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Expression of CD1d Under the Control of a MHC Class Ia Promoter Skews the Development of NKT Cells, But Not CD8⁺ T Cells ¹

Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology and Department of Pathology, University of Chicago, Chicago, IL 60637

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although CD1d and MHC class Ia share similar overall structure, they have distinct levels and patterns of surface expression. While the expression of CD1d1 is known to be essential for the development of NKT cells, the contribution of CD1d1 to the development of CD8⁺ T cells appears to be inconsequential. To investigate whether CD1d tissue distribution and expression levels confer differential capacity in selecting these two T cell subsets, we analyzed CD8 and NKT cell compartments in K^b-CD1d-transgenic mice that lack endogenous MHC class Ia and CD1d, respectively. We found that MHC class Ia-like expression pattern and tissue distribution are not sufficient for CD1d to rescue the development of CD8⁺ T cells, suggesting that unique structural features of CD1d preclude its active participation in selection of CD8⁺ T cells. Conversely, cell type-specific CD1d surface density is important for the selection of NKT cells, as the NKT cell compartment was only partially rescued by the K^b-CD1d transgene. We have previously demonstrated that increased CD1d expression on dendritic cells enhanced negative selection of NKT cells. In this study, we show that cell type-specific expression levels of CD1d establish a narrow window between positive and negative selection, suggesting that the distinct CD1d expression pattern may be selected evolutionarily to ensure optimal output of NKT cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The CD1 molecules are MHC-unlinked class Ib molecules that have been conserved throughout mammalian evolution (1, 2, 3). The mouse CD1d is relatively nonpolymorphic and widely expressed on cells of multiple hemopoietic lineages, including B and T cells, macrophages, and dendritic cells (DCs) ⁵ (4, 5, 6). Similar in structure to MHC class Ia, CD1d associates with ₂-microglobulin, has three extracellular domains (1, 2, and 3), a transmembrane region, and a short cytoplasmic tail. Unlike MHC class Ia however, which binds peptide ligand and presents Ag to conventional CD8⁺ T cells, CD1d binds and presents lipid and glycolipid Ag to NKT cells (7, 8, 9).

NKT cells (defined as NK1.1⁺TCR⁺ cells) are a unique subset of T cells that express several phenotypic markers usually associated with NK cells, such as NK1.1, CD122, and Ly49, and exhibit an activated/memory phenotype (HSA^low, CD62L^low, and CD44^high) (10). A major subset of NKT cells in the mouse is restricted by CD1d and these CD1d-restricted NKT cells localize preferentially to thymus and liver (11). CD1d-restricted NKT cells are almost exclusively either CD4⁺ or CD4^-CD8^- (double negative) (12, 13). The absence of CD8⁺ CD1d-restricted NKT cells is somewhat surprising, considering the structural homology between MHC class Ia and CD1d. Most of the CD1d-restricted NKT cells express an invariant TCR -chain (V14-J18) that preferentially pairs with V8.2, V7, or V2 (12, 14, 15, 16). Similar subsets of T cells are also present in humans (17). The majority of human NKT cells express the V24-J15 TCR, which has strong sequence homology to V14-J18 (17). The parallel conservation of both the invariant TCR -chain and the CD1d molecule suggests that this receptor-ligand interaction has an important immunologic function which has been maintained throughout evolution.

Although the physiological Ags for CD1d-restricted NKT cells remains to be defined, a marine sponge-derived glycolipid, -galactosylceramide (GalCer), has been shown to activate NKT cells (8). After GalCer stimulation, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN- (18, 19, 20, 21). This rapid response implicates NKT cells in a role branching innate and adaptive immunity. Indeed, NKT cells have been implicated in protective roles in bacterial and parasitic infection (22, 23, 24), tumor responses (25, 26), and prevention against various autoimmune diseases such as type I diabetes (27, 28, 29, 30) and multiple sclerosis (31, 32, 33).

The role of NKT cells in immunity to pathogens and tumors as well as in protection against autoimmune diseases implies that NKT cell development may be highly specialized. Studies of CD1d-deficient (CD1°) mice showed that the expression of CD1d is essential for the development of NKT cells, indicating that CD1d plays a nonredundant role in the selection of NKT cells. Development of both NKT cells and conventional T cells is thymus dependent (34, 35). Similar to conventional CD8⁺ T cells, the negative selection of NKT cells is enhanced when overall avidity is increased and is mediated by CD1d-expressing DCs, but not thymic epithelial cells (TECs) (36). Positive selection of NKT cells, conversely, is mediated by thymocytes, whereas CD8⁺ T cells are positively selected by thymic epithelial cells (35, 37, 38). Signaling requirements are also different for these two cell types, as NKT cell, but not conventional T cell, development is dependent on Fyn (39). The involvement of different cell types and signaling molecules in the positive selection of conventional CD8⁺ and NKT cells may in part contribute to the distinct characteristics associated with these two subsets of T cells.

The role of CD1d in the development of other T cell subsets is less well established. No detectable changes were observed in either CD4⁺ or CD8⁺ populations in CD1° mice (40, 41, 42), suggesting that the presence of MHC class Ia and class II molecules in these mice may obscure any role of CD1d in selection of conventional T cell populations. Indeed, the number of CD4⁺ T cells in CD1°II° mice was reduced significantly compared with the corresponding population in II° mice (43, 44). Although the observed differences between CD1°II° and II° mice were largely attributed to the loss of CD1d-restricted CD4⁺ NKT cells, a small population of CD4⁺NK1.1^- T cells was also reduced in CD1°II° mice. The majority of these CD1d-restricted CD4⁺NK1.1^- T cells express invariant V14 TCR. Interestingly, the remaining CD1d-restricted CD4⁺NK1.1^- cells also display limited TCR diversity (44). However, in CD1°TAP° mice, as compared with TAP° mice, no significant differences in the CD8⁺ T cell compartment could be detected (43, 44). Thus, despite the fact that CD8 seems to be a coreceptor for CD1d (45, 46), CD1d fails to contribute significantly to the development of CD8⁺ T cells.

Unlike MHC class Ia molecules, which are highly expressed on most cell types, including TECs and DCs, but minimally expressed on immature thymocytes, CD1d is moderately expressed on thymocytes, TECs, and DCs. Thus, it is possible that differential expression patterns of CD1d contribute to its unique role in the selection of NKT cells, while restricting the impact of CD1d in the selection of CD8⁺ T cells. To characterize the role of ligand density and expression pattern of CD1d in the selection of CD8⁺ T cells and NKT cells, we have generated CD1d-transgenic mice driven by the H2-K^b promoter in a MHC class Ia-deficient (K^boD^bo) background (K^b-CD1dTgK^boD^bo) and CD1° background (K^b-CD1dTgCD1°), respectively. In this study, we show that no quantitative or qualitative differences in the CD8⁺ T cell repertoire can be detected between K^b-CD1dTgK^boD^bo and K^boD^bo mice, suggesting that CD1d is not sufficient to mediate positive selection of CD8⁺ T cells, even when expressed at a density and pattern nearly identical to MHC class Ia. Furthermore, the NKT cell compartment is only partially reconstituted in K^b-CD1dTgCD1° mice, suggesting that altering cell type-specific CD1d expression ablates NKT cell development. Thus, the "epitope density threshold" defined by the differences in CD1d surface density on positive and negative selection cell types may be extremely important for efficient selection of NKT cells that express a restricted TCR repertoire.

	Materials and Methods

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Generation of K^b-CD1dTgK^boD^bo and K^b-CD1dTgCD1° mice

The K^b-CD1dTg mouse strains, originally generated in (BALB/c x C57BL/6)F₁, were backcrossed six to seven generations onto a C57BL/6 background and described in detailed previously (36). The K^b-CD1dTg mice were subsequently established on backgrounds deficient for expression of endogenous H2-class Ia (i.e., H2-K^b and H2-D^b) or CD1d by crossing transgenic (Tg) mice with either K^boD^bo mice (six generation backcrossed in B6 background) or CD1° mice (12 generations backcrossed in B6 background).

Cell preparation and flow cytometry

The following mAbs were purchased from BD PharMingen (San Diego, CA): FITC-conjugated mAbs specific for CD4 (RM4-5), TCR (H57-597), V2 (B20.6), V5 (MR9-4), V6 (RR4-7), V7 (TR310), and V8 (MR5-2); PE-conjugated mAbs specific for CD3 (145-2C11), B220 (RA3-6B2), CD8 (53-6.7), NK1.1 (PK136), H2-IA^b (M5114), and CD11c (HL3). Allophycocyanin-CD1d/GalCer tetramers were prepared as described by Matsuda et al. (47). The CD1d-specific mAb 5C6 (hamster IgG) has been described previously (5). Anti-H2-K^b, Y3, was obtained from the American Type Culture Collection (Manassas, VA). Single-cell suspensions from thymus and spleen were prepared by mechanical desegregation in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 20 mM HEPES, 50 µM 2-ME, nonessential amino acids, sodium pyruvate, penicillin, and streptomycin (RPMI 10). Lymphocytes from perfused liver were obtained according to the method described by Goossens et al. (48). Intraepithelial lymphocytes (IELs) were prepared and purified through discontinuous 40%/70% Percoll gradient centrifugation as described by Tagliabue et al. (49). Thymic stromal cell suspensions were prepared by digesting thymic lobes in 0.1% trypsin and 0.5 mM EDTA for 40 min at 37°C. Digestion was stopped by addition of immunofluorescence (IF) buffer (HBSS containing 2% FBS and 0.1% NaN₃). After mechanical disruption of the lobe, cells were harvested and washed twice with IF buffer before cell surface staining experiments. Cells were stained in IF buffer using combinations of fluorescent-conjugated Abs for 30 min at 4°C. When staining involved allophycocyanin-CD1d/GalCer tetramers, incubation time was extended to 1 h. The stained cells were analyzed by flow cytometry using a FACSCalibur (BD Biosciences, Mountain View, CA) with CellQuest software.

Immunohistochemistry

Thymi were embedded in Tissue-Tek OCT (Miles, Elkhart, IN) and frozen at -80°C. Sections measuring 5–7 µm were cut by using a Leica CM1800 cryostat (Leica, Heerbrugg, Switzerland), air dried at room temperature, and stained. For immunohistochemistry, sections were treated with a saturating concentration of the anti-mouse CD1d (1B1; BD PharMingen), and the Ab bindings were visualized using the ABC Elite system (Vector Laboratories, Burlingame, CA). Slides were counterstained with hematoxylin. Preparations were examined and photographed on an Axiophot 2 apparatus (Zeiss, Thornwood, NY).

Activation of NKT cells and analysis of cytokine production

For examining the functional activity of NKT cells, splenocytes or hepatic lymphocytes (5 x 10⁵ cells/well) from indicated mice were stimulated either with 100 ng/ml GalCer or vehicle (0.1% DMSO) in round-bottom microtiter wells in a final volume of 200 µl of RPMI 10. After 48 h, the supernatants were harvested and the levels of IFN- and IL-4 were quantitated by sandwich ELISA. For activation of NKT cell hybridomas, 5 x 10⁴ hybridoma cells were cultured together with irradiated thymocytes or splenocytes (5 x 10⁵ cells/well) in a total volume of 200 µl/well. After 24–48 h, culture supernatants were harvested and IL-2 release was quantitated by ELISA. Abs specific for cytokines and recombinant mouse cytokines were obtained from BD PharMingen and used according to the manufacturer’s directions.

Adoptive transfer

Bone marrow-derived cells were depleted of T cells by anti-Thy-1.2 (J1j.10; American Type Culture Collection) coupled with rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada). Recipient mice received 980 rad for 4 h before injection with donor bone marrow cells. A total of 1 x 10⁷ cells was injected i.v. into recipient mice. Seven weeks after adoptive transfer, splenocytes and hepatic lymphocytes were collected and the numbers of NKT cells were monitored by flow cytometry as described above.

Statistical analysis

Mean values were compared using Student’s t test for independent variables. Statistical significance was considered to be p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD1d expression in K^b-CD1dTg mice mimics H-2K^b expression

We and others have previously shown that CD1d does not contribute significantly to the development of CD8⁺ T cells (43, 44). One possible explanation for the limited impact of CD1d on the generation of the CD8⁺ T cell repertoire is relatively low surface expression and distinct tissue distribution as compared with MHC class Ia molecules. To address this possibility, we generated a K^b-CD1d-transgenic (Tg) mouse, in which the mouse CD1d1 gene expression is controlled by the classical class I (H-2K^b) promoter (36). Immunohistochemical analysis of thymi of K^b-CD1dTg mice and wild-type (WT) mice showed that K^b-CD1dTg thymic medulla expresses much higher levels of CD1d than WT thymus (Fig. 1A). Cortical thymic epithelial cells, which are required for CD8⁺ T cell selection, also express high levels of CD1d in Tg mice (Fig. 1A). Cell surface expression of H-2K^b and CD1d in the K^b-CD1dTg mice and WT controls was further determined by flow cytometric analysis (Fig. 1B). Compared with WT mice, the expression level of CD1d in K^b-CD1dTg mice is much higher in most of the cell types examined, including mature (CD3^high) thymocytes, splenic B and T cells, and thymic and intestinal epithelial cells. The levels CD1d expressed on CD3^low Tg cortical thymocytes, however, is comparable to the WT mice, as the contribution of the K^b-CD1d transgene is minimal on immature thymocytes (Fig. 1B). Overall, the expression pattern of CD1d in the K^b-CD1dTg mice is largely contributed by the K^b-CD1d transgene, which mimics the expression pattern of H-2K^b molecules.

View larger version (48K): [in this window] [in a new window]   FIGURE 1. CD1d expression in Kb-CD1dTg mice. A, Frozen thymic sections from Kb-CD1dTg mice and WT mice were stained with mAb 1B1 specific for CD1d (immunoperoxidase staining in brown) and counterstaining with hematoxylin. Kb-CD1dTg shows a reticular staining of cortical (C) epithelial cells and strong staining in the medulla (M) areas. A low level of CD1d is detected in the cortical and medulla regions in WT mice. No CD1d staining can be detected in CD1° mice (data not shown). Photographs were taken at x100 (a and b) and x200 (c and d). B, Flow cytometric analysis of cell surface expression of H-2Kb and CD1d in Kb-CD1dTg and WT mice. Specific fluorescence profiles (solid line) obtained with either anti-H2-Kb (Y3) or anti-CD1d (5C6) from indicated mice were overlayed onto background profiles (dotted line) obtained with isotype control Abs. Thymic epithelial cells were gated on I-Ab-positive and CD11c-negative cells. Results are representative of two experiments.

The K^b-CD1dTg mice were crossed with K^bD^b-deficient mice (K^boD^bo) and the effect of the CD1d transgene on the development of the CD8 compartment was examined. The absence of H-2 class Ia expression eliminates possible competition with the CD1d transgene that could limit CD1d-dependent thymic selection of CD8⁺ T cells. We examined the size of the CD4⁺ and CD8⁺ T cells pools in K^b-CD1dTgK^boD^bo and K^boD^bo mice by flow cytometric analysis. Compared with the WT control, the CD8⁺ population was significantly depleted in K^boD^bo mice. This CD8⁺ population was not reconstituted in K^b-CD1dTgK^boD^bo mice, as the number of residual CD8⁺ T cells in K^boD^bo mice is similar to that in K^b-CD1dTgK^boD^bo in all organs tested, including thymus, spleen, liver, and lymph nodes (Fig. 2 and Table I). The NKT cell population was decreased in K^b-CD1dTgK^boD^bo mice, presumably due to the enhanced negative selection, as previously reported (36). A decrease in the CD4 compartment in the liver of the K^b-CD1dTgK^boD^bo mice was also observed. This decrease is likely due to the loss of CD4⁺ CD1d-restricted NKT cells. In addition, no significant difference was found in the CD8TCR⁺ and CD8TCR⁺ IEL subpopulation between K^b-CD1dTgK^boD^bo and K^boD^bo mice (data not shown).

View larger version (60K): [in this window] [in a new window]   FIGURE 2. Expression of Kb-CD1d transgene in class Ia-deficient mice (KboDbo) does not enhance the selection/development of CD8+ T cells. Lymphocytes isolated from thymus, spleen, liver, and lymph nodes from the indicated strain were stained with FITC-anti-CD4 and PE-anti-CD8 and analyzed by flow cytometry. The numbers represent the percentage of CD8+ T cells in the gated lymphocyte population. Results represent the mean values from six mice per group.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Expression of Kb-CD1d transgene does not affect TCR V region usage of CD8+ T cells in Kb-CD1dTgKboDbo. Splenocytes from KboDbo and Kb-CD1dTgKboDbo littermate mice were stained and analyzed by flow cytometry for the expression of the indicated TCR V segments. The percentage of positive cells within the gated CD8+ populations of KboDbo and Kb-CD1dTgKboDbo mice are shown. Results represent the mean values from four mice per group.

We have previously demonstrated that the high ligand density of CD1d on hemopoietic-derived cells (e.g., DCs), but not on nonhemopoietic cells, can mediate negative selection of NKT cells (36). As a consequence of enhanced negative selection, K^b-CD1dTg mice have a greatly reduced NKT cell compartment as compared with WT mice. To determine whether expression of CD1d1 under the control of the H-2K^b promoter can rescue the positive selection of NKT cells, we crossed the K^b-CD1dTg mice onto the CD1d-deficient (CD1°) background. Since the K^b-CD1d transgene is expressed on most cell types tested, including double-positive (DP) thymocytes, one would expect that the expression of the K^b-CD1d transgene in CD1° mice could lead to positive selection of NKT cells. Indeed, a small but highly reproducible population of CD1d/GalCer tetramer-positive NKT cells was observed in K^b-CD1dTgCD1° mice but in not CD1° mice (Fig. 4A). However, this population by no means completely reconstitutes the NKT cell compartment. Furthermore, the reconstituted population in K^b-CD1dTgCD1° mice is significantly smaller than the residual population in K^b-CD1dTgCD1^wt mice (Fig. 4B). Thus, the incomplete reconstitution in K^b-CD1dTgCD1° mice cannot be explained solely by negative selection of the NKT cells. Endogenous CD1d must play some role in the efficient positive selection of NKT cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Expression of Kb-CD1dTg partially restores the NKT cell development in Kb-CD1dTgCD1° mice. A, Lymphocytes isolated from thymus, spleen, and liver of indicated mice were stained with FITC-anti-TCR and allophycocyanin-conjugated-CD1d/GalCer tetramers and analyzed by flow cytometry. B, The absolute number of NKT cells was calculated by percentage of tetramer+ T cells x total number of cells. Data shown represent mean ± SE of six mice in each group. No sizeable pool of CD1d/GalCer tetramer+ T cells can be detected in CD1° mice.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Recognition of Kb-CD1dTgCD1° thymocytes by NKT cell-derived hybridomas. Briefly, 5 x 104 cells of CD1d-specific NKT cell hybridomas (DN3A4 and D32.D3) were cultured with 5 x 105 thymocytes from Kb-CD1dTgCD1° (Tg+CD1°) mice or WT mice in the presence or absence of GalCer. IL-2 levels in the supernatant were detected by ELISA. NKT cell hybridomas cultured with thymocytes from CD1° mice did not secrete detectable amounts of IL-2 (<1 U/ml). Similar results were obtained when splenocytes from indicated mice were used as stimulators (data not shown). Bars represent means and SDs of duplicate determinations. Results are representative of two experiments.

View larger version (11K): [in this window] [in a new window]   FIGURE 6. Kb-CD1d expressed on bone marrow-derived cells determines the selection outcome of NKT cells. Bone marrow-derived cells (1 x 107) from WT, Tg+CD1°, and CD1° mice were transferred into lethally irradiated recipient mice (980 rad) by i.v. injection. After 7 wk, hepatic lymphocytes from bone marrow chimeras were isolated, stained with FITC- anti-TCR and PE-NK1.1, and analyzed by flow cytometry. Absolute NKT cell numbers of hepatic lymphocytes from each group of bone marrow chimeras were calculated as described above. Cytokine production by hepatic lymphocytes from each group of bone marrow chimeras was determined by culturing 5 x 105 hepatic lymphocytes from the indicated group of mice with 100 ng/ml GalCer for 48 h. IFN- and IL-4 levels in the supernatant were detected by ELISA. Data shown represent mean ± SE of three mice in each group.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Flow cytometric analysis of cell surface expression of H-2Kb and CD1d in WT, Kb-CD1dTg+ (Tg+CD1wt), Kb-CD1dTgCD1° (Tg+CD1°), and CD1° mice. Specific fluorescence profiles (filled histogram) obtained with anti-H2-Kb (Y3) from WT mice were overlayed onto background profiles (open histogram) obtained with isotype control Abs. Comparable levels of H2-Kb expression can be detected in Tg+CD1+, Tg+CD1°, and CD1° mice (data not shown). The level of CD1d surface expression was determined by the staining with anti-CD1d (5C6, solid line). Specific fluorescence profiles from WT mice were overlayed onto profiles obtained from CD1° mice (dotted line). Results are representative of four experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our finding that the expression of a K^b-CD1d transgene fails to lead to development of a significant CD8⁺ T cell population in class Ia-deficient mice is somewhat surprising, given that the overall structure of CD1d closely resembles MHC class Ia. Several recent studies have demonstrated the involvement of MHC class Ib molecules, such as H-2M3 and Qa-1, in the positive selection of CD8⁺ T cells (50, 51, 52). However, it is noteworthy that the sequence similarity between CD1d and MHC class Ia is only 30–35%, while other CD8 T cell-selecting MHC class Ib molecules share 60–70% sequence similarity with MHC class Ia. Several possible explanations could account for the failure of K^b-driven CD1d in participating in the selection/maintenance of the CD8⁺ T cell repertoire. First, CD1d may interact less effectively with CD8 than other class I molecules and, as a result, fail to select a significant number of developing T cells to the CD8 lineage. Mouse CD1d has been demonstrated to bind CD8 in redirected CTL assays (45); however, the affinity of CD1d for CD8 appears to be very low and difficult to measure (36). Second, the nonpolymorphic nature of CD1d suggests that CD1d may present a limited set of Ags, which in turn preclude selection of conventional CD8⁺ T cells that characteristically express a diverse TCR repertoire. Another intriguing, nonexclusive possibility is that CD1d may even structurally be inherently incapable of interacting with and selecting a broader array of TCRs. We cannot eliminate, however, the possibility that increased levels of CD1d in K^b-CD1dTg mice may actually result in negative selection of conventional CD8⁺ T cells. Yet, we do not detect an increased number of CD8TCR T cells in IELs, as has been demonstrated in negative selection models of conventional CD8⁺ T cells (53). Also, CD1d levels on negatively selecting thymic DCs in these mice are similar to the endogenous H2-K^b levels, which do not result in excessive negative selection of conventional CD8⁺ T cells.

In the present study, we also examine the effect of cell type-specific expression levels of CD1d on NKT cell development. We show here that expression of the K^b-CD1d transgene in CD1° mice only partially reconstitutes the NKT cell compartment. Differentiating the precise roles of positive and negative selection in this effect is complicated by the high level of expression of CD1d on multiple cell types. However, expression levels of CD1d on positively selecting DP thymocytes in K^b-CD1dTgCD1° mice are similar to endogenous CD1d expression levels in B6 mice. We have also previously shown that expression of the K^b-CD1d transgene on DP thymocytes did not enhance negative selection nor affect the positive selection of NKT cells (36). Thus, it would be predicted that NKT cells could be positively selected as efficiently in these mice as in WT mice. Indeed, a small but highly reproducible population of NKT cells is found in these mice as compared with CD1° mice, implying that positive selection does occur. Yet, the partial reconstitution of NKT cells in K^b-CD1dTgCD1° mice is not unexpected. The higher level of CD1d expression in K^b-CD1dTg mice has been previously demonstrated to result in negative selection of NKT cells (36). Thus, the high expression of CD1d on negatively selecting thymic DCs may, at least in part, explain the incomplete reconstitution of NKT cells in K^b-CD1dTgCD1° mice.

Interestingly, however, the reconstituted NKT cell population in K^b-CD1dTgCD1° is significantly and reproducibly smaller than the residual NKT cells in K^b-CD1dTgCD1^wt mice (Fig. 4B). It is unlikely that the K^b-driven CD1d is functionally different from endogenous CD1d since lymphocytes from K^b-CD1dTgCD1° mice can be recognized by and activate NKT cell hybridomas. In addition, hemopoietic cells from K^b-CD1dTgCD1° are capable of mediating positive selection of NKT cells in bone marrow chimeras. One explanation for the larger NKT cell population in K^b-CD1dTgCD1^wt is that the presence of endogenous CD1d must be required for efficient positive selection. Either positive selection is enhanced in K^b-CD1dTgCD1^wt mice due to greater levels of CD1d on thymocytes (Fig. 7) and/or positive selection is less effective in K^b-CD1dTgCD1° mice due to the differences in the pattern and timing of expression between endogenous and K^b-driven CD1d. During embryonic development, the levels of K^b expression decreases progressively on immature thymocytes between gestational day 16 and 1 wk old, and the low levels of K^b continue to be expressed on cortical thymocytes through adulthood (5). In contrast, no difference in the level of endogenous CD1d expression on thymocytes could be detected from gestational day 16 onward (5, 54). Since a significant number of CD1d-restricted NKT cells are not detected in thymus until 5 days after birth (55), the expression of endogenous CD1d may be crucial during early development for efficient NKT cell selection. Thus, it is possible there is a time window during the course of development for high efficiency positive selection of NKT cells.

Our data indicate that ligand density and cell type-specific expression of CD1d is important for NKT cell selection. A very tight regulation of NKT cell development is not surprising considering the broad role of NKT cells in controlling infection and autoimmunity as well as their rapid activation to secrete large amounts of IFN- and/or IL-4 (56). In our K^b-CD1d Tg system, even the difference between the presence and absence of endogenous CD1d results in differential selection of NKT cells. Because of the restricted TCR repertoire of NKT cells, the cell type-specific expression levels of CD1d may establish a narrow threshold for NKT cell development. It is possible that accessory cell surface molecules on different cell types could also attribute to this threshold, as could cell density and location.

Evolutionarily, the necessity of cell type-specific or time-specific expression of CD1d in NKT selection is supported by the differential regulation of CD1d. Despite structural and sequence similarity to MHC class Ia, class Ib, and even class II molecules, the region flanking the 5' transcriptional start site of CD1d shares little homology with these other molecules (Y. Geng and C.-R. Wang., unpublished results). Additionally, CD1d genes are located outside of the MHC in both mice and humans (3, 57). The segregation of these two gene complexes during evolution may facilitate the development of different regulatory mechanisms to control the expression of these Ag-presenting molecules. Our finding that changing the CD1d expression pattern in our K^b-CD1dTg mice results in loss of the majority of the NKT cell population suggests that there may be selective pressures for the different cell-specific regulation of CD1d separate from other Ag-presenting molecules. The distinct cell type-specific expression levels of CD1d, as well as structural divergence of CD1d from other MHC class I-like molecules, underlines the nonredundant role of CD1d and MHC molecules in the development of the T cell repertoire.

	Acknowledgments

 
We thank Kirin Brewery Co. Ltd. (Gunma, Japan) for providing GalCer, Dr. M. Kronenberg for providing recombinant CD1d baculovirus, Dr. M. Zimmer for critical reading for this manuscript, and T. King for help in maintenance and genotyping the mice.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI43407 (to C.-R.W.) and a Cancer Research Institute postdoctoral fellowship (to T.C.).

² H.X., T.C., and A.P. contributed equally to this work.

³ Current address: Department of Microbiology and Immunology, School of Medicine, Hanyang University, Seoul, South Korea.

⁴ Address correspondence and reprint requests to Dr. Chyung-Ru Wang, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Room 412, 924 East 57th Street, Chicago, IL 60637-5420. E-mail address: cwang{at}uchicago.edu

⁵ Abbreviations used in this paper: DC, dendritic cell; WT, wild type; GalCer, -galactosylceramide; TEC, thymic epithelial cell; Tg, transgenic; DP, double positive; IF, immunofluorescence.

Received for publication May 2, 2003. Accepted for publication August 5, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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