocyte precursors. Immunity 22: 705-716. [Medline] superfamily members. Cell 85: 331-343. [Medline] versus T cell fate determination is initiated independently of T cell receptor signaling. J. Exp. Med. 193: 689-698. This article has been cited by other articles:
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  H. Wakao, R. Wakao, S. Sakata, K. Iwabuchi, A. Oda, and H. Fujita In vitro induction of natural killer T cells from embryonic stem cells prepared using somatic cell nuclear transfer FASEB J, July 1, 2008; 22(7): 2223 - 2231. [Abstract] [Full Text] [PDF]
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, probe used for Southern blot.
