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The Journal of Immunology, 2006, 176: 1421-1430.
Copyright © 2006 by The American Association of Immunologists

A Cell-Type Specific CD1d Expression Program Modulates Invariant NKT Cell Development and Function1

Michael I. Zimmer*, Angela Colmone*, Kyrie Felio*, Honglin Xu*, Averil Ma{dagger} and Chyung-Ru Wang2,*

* Department of Pathology, University of Chicago, Chicago, IL 60637; and {dagger} Department of Medicine, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Invariant NK T (iNKT) cells are a distinct subset of T cells that rapidly produce an array of immunoregulatory cytokines upon activation. Cytokines produced by iNKT cells subsequently transactivate other leukocytes and elicit their respective effector functions. In this way, iNKT cells play a central role in coordinating the development of immune responses in a variety of settings. However, the mechanisms governing the quality of the iNKT cell response elicited remain poorly defined. To address whether changes in the CD1d expression pattern could regulate iNKT cell function, we generated a transgenic (Tg) mouse model in which thymocytes and peripheral T cells express high levels of CD1d (Lck-CD1d Tg+ mice). The expression of CD1d by T cells was sufficient to rescue development of iNKT cells in mice deficient of endogenous CD1d. However, the relative proportions of iNKT cell subsets in Lck-CD1d Tg+ mice were distinctly different from those in wild-type mice, suggesting an altered developmental program. Additionally, iNKT cells were hyporesponsive to antigenic stimulation in vivo. Interestingly, Lck-CD1d Tg+ mice develop liver pathology in the absence of any exogenous manipulation. The results of these studies suggest that changes to the CD1d expression program modulate iNKT cell development and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD1 family of MHC-unlinked class Ib molecules is conserved across mammalian species. The sole murine family member, CD1d, is widely expressed on cells of multiple hemopoietic lineages, including B and T cells, macrophages, and dendritic cells (DCs)3 (1, 2, 3). Recent studies have shown that CD1d presents lipid/glycolipid Ags to T cells (4). These Ags include endogenous lipids, such as isoglobotrihexosylceramide, disialoganglioside GD3, phosphoethanolamine, and sulfatide (5, 6, 7, 8), and microbial lipids, such as mycobacterial phosphatidylinositol mannoside and sphingomonas glycosphingolipids (9, 10).

Mice deficient in CD1d are impaired in the development of a unique subset of T cells, NKT cells (11, 12, 13). The CD1d-restricted NKT cells can be divided into two groups: variant NKT cells, which display diverse TCR repertoire, and invariant NKT (iNKT) cells, which express an invariant TCR {alpha}-chain (V{alpha}14-J{alpha}18) (14, 15). Most iNKT cells express NK cell receptors, are either CD4+ or double negative (DN), and exhibit an activated/memory phenotype (16). A homologous T cell population, which expresses an invariant V{alpha}24-J{alpha}15 TCR, is also present in humans (17). The highly conserved nature of both CD1d and iNKT cells suggests a critical role in the immune response.

A marine sponge-derived glycolipid, {alpha}-galactosylceramide ({alpha}-GalCer), has been shown to activate iNKT cells in a CD1d-restricted manner (4). After {alpha}-GalCer stimulation, iNKT cells quickly secrete considerable quantities of both Th1 and Th2 cytokines, including IFN-{gamma} and IL-4. Invariant NKT cell activation can subsequently activate NK cells, B cells, DCs, and CD8+ T cells, suggesting that iNKT may play a role in regulating innate and adaptive immunity (18, 19, 20). Invariant NKT cells have been implicated in both stimulation and inhibition of the immune response, including protective roles in microbial infection, tumor immunity, and prevention against various autoimmune diseases (21). Conversely, a role for NKT cells in liver pathogenesis has been described in suppressor of cytokine signaling-1-deficient mice (22) and the Con A-induced hepatitis model (23, 24).

The mechanism(s) regulating iNKT function is unclear. Functional differences in iNKT cells in various disease models may be mediated by the quality of the stimulating signal, which may be attributable to differential TCR/ligand interactions (25, 26) and selective presentation of Ag by different CD1d-expressing cell types (27). Activation of phenotypically and/or anatomically distinct subsets of iNKT cells may also account for differences in iNKT function. In humans, the CD4+ subset of iNKT cells has been shown to secrete more IL-4 than the DN subset upon stimulation (28, 29), but convincing evidence for functional subsets of mouse iNKT cells is lacking. Additionally, the expression of inhibitory NK cell receptors of the Ly49 family has been shown to inversely correlate with NKT cell activation, resulting in iNKT cell tolerance in a chronic activation model (30, 31).

Invariant NKT cell selection and development are highly regulated processes. Invariant NKT cannot be detected in the periphery until day 7 after birth, expand significantly after wk 3, and reach adult levels at ~6 wk of age (32). The majority of early thymic emigrants are often NK1.1–, and they complete maturation in the periphery (32, 33). The development of iNKT cells may parallel, but be distinct from, those of other self-reactive T cells such as CD8{alpha} and CD4+CD25+ (regulatory) T cells and conventional T cells (34). Similar to conventional T cells, DCs, but not thymic epithelial cells, are sufficient for negative selection of iNKTs cell when overall avidity is increased (35). Agonist selection of the nonconventional T cells, including iNKT cells, may be responsible for their activated phenotype (15, 34). Positive selection of NKT cells, however, is mediated by thymocytes, whereas conventional and regulatory T cells are positively selected by thymic epithelial cells (36, 37). Signaling requirements are also different for these cell types (21). The involvement of different cell types and signaling molecules in the positive selection of conventional compared with iNKT cells and other nonconventional T cells may in part contribute to the distinct phenotypic and functional characteristics associated with these subsets of T cells.

The expression pattern of CD1d has been shown to be very important for iNKT cell development. Altering CD1d expression to mimic either MHC class Ia or MHC class II expression results in inefficient, if any, selection of iNKT cells (38, 39). To further explore the effects of altered CD1d expression on NKT cell development and subset differentiation, we have directed CD1d expression to thymocytes and mature T cells using an Lck promoter. We report that T cell-restricted CD1d expression is sufficient for development of iNKT cells. However, the ratio of various CD4/NK1.1 iNKT subsets is altered in Lck-CD1d transgenic (Tg) mice. Tg+ iNKT cells are also hyporesponsive to Ag stimulation in vivo, and Tg+ mice exhibit liver pathology characterized by increased leukocyte cellularity and hepatomegaly. Taken together, our results suggest that modification of CD1d expression directly effects NKT cell development and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and CD1d tetramers

The following Abs used in this study were purchased from BD Pharmingen; FITC-conjugated mAbs specific for hamster IgG, CD4 (GK1.5), and TCRbeta (H57-597); PE-conjugated mAbs specific for CD8{alpha} (53-6.7), NK1.1 (PK136), CD11c (HL3), F4/80, B220 (RA3-6B2), and CD69 (H1.2F3); biotinylated mAbs specific for CD4 (RM4-5); and allophycocyanin-conjugated mAbs specific for TCRbeta (H57-597). Staining with biotinylated mAbs was revealed using PerCP-conjugated streptavidin. The CD1d-specific mAb 5C6 and the generation of CD1d/{alpha}-GalCer tetramers have been described previously (2, 40)

Mice

C57BL/6 mice were purchased from The Jackson Laboratory. Generation of CD1d-deficient (CD1°) mice has been described previously (11). The CD1° mice used in this study were backcrossed onto the C57BL/6 background for >12 generations. All animal work was approved by the University of Chicago institutional animal care and use committee.

Cell preparations and FACS analysis

Single-cell suspensions from thymus and spleen were prepared by mechanical disruption in immunofluorescence buffer (HBSS containing 2% FBS (HyClone)). Lymphocytes from perfused liver were isolated according to the method described by Goossens et al. (41). Cells were stained in immunofluorescence buffer for 1 h at 4°C. After mAb staining, cells were analyzed by flow cytometry using a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Generation of Lck-CD1d Tg mice

We engineered a construct in which CD1d expression is driven by the T cell-specific Lck promoter (42). Full-length CD1d1 cDNA was inserted into the AcsI site of the plck-E2 plasmid (provided by Dr. T. Hettmann, University of Chicago, Chicago, IL). A 9-kb fragment was excised by NotI and used for subsequent microinjection. Three to 5 ng of DNA was injected into the male pronucleus of each single-cell B6 embryo; injected embryos were then implanted into CBA pseudopregnant foster mothers. Tg mice were identified by PCR analysis using primers specific for the lck promoter (5'-GCTGATGGTGGCTGAGTCATTAC-3') and the CD1D1 gene (5'-CAGGGGAGTTGTAATGA AGAGGGACA-3'). Two positive founders were generated and designated 9987 (line 1) and 9980 (line 2). The Lck-CD1d Tg mice were subsequently established on a CD1d-deficient background (Lck-CD1d TgCD1°) by crossing Lck-CD1d Tg mice with CD1° mice. All data shown are from Tg line 1 unless indicated otherwise.

Histology

Liver tissue was fixed overnight in 70% ethanol and subsequently embedded in paraffin. Sections measuring 5–7 µm were cut and stained with H&E to examine tissue architecture. Images were examined and photographed on an Axiophot 2 apparatus (Zeiss).

DC culture and in vivo stimulation

DC were generated from bone marrow progenitors as previously described (35). On day 6 of culture, cells were harvested, and CD11c+ DC were MACS purified (Miltenyi Biotec). Purified CD11c+ DC (>95% purity) were pulsed overnight in the presence of 100 ng/ml {alpha}-GalCer. For in vivo stimulation, each mouse received a total of 6 x 105 {alpha}-GalCer-loaded DC in a total volume of 200 µl. Mice were bled 24 h after injection to isolate serum for cytokine analysis and were killed 72 h after injection to analyze iNKT cells in spleen and liver.

In vivo iNKT stimulation with Ags

For flow-based cytokine secretion assays, mice were injected i.v. with either 100 µg/kg {alpha}-GalCer or 2 µg of anti-CD3 mAb (2C11). Mice were killed 1 h (for {alpha}-GalCer) or 20 min (for anti-CD3) after injection, and sera, spleens, and livers were harvested and processed for analysis of cytokines as described below. For {alpha}-GalCer-induced proliferation/activation experiments, mice were injected i.p. with 0.2 ml of PBS containing either 5 µg of {alpha}-GalCer or control vehicle. Mice were killed 72 h after injection, and spleens and livers were harvested and processed for flow cytometric analysis.

Cytokine analysis

IFN-{gamma} and IL-4 production by iNKT cells was measured using Mouse IFN-{gamma} or IL-4 Secretion Assay Detection Kits (Miltenyi Biotec). The assay was performed according to the manufacturer’s instructions. Results were quantitated by flow cytometry and were analyzed using FlowJo software. Serum levels of IFN-{gamma} and IL-4 were quantitated using a sandwich ELISA (BD Pharmingen).

Statistical analysis

Mean values were compared using unpaired Student’s t test. All statistical analyses were performed with the PRISM program (GraphPad). Statistically significant differences are noted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD1d expression by T cells in Lck-CD1d Tg mice

To examine the role of CD1d-expressing T cells in iNKT cell development, we generated CD1d Tg mice in which CD1d expression is driven by the proximal Lck promoter (Fig. 1A). This strategy has been used successfully in other Tg models to obtain T cell-specific expression (42, 43). Two independent lines of Lck-CD1d Tg mice were obtained and designated lines 9987 (line 1) and 9980 (line 2). Both lines of Lck-CD1d Tg mice were backcrossed onto CD1° mice to investigate the effects of the transgene on iNKT cell development and function in the absence of the endogenous CD1d expression program. Flow cytometric analysis of CD1d expression on various leukocyte populations from Lck-CD1d TgCD1° mice revealed enhanced CD1d expression by TCRbeta+ T cells in the thymus and peripheral organs, including spleen, liver, and lymph nodes, compared with wild-type (WT) mice (Fig. 1B and data not shown). In contrast, the expression of CD1d was reduced in other leukocyte populations analyzed, including B cells, macrophages, and DCs (Fig. 1, B and C). In Lck-CD1d TgCD1° mice, equivalent levels of CD1d were detected among immature TCRbetalow and mature TCRbetahigh thymocytes, whereas splenic T cells were found to express relatively higher levels of CD1d. Although the overall levels of CD1d differ between the two Lck-CD1d Tg lines, the patterns of CD1d expression observed in the thymus and periphery are similar.


Figure 1
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  		FIGURE 1. Enhanced expression of CD1d on T cells in Lck-CD1d Tg+CD1° mice. A, Schematic diagram of the CD1D1 Tg construct used to generate Lck-CD1d Tg+ mice. The transgene consists of the Lck proximal promoter, mouse CD1D1, human growth hormone gene polyadenylation site, and locus control region elements from the human CD2 gene. B, Cells from WT and Lck-CD1d Tg+CD1° mice were stained with mAbs to CD1d and TCRbeta, B220, F4/80, or CD11c to identify T cells, B cells, macrophages, and DCs, respectively, and were analyzed by flow cytometry. Shown is the expression of CD1d in Tg+ leukocytes relative to WT mice. Results are representative of three separate experiments. C, Representative histogram of CD1d expression on the indicated cell types (thin line, CD1°; thick line, WT or Lck-CD1d Tg+CD1° line 1).

 
Invariant NKT cell development in Lck-CD1d TgCD1° mice

To evaluate iNKT cell development in Lck-CD1d Tg mice, we stained lymphocyte populations from the thymus, spleen, and liver of CD1°, WT, and Lck-CD1d TgCD1° mice with {alpha}-GalCer-loaded CD1d tetramers and analyzed the results by flow cytometry. Comparable proportions of CD1d tetramer-reactive iNKT cells were detected in the thymus and spleen of WT and Lck-CD1d TgCD1° mice (Fig. 2, A and B). Total thymocyte and splenocyte cell numbers were similar among WT and Lck-CD1d TgCD1° mice, resulting in comparable total iNKT cell numbers in these organs (Fig. 2C). In contrast to these organs, the percentage of iNKT cells in the liver of Lck-CD1d TgCD1° mice was reduced by 50–75% compared with WT age-matched controls (Fig. 2, A and B). However, total liver leukocyte cell numbers were increased 2- to 3-fold in Lck-CD1d TgCD1° livers compared with WT mice, resulting in similar total iNKT cell numbers among WT and Lck-CD1d TgCD1° mice (Fig. 2C). These results demonstrate that the expression of CD1d by Tg+ T cells is sufficient to rescue the development of iNKT cells in CD1° mice, enabling us to examine the effects of changes to the CD1d expression program on iNKT cell development and function.


Figure 2
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  		FIGURE 2. Development of iNKT cells in Lck-CD1d Tg+CD1° mice. A, Cells from CD1°, WT, and Lck-CD1d Tg+CD1° mice were stained with an anti-TCRbeta mAb and CD1d/{alpha}-GalCer tetramers and analyzed by flow cytometry. TCRbeta+ tetramer+ iNKT cells are highlighted by the gate, and the proportion of gated cells is indicated. The lack of staining in the CD1° samples confirms the specificity of tetramer staining. B and C, Bar graphs depict the mean and SD for the proportion of iNKT cells (B) and absolute iNKT cell numbers (C) for WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice. ***, Statistically significant differences (p < 0.001).

 
To examine the phenotype of Lck-CD1d TgCD1° iNKT cells, we quantitated CD1d/{alpha}-GalCer tetramer+ iNKT cell subsets based on differential expression of the cell surface markers CD4, CD8, and NK1.1. The majority of CD1d-restricted iNKT cells in the mouse exhibit a CD4+ or CD4–CD8– (DN) phenotype (16). We did not observe statistically significant differences in the proportions of CD4+ and DN subsets in thymic or splenic CD1d/{alpha}-GalCer tetramer+ iNKT cells between WT and Lck-CD1d TgCD1° mice (Fig. 3A). However, the proportion of the DN subset of iNKT cells was significantly higher in the livers of Lck-CD1d TgCD1° mice compared with WT mice (Fig. 3A). Segregation of tetramer+ iNKT cell subsets using CD4 and NK1.1 expression revealed a 2- to 3-fold reduction of the CD4+NK1.1+ subset of iNKT cells in the liver of Lck-CD1d TgCD1° mice (18.5 ± 1.2 vs 61.5 ± 2.7%) and was associated with a corresponding significant increase in the proportion of the CD4+NK1.1– and CD4–NK1.1– subsets of iNKT cells (Fig. 3B). The decrease in the CD4+NK1.1+ subset and reciprocal increase in the CD4+NK1.1– subset of iNKT cells was also apparent in the spleen of Lck-CD1d TgCD1° mice. However, the reduction of CD4+NK1.1+ iNKT cells is much more subtle in the thymus of Lck-CD1d TgCD1° mice (42.5 ± 1.4 vs 48.9 ± 1.5%). Importantly, changes in the iNKT cell subsets described above were evident in both lines of Lck-CD1d TgCD1° mice. Taken together, these results demonstrate that CD1d-expressing T cells are sufficient to mediate the development of iNKT cells. However, the iNKT cells that develop in Lck-CD1d TgCD1° mice are phenotypically distinct from those in WT animals.


Figure 3
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  		FIGURE 3. Analysis of iNKT cell subsets in Lck-CD1d Tg+CD1° mice. A, Cells were stained with CD1d/{alpha}-GalCer tetramers and mAb to TCRbeta, CD4, and CD8 or NK1.1 and analyzed by flow cytometry. The relative proportion of each subset is indicated in the respective quadrant. Values that are significantly different between WT and Lck-CD1d Tg+CD1° mice are highlighted in bold. Results are representative of a total of six mice per group. B, Bar graphs depict the mean and SE for various iNKT cell subsets in the thymus, spleen, and liver for WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Ontogeny of iNKT cells in Lck-CD1d TgCD1° mice

Our phenotypic analysis of iNKT cells suggested that the developmental process is somehow different in Lck-CD1d TgCD1° mice compared with WT animals. The differences in the iNKT cell populations of Lck-CD1d TgCD1° mice might result from unique thymic instruction, intercellular interactions in the periphery, or a combination of these. To explore this issue, we performed a phenotypic analysis of iNKT cell populations in WT and Lck-CD1d TgCD1° mice at various ages. We focused our efforts on the liver, because it was in this organ that we observed the largest differences in iNKT cell populations between WT and Lck-CD1d TgCD1° mice (Fig. 2, A and B, and Fig. 3). Although not statistically significant, we consistently observed a trend toward a lower proportion of liver iNKT cells in Lck-CD1d TgCD1° mice than WT mice as early as 3 and 4 wk of age (Fig. 4). The relative proportions of all four CD4/NK1.1 iNKT cell subpopulations at 3 wk of age were comparable between WT and Lck-CD1d TgCD1° mice. However, significant differences in the proportions of CD4+NK1.1+ and CD4+NK1.1– iNKT subsets between WT and Lck-CD1d TgCD1° mice were clearly evident by 4 wk of age (Fig. 4). In particular, the proportion of the CD4+NK1.1+ iNKT cell subset in Lck-CD1d TgCD1° liver did not appear to increase as profoundly as that in WT liver.


Figure 4
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  		FIGURE 4. Ontogeny of iNKT cells in WT and Lck-CD1d Tg+CD1° mice. Liver leukocytes were isolated from WT and Lck-CD1d Tg+CD1° mice at 3, 4, 10, and 16 wk of age, and cells were stained with CD1d/{alpha}-GalCer tetramers and mAb to TCRbeta, CD4, and NK1.1. Left panel, Representative dot plots of TCRbeta+ tetramer+ iNKT cells from WT and Lck-CD1d Tg+CD1° mice at the indicated time points. Right panel, Analysis of CD4 and NK1.1 expression on iNKT cell populations at the indicated time points. The relative proportion of each subset is indicated in the respective quadrant. Values that are significantly different between WT and Lck-CD1d Tg+CD1° mice are highlighted in bold. Data shown are rep resentative of three to six mice in each age group.

 
In WT mice, the percentage and total number of hepatic iNKT cells increase steadily as the animal matures. In contrast, in Lck-CD1d TgCD1° mice, the total number of hepatic iNKT cell increases similarly to that in WT mice, whereas the percentage of iNKT cells remains relatively constant. This difference can be explained by an abnormal increase in total hepatic leukocyte cellularity. Analysis of iNKT cells from the liver of WT and Lck-CD1d TgCD1° mice at 10 wk of age revealed statistically significant differences in the percentage of iNKT cells. In addition, the differences in the proportions of CD4+NK1.1+, CD4+NK1.1–, and CD4–NK1.1– iNKT cell subsets between WT and Lck-CD1d TgCD1° mice described previously were clearly established at this time. Similar results were obtained from analysis at 16 wk of age. These results demonstrate that the phenotypic differences between WT and Lck-CD1d TgCD1° iNKT population can be detected early in iNKT cell ontogeny and are accentuated with age. Unlike the iNKT cell population in the liver, the proportion of iNKT cell subsets in the thymus of Lck-CD1d TgCD1° mice did not differ considerably from WT (Fig. 3A and data not shown), suggesting that the peripheral environment may modulate the development and differentiation of iNKT cells in Lck-CD1d TgCD1° mice.

Liver pathology in Lck-CD1d TgCD1° mice

During the course of our phenotypic analyses, we noted dramatic differences in liver size and morphology between WT and Lck-CD1d TgCD1° mice. The abnormal phenotype of Lck-CD1d TgCD1° livers was more obvious in older mice than in younger ones, suggesting that pathology develops as mice mature. Liver homeostasis is a highly regulated process (44). In a healthy adult WT mouse, the liver weight is ~5% of the total body weight (44). In agreement with this, we found that liver weights were 5.5 ± 0.7 and 5.3 ± 0.4% of the total body weight in WT and CD1° mice, respectively (Fig. 5A). However, in Lck-CD1d TgCD1° mice, liver weights were 7.2 ± 1.1 and 6.4 ± 0.6% of total body weight in Lck-CD1d TgCD1° lines 1 and 2, respectively (Fig. 5A). Histological analysis of liver tissue sections from Lck-CD1d TgCD1° mice revealed focal leukocyte infiltration and enlarged hepatocytes that exhibited karyomegaly (Fig. 5B). We did not observe preferential recruitment of any specific leukocyte subset into the Lck-CD1dTg liver in phenotypic analysis using flow cytometry (data not shown). Instead, the increased leukocyte cell numbers documented in the liver of Lck-CD1dTg mice seem to result from an enrichment of all leukocytes. It is highly unlikely that the liver pathology observed in Lck-CD1d TgCD1° mice results from aberrant transgene integration, because pathology develops in both lines of Lck-CD1d Tg+ mice in both CD1° and WT backgrounds (Fig. 5A and data not shown). It is also important to note that liver pathology develops in the absence of any exogenous treatment or manipulation. Because the liver is the organ with the highest proportion of iNKT cells, and iNKT cell development appears to be distinctly different in Lck-CD1d TgCD1° mice compared with WT mice, the liver pathology observed in Lck-CD1d TgCD1° mice suggests that iNKT cell function may also be altered.


Figure 5
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  		FIGURE 5. Lck-CD1d Tg+ mice develop enlarged livers with age. A, Bar graphs depict the mean and SE liver weight as a proportion of the total body weight from CD1°, WT, and two lines of Lck-CD1d Tg+CD1° mice. Statistically significant differences between WT and Lck-CD1d Tg+CD1° mice are indicated. B, Representative images from the liver of WT and Lck-CD1d Tg+CD1° mice (line 1) at 1 and 4 mo of age. Tissue sections were stained with H&E. Arrows highlight pockets of cellular infiltration.

 
Lck-CD1d Tg+ iNKT cells produce less cytokine after activation

To assess the integrity of iNKT cell function in Lck-CD1d TgCD1° mice, we tested their ability to produce the immunoregulatory cytokines IFN-{gamma} and IL-4 after stimulation in vivo with {alpha}-GalCer. The production of IFN-{gamma} and IL-4 by iNKT cells after stimulation with {alpha}-GalCer is a relatively rapid response and can be detected within 1 h of stimulation (40). To examine cytokines produced specifically by iNKT cells, we used a flow cytometry-based assay in which secreted cytokines are captured at the cell surface. WT and Lck-CD1d TgCD1° mice were injected i.v. with {alpha}-GalCer, and after 1 h, we harvested serum and isolated splenocytes and liver leukocytes to analyze the production of IFN-{gamma} and IL-4 by CD1d/{alpha}-GalCer tetramer+ iNKT cells in these organs. The iNKT cells from the spleen of WT and Lck-CD1d TgCD1° animals produced comparable amounts of IFN-{gamma} after {alpha}-GalCer stimulation (Fig. 6A). The production of IL-4 by WT and Lck-CD1d TgCD1° iNKT cells in the spleen was also similar, although we did consistently find slightly lower levels of IL-4 production in Lck-CD1d TgCD1° mice. In contrast to the spleen, the cytokine production profile of iNKT cells from Lck-CD1d TgCD1° livers was significantly different from that in WT mice. Although ~60% of WT iNKT cells produced IFN-{gamma}, in Lck-CD1d TgCD1° mice, only ~30% of liver iNKT cells were IFN-{gamma}+. Moreover, although >90% of WT iNKT cells produced IL-4, only ~30% of liver iNKT cells from Lck-CD1d TgCD1° mice were IL-4+. Consistent with results in the liver, we found significantly lower levels of IFN-{gamma} and IL-4 in the serum of Lck-CD1d TgCD1° mice compared with WT mice (Fig. 6B). Defects in the production of IFN-{gamma} and IL-4 by liver iNKT cells after {alpha}-GalCer stimulation were also observed in Lck-CD1d TgCD1+ mice (data not shown). Hence, iNKT cells from Lck-CD1d Tg+ mice, in particular from the liver, produce less cytokine than WT iNKT cells.


Figure 6
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  		FIGURE 6. Inferior cytokine production by Tg+CD1° iNKT cells after in vivo {alpha}-GalCer stimulation. A, WT and Lck-CD1d Tg+CD1° mice were injected i.v. with 100 µg/kg {alpha}-GalCer. After 1 h, cells were stained with CD1d/{alpha}-GalCer tetramers, mAb to TCRbeta, and a specific cytokine capture reagent to identify iNKT cells producing IFN-{gamma} or IL-4. Histograms depict cytokine production from TCRbeta+ tetramer+ iNKT cells. Cytokine histograms of iNKT cells from unstimulated control mice were negative (limited to the first decade of the log scale histogram plot), reflecting the lack of cytokine production, secretion, and detection in these samples. B, The amounts of IFN-{gamma} and IL-4 present in the serum of WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice after 1 h of {alpha}-GalCer stimulation were quantitated by sandwich ELISA. Serum cytokine levels from unstimulated control mice were below the limits of detection. Bar graphs depict means obtained from duplicate wells. The data shown are representative of four independent experiments.

 
Inefficient Ag presentation cannot account for the hyporesponsiveness of Tg+ iNKT cells

The results obtained from the in vivo {alpha}-GalCer stimulation assays suggest that iNKT cells from Lck-CD1d TgCD1° mice may have intrinsic defects with respect to their ability to respond to glycolipid Ag stimulation. Alternatively, the lower levels of cytokine production by Lck-CD1d TgCD1° iNKT cells may reflect poor Ag presentation. In the latter scenario, {alpha}-GalCer may be presented by T cells, a nonprofessional APC population that expresses high levels of CD1d (Fig. 1B) and simply fails to elicit an efficient iNKT cell response. To begin to investigate the mechanism(s) resulting in the inferior response of Tg+ iNKT cells to stimulation, we conducted a series of experiments, each designed to address a specific aspect of the APC/iNKT cell interaction. First, we repeated the in vivo functional assays using anti-CD3 mAb stimulation. The use of anti-CD3 mAb to activate iNKT cells bypasses the requirement for CD1d-mediated ligand presentation by a specific APC. Similar to the {alpha}-GalCer experiments, production of IFN-{gamma} was comparable in WT and Lck-CD1d TgCD1° iNKT cells in the spleen (Fig. 7A). In contrast to previous results, we did not find significant differences in the production of IFN-{gamma} by WT and Lck-CD1d TgCD1° iNKT cells in the liver. However, in both the spleen and liver, we found that iNKT cells from Lck-CD1d TgCD1° mice produced less IL-4 than WT. Consistent with these results, we found significantly lower levels of IL-4 in the serum of Lck-CD1d TgCD1° mice than WT mice, whereas the amounts of IFN-{gamma} were comparable, possibly due to the production of IFN-{gamma} by conventional T cells in this system (Fig. 7B). Hence, after direct ligation of the TCR by anti-CD3 mAb, iNKT cells in Tg+ mice fail to realize maximum cytokine production potential.


Figure 7
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  		FIGURE 7. Inefficient Ag presentation cannot account for the hyporesponsiveness of Tg+ iNKT cells. A, WT and Lck-CD1d Tg+CD1° mice were injected i.v. with 2 µg of anti-CD3 mAb (2C11) in a total volume of 200 µl of PBS. After 20 min, cytokine secretion by splenic and hepatic iNKT cells was determined as described in Fig. 6. Cytokine histograms of iNKT cells from unstimulated control mice were negative (limited to the first decade of the log scale histogram plot), reflecting the lack of cytokine production, secretion, and detection in these samples. B, The amounts of IFN-{gamma} and IL-4 present in the serum of WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice after 1 h of anti-CD3 stimulation were quantitated by sandwich ELISA. Serum cytokine levels from unstimulated control mice were below the limits of detection. C, The amount of IFN-{gamma} in the serum of WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice 24 h after stimulation with 6 x 105 {alpha}-GalCer-pulsed DC was quantitated by sandwich ELISA. Serum cytokine levels from control mice stimulated with unpulsed WT DC were below the limits of detection. D, Total number of TCRbeta+ CD1d/{alpha}-GalCer tetramer+ iNKT cells present in the spleens and livers of WT ({square}) and Lck-CD1d Tg+CD1° ({blacksquare}) mice 72 h after stimulation with either unpulsed (Un) or {alpha}-GalCer-pulsed ({alpha}GC) DC. Bar graphs depict means obtained from duplicate wells. The data shown are representative of three independent experiments.

 
We next addressed the role of APC in the reduced functional response by iNKT cells in Lck-CD1d TgCD1° mice. To do this, we used exogenous WT bone marrow-derived DCs pulsed with {alpha}-GalCer to activate iNKT cells in vivo. In this way we could compare the responses of WT and Lck-CD1d TgCD1° iNKT cells to activation mediated by the same APC population. It has been shown that administration of in vitro-derived DCs pulsed with {alpha}-GalCer induces a robust IFN-{gamma} response (45). Consistent with previous reports, we found high levels of IFN-{gamma} in the serum of WT mice 24 h after {alpha}-GalCer-pulsed DC administration (Fig. 7C). However, the amount of IFN-{gamma} in the serum of Lck-CD1d TgCD1° mice was significantly less than that in WT mice (Fig. 7C). In addition to superior IFN-{gamma} production, we found increased numbers of iNKT cells in the spleens and livers of WT mice 72 h after DC stimulation, indicating that WT iNKT cells proliferated after activation (Fig. 7D). In contrast, significant iNKT cell expansion was not observed in the spleens or livers of Lck-CD1d TgCD1° animals (Fig. 7D). The difference in the proliferative response between WT and Lck-CD1d TgCD1° mice after DC-mediated activation resulted in a significant difference in the total number of iNKT cells present in the spleens and livers of these mice that is not seen otherwise (Figs. 7D and 2C). Results from these experiments suggest that the hyporesponse of Tg+ iNKT cells is not the result of improper Ag presentation by CD1d+ T cells in Lck-CD1d TgCD1° mice.

CD1d expression is heterogeneous with respect to cell type and overall expression levels and is found on both hemopoietic and nonhemopoietic cell lineages (21). Although special roles for tissue-specific APCs in activating iNKT cells have recently been described, in theory any CD1d+ cell is capable of eliciting some type of iNKT cell response (27). To address the possibility that the observed Lck-CD1d Tg+ iNKT cell phenotype is due to the selective absence of CD1d on some cell types, we repeated the in vivo {alpha}-GalCer stimulation assays in both Lck-CD1d TgCD1° and Lck-CD1d TgCD1+ mice, the latter of which maintain the endogenous CD1d expression program. As a functional readout, we examined the ability of iNKT cells to proliferate and transactivate B cells in response to in vivo {alpha}-GalCer stimulation. Seventy-two hours after stimulation, the proportion of iNKT cells from WT mice increased ~5- and 2-fold for spleen and liver, respectively, consistent with published reports (46) (Fig. 8). In addition, a substantial proportion of B220+ B cells in both the spleen and liver of WT mice expressed the early activation marker CD69. In contrast to results obtained from WT mice, splenic and liver iNKT cells from both Lck-CD1d TgCD1+ and Lck-CD1d TgCD1° mice failed to expand in response to {alpha}-GalCer stimulation (Fig. 8). In fact, the percentage of CD1d/{alpha}-GalCer tetramer+ iNKT cells in the spleen and liver of Lck-CD1d Tg+CD1° mice was decreased in {alpha}-GalCer-treated mice compared with vehicle-treated controls. Consistent with a poor response to {alpha}-GalCer stimulation by Tg+ iNKT cells, we did not observe significant CD69 expression by B220+ B cells in the spleen or liver. These results also support the idea that the impaired response to antigenic stimulation by iNKT cells in Lck-CD1d TgCD1° is not simply due to the lack of CD1d expression on the professional APCs; rather, iNKT cells in Lck-CD1d Tg mice may have an intrinsic functional deficit that may result from an altered developmental program.


Figure 8
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  		FIGURE 8. Lack of expansion and inferior transactivation by Tg+ iNKT cells after in vivo {alpha}-GalCer stimulation. WT, Lck-CD1d Tg+CD1+, and Lck-CD1d Tg+CD1° mice were injected i.p. with vehicle or 5 µg of {alpha}-GalCer. Seventy-two hours later, cells were stained with mAb to TCRbeta and CD1d/{alpha}-GalCer tetramers to identify iNKT cells or with mAb to B220 and CD69 to identify activated B cells. The data shown are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Results from recent studies using Tg mouse models in which CD1d expression is directed by either the classical MHC class I (Kb-CD1d Tg) or the class II promoter suggest that a specific pattern of CD1d expression is required for proper selection and development of NKT cells (38, 39). However, both models fail to restore sufficient numbers of iNKT cells to address the question of whether a cell type-specific CD1d expression pattern is important for the differentiation and maintenance of various iNKT cell subsets, which may, in turn, affect the quality of the iNKT cell-mediated immune responses. To test whether alterations in CD1d expression levels modulate iNKT cell function, we generated a new Tg mouse model in which CD1d expression is enhanced in T cells (Lck-CD1d TgCD1° mice). Using this model, we demonstrated that CD1d expression by T cells is sufficient to mediate the development of iNKT cells. However, the composition and phenotype of iNKT cell populations are distinctly different in Lck-CD1d TgCD1° and WT mice, suggesting a unique iNKT cell developmental program in Lck-CD1d TgCD1° mice. With age, Lck-CD1d TgCD1° mice spontaneously develop pathology in the liver, the organ with the highest proportion of iNKT cells. Upon Ag stimulation, iNKT in Lck-CD1d TgCD1° mice appear to be hyporesponsive in terms of their capacity to produce cytokines, proliferate, and transactivate other cell types, such as B cells, compared with WT iNKT cells. Taken together, these results suggest that changes in CD1d expression levels qualitatively affect iNKT cell development.

Our finding that the iNKT cell compartment is efficiently restored in Lck-CD1d TgCD1° mice is consistent with previous work demonstrating a critical role for double-positive thymocytes in the positive selection of NKT cells (37). We previously demonstrated that the expression of Kb-CD1d transgene rescues only a small proportion of iNKT cells in Kb-CD1d TgCD1° mice (38). The paucity of iNKT cells in Kb-CD1d TgCD1° is thought to be due primarily to excessive negative selection by thymic DCs expressing high levels of CD1d. The total iNKT cell numbers among WT and Lck-CD1d TgCD1° mice were comparable despite the fact that thymic DCs from Lck-CD1d TgCD1° mice do not express appreciable amounts of CD1d. Hence, the levels of CD1d expressed on thymocytes in Lck-CD1d TgCD1° mice may be sufficient for negative selection to occur, thereby preventing overt iNKT cell expansion and autoimmunity. The enhanced CD1d expression on thymocytes in Lck-CD1d TgCD1° mice is quite heterogeneous. In fact, a significant percentage of thymocytes express 15- to 30-fold higher levels of CD1d than WT, comparable to the CD1d levels expressed by DCs in Kb-CD1d TgCD1° mice. The lack of excessive negative selection in Lck-CD1d TgCD1° mice may be indicative of qualitative differences in the capacity of these two cell types to mediate negative selection.

Analysis of iNKT cell subsets in Lck-CD1d TgCD1° mice revealed significant differences in the relative prevalence of various iNKT cell populations based on their expression patterns of CD4 and NK1.1. Although only subtle differences in the proportion of CD4+ NK1.1+ iNKT subsets between adult WT and Lck-CD1d TgCD1° mice were observed in the thymus, these differences were much more profound in the peripheral tissues (Fig. 3B). Although at this time we cannot exclude the potential for thymic export to be distinct from that in WT mice, the differences in iNKT cell subsets observed between WT and Lck-CD1d TgCD1° mice most likely result from differential expansion and/or maturation in the periphery. In support of this idea, the proportion of the CD4+NK1.1+ iNKT cell subset in the liver increases significantly in WT mice between 3 and 4 wk of age (Fig. 4). In contrast, expansion of this subset was not observed in Lck-CD1d TgCD1° livers. One potential reason for the inefficient expansion of CD4+NK1.1+ iNKT cells in Lck-CD1d TgCD1° mice could be the lack of some essential cell type-specific, CD1d-mediated signal. However, similar changes in the proportions of iNKT cell subsets were observed in Lck-CD1d TgCD1+ mice which maintain the WT CD1d expression pattern, arguing against this possibility. Alternatively, upon arriving in the spleen or liver, maturing iNKT cells encounter high levels of CD1d expressed on T cells, possibly resulting in chronic TCR stimulation. Such constant stimulation may be more beneficial for some iNKT cells subsets than others, providing a selective advantage and niche that can be occupied. Another possible explanation, although not mutually exclusive, is that chronic stimulation may down-regulate NK1.1 expression on iNKT cells in Lck-CD1d TgCD1° mice. However, the proportion of the CD4– NK1.1+ iNKT cell subset in Lck-CD1d TgCD1° mice is comparable to that in WT mice and remains relatively constant throughout development.

The phenotypic differences observed in Tg+ iNKT cells were associated with altered function. Results from the functional studies strongly suggest that inefficient Ag presentation cannot fully account for the inferior cytokine production by iNKT cells in Lck-CD1d TgCD1° mice, but, instead, result from true intrinsic functional defects in Tg+ iNKT cells. The functional defects observed may be due to differences in the relative proportions of iNKT cell subsets in Lck-CD1d TgCD1° mice. Alternatively, Lck-CD1d TgCD1° iNKT cells may be hyporesponsive due to chronic exposure to high levels of CD1d/endogenous Ag complexes on T cells. Stimulation by T cells, a nonprofessional APC population that lacks expression of costimulatory molecules, may induce constant, weak signals by the iNKT cells, such that subsequent antigenic challenge elicits poor functional responses. In support of this, inefficient Ag presentation is known to induce anergy in conventional T cells (47). It has recently been reported than chronic glycolipid Ag stimulation results in the up-regulation of Ly49 inhibitory receptor expression on iNKT cells (31). However, we did not observe significant differences in expression of Ly49 family members between WT and Lck-CD1d TgCD1° iNKT cells, suggesting that signals induced by CD1d/endogenous Ag complexes may be distinct from those induced by high affinity ligands such as {alpha}-GalCer. Nevertheless, we cannot at this time rule out the possibility that other, as yet unidentified, NK inhibitory or activating receptors may contribute to the iNKT cell phenotype in Lck-CD1dTg mice.

One of the more interesting observations of Lck-CD1d TgCD1° mice is the development of liver pathology characterized by hepatomegaly, abnormal hepatocyte morphology, increased leukocyte infiltrate accompanied by a decrease in the proportion of iNKT cells, and changes to iNKT cell composition and function. It is important to emphasize that the liver pathology described above spontaneously develops in both lines of Lck-CD1d TgCD1° mice, arguing against the possibility that these changes result simply from aberrant transgene integration. Because only CD1d expression has been altered in Lck-CD1d TgCD1° mice, we hypothesize that liver pathology develops due to altered function of CD1d-resticted NKT cells. Liver pathology in Lck-CD1d TgCD1° mice may result directly from changes in the NKT cell-derived cytokines and/or indirectly by modulating the function of recruited leukocytes. That liver pathology develops in the absence of any exogenous manipulation suggests that endogenous CD1d-presented ligands are sufficient to modulate iNKT cell function. Another group has recently demonstrated, using a similar Lck-CD1d transgenic approach, the crucial role of CD1d-expressing T cells in iNKT cell development, but did not report finding liver pathology (48). The lack of liver pathology in this other model system may reflect differences in peripheral CD1d expression between the two models. Specifically, only ~10% of the thymic and peripheral T cells are CD1d+ in the model presented in the report by Wei et al. (48), whereas >90% of the T cells in our model express CD1d (Fig. 1B). Moreover, CD1d expression levels in our Lck-CD1dTg+ mice are more heterogeneous than those presented in the model by Wei et al. (48) (Fig. 1, B and C). The differences in CD1d expression and liver phenotype between these two model systems are consistent with the hypothesis that CD1d expression levels modulate NKT cell function. To directly address whether altered function of iNKT cells contributes to liver pathogenesis, we are currently breeding Lck-CD1d TgCD1° mice with J{alpha}18° mice, which specifically lack iNKT cells. These mice will allow us not only to address the role of iNKT cells, but also examine CD1d-restricted variant or nonclassical NKT cells as well.

Invariant NKT cells are unique immunoregulatory lymphocytes capable of enhancing or suppressing immunity in a variety of settings. Hence, the mechanisms governing the phenotypic and functional changes in iNKT cells during development must be highly regulated to maintain the proper balance of various iNKT cell subsets. Results from our studies demonstrate that cell type-specific CD1d expression not only affects the selection of iNKT cells in the thymus, but also the differentiation and function of iNKT cells in the periphery. Moreover, endogenous ligands presented in the context of CD1d are sufficient to modulate iNKT cell function. Interestingly, a number of studies have documented increased levels of CD1d expression in a variety of pathological settings. For example, up-regulated levels of CD1d have been found in hepatitis C virus-infected livers, on cardiac endothelial cells during coxsackievirus infection, on B cells residing in inflamed intestine, on primary myeloma tumor cells derived from human patients, and on macrophages and T cells invading the CNS in experimental autoimmune encephalomyelitis (49, 50, 51, 52, 53). Our study suggests that the effects of increased CD1d expression levels on iNKT cell function in these settings warrant additional investigation, because changes to the CD1d expression program may provide an additional mechanism of how iNKT cell subset composition and function can be altered.


   Acknowledgments
 
We thank Dr. Robert Enders for assistance documenting the liver pathology in Lck-CD1d Tg+ mice, Kirin Brewery (Gunma, Japan) for providing {alpha}-GalCer, and Tara King, Ashley Rohr, and Chunting Yang for technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01AI43407 (to C.-R.W.). M.I.Z. was supported by a postdoctoral fellowship from a cardiovascular training grant (National Institutes of Health Grant T32HL07237). Back

2 Address correspondence and reprint requests to Dr. Chyung-Ru Wang, Department of Pathology, University of Chicago, 924 East 57th Street, BSLC R412, Chicago, IL 60637-5420. E-mail address: cwang{at}uchicago.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; DN, double negative; {alpha}-GalCer, {alpha}-galactosylceramide; iNKT, invariant NKT; Tg, transgenic. Back

Received for publication June 30, 2005. Accepted for publication November 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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