CD1d-restricted NKT cells expressing invariant TCR α-chain rearrangements (iNKT cells) have been reported to be deficient in humans with a variety of autoimmune syndromes and in certain strains of autoimmune mice. In addition, injection of mice with α-galactosylceramide, a specific glycolipid agonist of iNKT cells, activates these T cells and ameliorates autoimmunity in several different disease models. Thus, deficiency and reduced function in iNKT cells are considered to be risk factors for the development of such diseases. In this study we report that the development of systemic lupus erythematosus in (New Zealand Black (NZB) × New Zealand White (NZW))F1 mice was paradoxically associated with an expansion and activation of iNKT cells. Although young (NZB × NZW)F1 mice had normal levels of iNKT cells, these expanded with age and became phenotypically and functionally hyperactive. Activation of iNKT cells in (NZB × NZW)F1 mice in vivo or in vitro with α-galactosylceramide indicated that the immunoregulatory role of iNKT cells varied over time, revealing a marked increase in their potential to contribute to production of IFN-γ with advancing age and disease progression. This evolution of iNKT cell function during the progression of autoimmunity may have important implications for the mechanism of disease in this model of systemic lupus erythematosus and for the development of therapies using iNKT cell agonists.

Invariant NKT cells (iNKT cells)3 constitute a unique subset of lymphocytes with surface markers characteristic of both NK cells (NK1.1 or CD161) and conventional T cells (CD3 and TCRαβ). They display an invariant TCR α-chain (Vα14-Jα18 in mice and Vα24-Jα18 in humans) paired with a limited set of TCR β-chains and recognize self and exogenous lipids in association with CD1d, a nonclassical MHC class I-like Ag-presenting molecule (1). Although the natural ligands of CD1d are still poorly defined, certain forms of α-galactosylceramide (αGalCer) bind strongly to CD1d and are specifically recognized by the TCRs of iNKT cells (2). Upon activation, iNKT cells rapidly and abundantly produce various immunoregulatory cytokines, including IL-4 and IFN-γ, and trigger the activation and differentiation of a variety of other leukocytes (3, 4).

Multiple lines of evidence implicate iNKT cells in the regulation of autoimmunity (5). Circulating iNKT cells are reported to be reduced in several human autoimmune diseases, including type 1 diabetes, systemic sclerosis, rheumatoid arthritis, and multiple sclerosis (6, 7, 8, 9, 10). In diabetes-prone NOD mice, iNKT cells are deficient in number and function before disease onset (11, 12, 13). Moreover, both spontaneous diabetes in the NOD mouse and experimental allergic encephalomyelitis in nonautoimmune mouse strains can be prevented by repeated injection of αGalCer or by augmentation of iNKT cells (14, 15, 16, 17). Together, these data suggest that a deficiency in iNKT cells may be a primary immune defect leading to the emergence of autoimmunity.

In systemic lupus erythematosus (SLE), a relatively common and potentially severe autoimmune disease, a detailed analysis and a precise understanding of the role of iNKT cells during the development and progression of disease are still lacking. Humans with SLE have shown reduced numbers of circulating iNKT cells (6, 8, 9), whereas studies in lupus-prone mouse strains have yielded conflicting data, suggesting both a protective and a deleterious role for iNKT cells (18, 19). The current study was thus undertaken to document the natural history of changes in the iNKT cell population of (New Zealand Black (NZB) × New Zealand White (NZW))F1 mice, a strain that spontaneously develops systemic autoimmunity with severe glomerulonephritis that closely resembles SLE in humans (20).

Using αGalCer-loaded CD1d tetramers to achieve accurate quantitation of iNKT cells, we showed that, in contrast to diabetes-prone NOD mice, young lupus-prone (NZB × NZW)F1 mice did not display numerical or functional deficiencies in these cells. Instead, this model of murine SLE was associated with a massive expansion of iNKT cells that displayed signs of spontaneous activation in vivo. The expanded iNKT cells showed markedly enhanced cytokine production, with a progressive shift toward increasing secretion of IFN-γ as the animals aged. Isolated iNKT cells from (NZB × NZW)F1 mice were able to strongly augment B cell activation in vitro, inducing polyclonal B cell proliferation and secretion of Igs. Our data support a temporally variable role of iNKT cells in SLE, with a potentially protective role in the early stage of the disease before the occurrence of marked iNKT cell expansion and hyperactivity, and a pathogenic role in the later stages of active lupus.

(NZB × NZW)F1 female mice were purchased from Harlan Sprague Dawley and The Jackson Laboratory. C57BL/6 and NOD/ltj female mice were obtained from The Jackson Laboratory. Mice were housed at the animal facility at the Albert Einstein College of Medicine under strictly controlled, specific pathogen-free conditions.

The αGalCer used in these studies was produced by a synthetic method described previously (21). Its structure is identical with that of KRN7000, except that the fatty acid chain of this synthetic αGalCer is two carbons shorter. αGalCer was dissolved in PBS containing 0.5% Tween 20, heated to 80°C for 10 min, and sonicated for 5 min in a water bath sonicator (model 2510; Branson Ultrasonic) before use.

Single-cell suspensions of thymus, spleen, liver, and kidneys were prepared. Lymphoid cells from PBS-perfused liver and kidneys were separated by centrifugation with Lympholyte M (Cedarlane Laboratories). FcγRs were blocked by incubation with 2.4G2 mAb (BD Pharmingen), and cells were stained at 4°C in PBS/1% BSA. Fluorochrome-conjugated Abs specific for CD3, pan-TCRβ, B220, NK1.1, CD4, CD8, CD69, CD44, IL-4, and IFN-γ were purchased from BD Pharmingen. The iNKT cells were stained by incubating them for 1 h at room temperature with αGalCer-loaded CD1d tetramers, produced using a baculovirus expression system (22). For intracellular cytokine staining, splenocytes were stimulated in vitro by a combination of PMA (100 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) for 8 h in the presence of monensin (GolgiStop; BD Pharmingen). Cells were surface stained, fixed for 10 min with the Cytofix buffer, permeabilized using the Wash/Perm buffer (BD Pharmingen), and finally stained for the cytokine of interest. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest software (BD Biosciences).

Splenocytes (3 × 105) were plated in 96-well, flat-bottom plates in RPMI 1640 containing 10% FBS (HyClone), l-glutamine, penicillin-streptomycin, 2-ME, and HEPES (Invitrogen Life Technologies). Cells were stimulated for 72 h at 37°C in a 5% CO2 incubator with various concentrations of αGalCer (0.1–100 nM) or medium alone. Proliferation was measured by adding [3H]thymidine (1 μCi/well; NEN) for the last 12 h of the culture. Cells were harvested with a TomTech cell harvester, and [3H]thymidine incorporation was determined using a Microbeta Trilux scintillation counter (Wallac). Supernatants levels of IL-4 and IFN-γ were measured by standard ELISA. The following pairs of mAbs were used for capture and detection: 11B11 and BVD6-24G2 for IL-4, R4-6A2 and XMG1.2 for IFN-γ, and JES6-1A12 and JES6-5H4 for IL-2 (BD Pharmingen). Alkaline phosphatase-streptavidin (Zymed Laboratories) was used as the secondary reagent and developed using p-nitrophenyl phosphate substrate (Sigma-Aldrich). The rIL-4, rIL-2, and rIFN-γ standards were obtained from PeproTech.

Mice were given an i.p. injection of 4 μg of αGalCer or vehicle (PBS and 0.5% Tween 20) alone. Two or 23 h after injection, blood was collected from the retro-orbital plexus. Serum samples were diluted 1/5 with PBS and tested for IL-4 and IFN-γ by ELISA as described above.

iNKT cell lines were derived from spleens of female (NZB × NZW)F1 mice. Splenocytes were collected, plated in 24-well plates at 3 × 106 cells/ml, and cultured with αGalCer (100 ng/ml). On the fourth day of culture, human IL-2 was added (20 U/ml). Cells were fed every 2 days with IL-2 and were restimulated weekly with αGalCer plus irradiated syngeneic splenocytes (2500 rad). After 2 wk of culture, cells that costained with anti-TCRβ and αGalCer-loaded CD1d tetramers were purified by cell sorting using a MoFlo high speed cell sorter (DakoCytomation).

Splenic B cells were positively selected using anti-B220-coated Dynal beads (Dynal Biotech) and were incubated (5 × 104 cells/well) with the irradiated (3000 rad) iNKT cell line (3 × 105 cells/well) in 96-well, flat-bottom plates previously coated, or not, with anti-CD3 Ab at 5 μg/ml (BD Pharmingen). B cell proliferation was measured by [3H]thymidine incorporation (added for the last 12 h, 1 μCi/well) after 5 days of culture in medium at 37°C 5% CO2. Total IgM, IgG, and IgG2a were measured in culture supernatants after 5 and 10 days in culture using an ELISA with goat anti-mouse IgM or IgG (H+L chain) Abs (Southern Biotechnology Associates) to capture mouse IgM and IgG, and alkaline phosphatase-labeled, affinity-purified goat Abs specific for mouse Ig isotypes for detection (Southern Biotechnology Associates). IgM and IgG anti-dsDNA Abs were captured using sonicated calf thymus DNA (Calbiochem), and ELISA was performed as described previously (23). Anti-dsDNA titers were expressed as units per milliliter using a reference-positive standard of pooled serum from 7- to 8-mo-old (NZB × NZW)F1 mice. A 1/200 dilution of this standard serum was arbitrarily assigned a value of 100 U/ml.

Statistical analyses were performed using PRISM software (GraphPad). Lymphocyte percentages and numbers were compared by ANOVA with Bonferroni correction. Differences in proteinuria onset and survival time of groups were analyzed using the log-rank test.

We evaluated the frequency and absolute number of iNKT cells by αGalCer-loaded CD1d tetramer staining before lupus development in thymus, spleen, liver, and kidney of 4-wk-old (NZB × NZW)F1 mice (Fig. 1). As expected, NOD mice displayed a significantly reduced iNKT cell population compared with normal C57BL/6 mice (p < 0.05 in spleen and liver). In contrast, (NZB × NZW)F1 mice showed similar percentages and numbers of iNKT cells in the kidney, liver, and spleen compared with C57BL/6 mice and significantly higher percentages than C57BL/6 mice in the blood (p < 0.05). Thus, at a young age, (NZB × NZW)F1 mice did not show a numerical deficiency of immunoregulatory iNKT cells.

FIGURE 1.

Normal size of the iNKT cell population in various tissues of young (NZB × NZW)F1 mice. Flow cytometric analysis of thymus, spleen, liver, kidneys, and blood mononuclear cells from 4- to 6-wk-old nonautoimmune C57BL/6, diabetes-prone NOD, and lupus-prone (NZB × NZW)F1 female mice. Cells were costained with anti-TCRβ and αGalCer-loaded CD1 tetramers. A, Dot plots showing percentages of iNKT cells in various organs from one representative mouse. Numbers show percentages of tetramer-positive cells in circled regions. B, Absolute numbers of iNKT cells. Each symbol corresponds to one mouse. Horizontal bars represent the mean for each group. Statistical analysis was calculated by one-way ANOVA with Bonferroni correction (∗, p < 0.05 compared with normal C57BL/6 mice). B6, C57BL/6; B/WF1, (NZB × NZW)F1.

FIGURE 1.

Normal size of the iNKT cell population in various tissues of young (NZB × NZW)F1 mice. Flow cytometric analysis of thymus, spleen, liver, kidneys, and blood mononuclear cells from 4- to 6-wk-old nonautoimmune C57BL/6, diabetes-prone NOD, and lupus-prone (NZB × NZW)F1 female mice. Cells were costained with anti-TCRβ and αGalCer-loaded CD1 tetramers. A, Dot plots showing percentages of iNKT cells in various organs from one representative mouse. Numbers show percentages of tetramer-positive cells in circled regions. B, Absolute numbers of iNKT cells. Each symbol corresponds to one mouse. Horizontal bars represent the mean for each group. Statistical analysis was calculated by one-way ANOVA with Bonferroni correction (∗, p < 0.05 compared with normal C57BL/6 mice). B6, C57BL/6; B/WF1, (NZB × NZW)F1.

Close modal

In contrast, as the mice aged, we observed a striking expansion of iNKT cells in multiple tissues of (NZB × NZW)F1 mice (Fig. 2). At 14 wk of age, a time at which these mice have not yet developed significant clinical disease, both the percentage and the absolute number of iNKT cells were increased in the thymus, spleen, and liver compared with 4-wk-old mice. At 14 wk of age, the enrichment of iNKT cells in the kidney was highly significant. At 34 wk of age, after the onset of clinically apparent SLE (e.g., ∼50% of the animals positive for proteinuria), the number of iNKT cells was significantly increased in the thymus, liver, and spleen compared with 14-wk-old mice (Fig. 2,B). In the spleen, continuing expansion of the absolute number of iNKT cells was not reflected as an increase in percentage because of the remarkable hypercellularity that developed in this organ during the progression of SLE (Fig. 2). In contrast, no statistically significant changes were observed in the numbers of iNKT cells in the spleen, thymus, or liver lymphocytes of C57BL/6 mice. Although a slight trend toward increasing iNKT cells was observed in the livers of these normal control animals, analyses of the spleen and thymus mononuclear cell populations did not reveal even the slightest suggestion of an age-related expansion comparable to that observed in (NZB × NZW)F1 mice (Fig. 2 B). Thus, the specific enrichment of iNKT cells in (NZB × NZW)F1 mice appeared to be an early feature of the immunopathology that preceded the onset of overt autoimmunity, and this became further amplified after the development of clinical disease.

FIGURE 2.

Expansion of iNKT cells in (NZB × NZW)F1 mice with increasing age and disease progression. iNKT cells from (NZB × NZW)F1 and C57BL/6 mice were quantified by flow cytometry using anti-TCRβ and αGalCer-loaded CD1 tetramer double staining. A, Representative dot plot for individual (NZB × NZW)F1 mice at 4, 14, and 34 wk of age. Numbers show percentages of tetramer-positive cells in the respective circled regions. B, Total numbers of iNKT cells in spleen, liver, thymus, and kidney are plotted for mice in different age groups. The x-axis indicates the mean age of the mice in each group (ranges were 4–5 wk for the group with mean age of 4 wk, 10–18 wk for the group with mean age of 14 wk, and 25–35 wk for the group with mean age of 30 wk). Each symbol corresponds to one mouse. Horizontal bars represent the mean for each group. Statistical analysis was performed using one-way ANOVA with Bonferroni correction (∗, p < 0.001; ∗∗, p < 0.05; (compared with age-matched normal C57BL/6 mice)).

FIGURE 2.

Expansion of iNKT cells in (NZB × NZW)F1 mice with increasing age and disease progression. iNKT cells from (NZB × NZW)F1 and C57BL/6 mice were quantified by flow cytometry using anti-TCRβ and αGalCer-loaded CD1 tetramer double staining. A, Representative dot plot for individual (NZB × NZW)F1 mice at 4, 14, and 34 wk of age. Numbers show percentages of tetramer-positive cells in the respective circled regions. B, Total numbers of iNKT cells in spleen, liver, thymus, and kidney are plotted for mice in different age groups. The x-axis indicates the mean age of the mice in each group (ranges were 4–5 wk for the group with mean age of 4 wk, 10–18 wk for the group with mean age of 14 wk, and 25–35 wk for the group with mean age of 30 wk). Each symbol corresponds to one mouse. Horizontal bars represent the mean for each group. Statistical analysis was performed using one-way ANOVA with Bonferroni correction (∗, p < 0.001; ∗∗, p < 0.05; (compared with age-matched normal C57BL/6 mice)).

Close modal

To assess the activation status of iNKT cells from (NZB × NZW)F1 mice, we evaluated the expression of NK1.1, CD44 (a late activation marker typical of mature iNKT cells) and CD69 (an early activation marker on iNΚΤ cells). Independent of age, NK1.1 expression was significantly reduced on iNKT cells from the spleens (p < 0.001) and kidneys (p < 0.01) of (NZB × NZW)F1 mice compared with age-matched C57BL/6 mice (Fig. 3). Interestingly, NK1.1 levels were normal in the hepatic and thymic iNKT cells of (NZB × NZW)F1 mice (Fig. 3,A). A uniformly high expression of CD44 was observed at the surface of splenic iNKT cells of both C57BL/6 and (NZB × NZW)F1 mice, whereas CD69 expression was markedly reduced in this organ (Fig. 3 B). The specific down-regulation of NK1.1 in the spleen and kidney and CD69 in the kidney combined with a normal expression of CD44 were strongly consistent with a recent acute or ongoing chronic activation of these cells in organs affected by the pathological process in SLE (24, 25).

FIGURE 3.

Reduced expression of NK1.1 and CD69 on iNKT cells from the spleen and kidneys of (NZB × NZW)F1 mice. Spleen, kidney, liver, and thymus from 4- to 8-wk-old (NZB × NZW)F1 mice were harvested, and iNKT cells were analyzed by flow cytometry for their expression of NK1.1, CD69, and CD44 markers. A, Histogram plots show NK1.1 expression on iNKT cells, gated by double staining with anti-TCRβ- and αGalCer-loaded CD1d tetramers. Numbers represent percentages of positive cells in the indicated region of the histogram plots. B, Percentages of NK1.1-, CD69-, and CD44-positive cells among iNKT cells in the spleen and kidney. Each symbol represents one mouse. Horizontal bars represent the mean for each group. Statistical analysis was performed using one-way ANOVA with Bonferroni correction (∗, p < 0.001 compared with C57BL/6 mice).

FIGURE 3.

Reduced expression of NK1.1 and CD69 on iNKT cells from the spleen and kidneys of (NZB × NZW)F1 mice. Spleen, kidney, liver, and thymus from 4- to 8-wk-old (NZB × NZW)F1 mice were harvested, and iNKT cells were analyzed by flow cytometry for their expression of NK1.1, CD69, and CD44 markers. A, Histogram plots show NK1.1 expression on iNKT cells, gated by double staining with anti-TCRβ- and αGalCer-loaded CD1d tetramers. Numbers represent percentages of positive cells in the indicated region of the histogram plots. B, Percentages of NK1.1-, CD69-, and CD44-positive cells among iNKT cells in the spleen and kidney. Each symbol represents one mouse. Horizontal bars represent the mean for each group. Statistical analysis was performed using one-way ANOVA with Bonferroni correction (∗, p < 0.001 compared with C57BL/6 mice).

Close modal

We examined the functional properties of iNKT cells by assessing their capacity for cytokine production at the single-cell level by intracellular staining. The iNKT cells from 6- and 20-wk-old (NZB × NZW)F1 mice produced increased levels of IFN-γ and IL-4 and displayed greater frequencies of cytokine-producing cells compared with age-matched C57BL/6 mice (Fig. 4). Although the cytokine levels produced by iNKT cells from C57BL/6 mice were comparable at different ages, iNKT cells from (NZB × NZW)F1 mice showed a marked increase in production of both cytokines as the mice aged. Thus, the increasing hyperactivity of iNKT with advancing age in (NZB × NZW)F1 mice appeared to be associated with the predisposition of these mice to develop SLE.

FIGURE 4.

High frequency of cytokine-producing cells among splenic iNKT cells in (NZB × NZW)F1 mice. A, Dot plot shows the identification of iNKT cells as the population positively costained with anti-TCRβ- and αGalCer-loaded CD1d tetramers. Histogram plots show production of IFN-γ and IL-4 by gated splenic iNKT cells from representative 6-wk-old C57BL/6, 6-wk-old (NZB × NZW)F1, and 20-wk-old (NZB × NZW)F1 mice after stimulation with PMA and ionomycin in vitro. The percentages of cytokine-positive cells were determined for the histogram regions shown, which were selected based on background staining levels with isotype-matched control Abs. B, Summary of intracellular cytokine staining of iNKT cells activated with PMA plus ionomycin. Bars display the means and SDs of the frequencies of IFN-γ- and IL-4-positive iNKT cells for analyses of three mice of each strain and age group, measured by flow cytometry as shown in A.

FIGURE 4.

High frequency of cytokine-producing cells among splenic iNKT cells in (NZB × NZW)F1 mice. A, Dot plot shows the identification of iNKT cells as the population positively costained with anti-TCRβ- and αGalCer-loaded CD1d tetramers. Histogram plots show production of IFN-γ and IL-4 by gated splenic iNKT cells from representative 6-wk-old C57BL/6, 6-wk-old (NZB × NZW)F1, and 20-wk-old (NZB × NZW)F1 mice after stimulation with PMA and ionomycin in vitro. The percentages of cytokine-positive cells were determined for the histogram regions shown, which were selected based on background staining levels with isotype-matched control Abs. B, Summary of intracellular cytokine staining of iNKT cells activated with PMA plus ionomycin. Bars display the means and SDs of the frequencies of IFN-γ- and IL-4-positive iNKT cells for analyses of three mice of each strain and age group, measured by flow cytometry as shown in A.

Close modal

We then measured ex vivo responses of whole splenocyte populations from (NZB × NZW)F1 mice to αGalCer stimulation and compared these with the responses in age-matched C57BL/6 mice (Fig. 5,A). Both 6- and 20-wk-old (NZB × NZW)F1 splenocytes showed a strong increase in production of IL-4 compared with C57BL/6 mice. However, although 6-wk-old (NZB × NZW)F1 splenocytes secreted a level of IFN-γ similar to that of control mice, 20-wk-old (NZB × NZW)F1 splenocytes showed a significant increase in the production of IFN-γ compared with age-matched controls. Although the IFN-γ secreted in this whole splenocyte culture system is known to originate largely from NK cells that are activated as a result of downstream events after iNKT cell activation, the initiation of this response by αGalCer is entirely dependent on iNKT cells (4, 21). Thus, the increased IFN-γ production in this analysis is another reflection of the hyperactivity of the iNKT cell population in (NZB × NZW)F1 mice and indicates a heightened Th1 bias of adult SLE-prone mice in response to αGalCer stimulation. This phenomenon was specific to (NZB × NZW)F1 mice, because the response of C57BL/6 mice splenocytes to αGalCer did not significantly vary with age (Fig. 5 B).

FIGURE 5.

Enhanced responses of (NZB × NZW)F1 splenocytes to in vitro αGalCer stimulation. Splenocytes from 6- or 20-wk-old C57BL/6 or (NZB × NZW)F1 mice were stimulated with various doses of αGalCer. A, Proliferation was measured by [3H]thymidine incorporation. B, Cytokine production was quantified by ELISA performed on the culture supernatant. Graphs show the means and SDs for measurements performed in triplicate. Results are representative of five independent experiments.

FIGURE 5.

Enhanced responses of (NZB × NZW)F1 splenocytes to in vitro αGalCer stimulation. Splenocytes from 6- or 20-wk-old C57BL/6 or (NZB × NZW)F1 mice were stimulated with various doses of αGalCer. A, Proliferation was measured by [3H]thymidine incorporation. B, Cytokine production was quantified by ELISA performed on the culture supernatant. Graphs show the means and SDs for measurements performed in triplicate. Results are representative of five independent experiments.

Close modal

An additional assessment of the cytokine production resulting from activation of iNKT cells in (NZB × NZW)F1 mice was conducted by measurement of serum cytokine levels after in vivo administration of αGalCer. By this approach, no significant differences between young and old (NZB × NZW)F1 and aged-matched C57BL/6 mice in terms of circulating IL-4 were observed 2 h after αGalCer injection (Fig. 6,A). In contrast, serum IFN-γ was strikingly increased in 35-wk-old SLE-prone mice compared with 6-wk-old (NZB × NZW)F1 mice or 6- to 20-wk-old C57BL/6 mice (Fig. 6 B). As for αGalCer-stimulated splenocyte cultures, the IFN-γ measured in the serum at 23 h most likely reflects both the direct activation of iNKT cells and the secondary activation of other cells, such as NK cells. However, the serum cytokine response to αGalCer has previously been shown to be entirely iNKT cell dependent (26), confirming the augmented Th1 cytokine profile of cytokine secretion that occurs in adult SLE-prone mice after iNKT cell stimulation in vivo.

FIGURE 6.

Th1 bias in the serum cytokine levels of adult (NZB × NZW)F1 mice after a single injection of αGalCer. Levels of IL-4 (A) and IFN-γ (Β) were measured in the serum 2 and 23 h after i.p. injection of αGalCer into C57BL/6 and (NZB × NZW)F1 mice. Two sets of mice for each strain were analyzed; one set consisted of young animals <8 wk of age (range, 4–8 wk), and the other of older animals >27 wk of age (range, 27–35 wk). Bars show the mean ± 1 SD for three mice. Results are representative of two independent experiments.

FIGURE 6.

Th1 bias in the serum cytokine levels of adult (NZB × NZW)F1 mice after a single injection of αGalCer. Levels of IL-4 (A) and IFN-γ (Β) were measured in the serum 2 and 23 h after i.p. injection of αGalCer into C57BL/6 and (NZB × NZW)F1 mice. Two sets of mice for each strain were analyzed; one set consisted of young animals <8 wk of age (range, 4–8 wk), and the other of older animals >27 wk of age (range, 27–35 wk). Bars show the mean ± 1 SD for three mice. Results are representative of two independent experiments.

Close modal

Cytokines such as IFN-γ and IL-4 have been directly implicated in the development of autoantibodies, a characteristic of the lupus-like disease in (NZB × NZW)F1 mice (27). Because iNKT cells of (NZB × NZW)F1 mice showed markedly exaggerated secretion of both cytokines, we examined the role of iNKT cells in B cell function using an in vitro culture system. We derived a short-term cultured iNKT cell line from (NZB × NZW)F1 mice and cocultured it with splenic B cells purified from 12-wk-old syngeneic animals (Fig. 7). Cultured iNKT cells enhanced B cell proliferation (Fig. 7,A) and augmented the secretion of IgM anti-dsDNA Ab and polyclonal total IgM and IgG by ∼10-, 50-, and 100-fold, respectively (Fig. 7, B and C). Interestingly, to provide B cell help, cultured iNKT cells required prior activation with either anti-CD3 mAb or αGalCer. This effect was independent of the age and extent of disease of the B cell donors, because similar results were obtained with B cells from 34-wk-old (NZB × NZW)F1 mice with established proteinuria (data not shown). However, in this culture system, iNKT cells did not induce the production of IgG anti-dsDNA Abs and did not stimulate detectable increases in the levels of total IgG2a (not shown). These findings suggested that the hyperactivated iNKT cells of (NZB × NZW)F1 mice may be involved in the development of some of the abnormal B cell responses characteristic of the lupus-like disease in these animals.

FIGURE 7.

Enhancement of B cell function by activated iNKT cells. B cells purified from (NZB × NZW)F1 splenocytes were cocultured with iNKT cells sorted from a line derived from the spleen of a 5-wk-old (NZB × NZW)F1 mouse. The sorted iNKT cells were previously irradiated then activated, or not, with anti-CD3. A, Proliferation of B cells was measured by [3H]thymidine incorporation after 5 days of incubation. B, Total IgM and IgG Ab production was measured in the culture supernatant by ELISA after 10 days in culture. C, Levels of IgM anti-dsDNA were also measured by ELISA in day 10 culture supernatants. Results are expressed as the mean ± SE from a representative of three independent experiments. BD, below detection (detection limit values were 2 and 4 ng/ml for the IgM and IgG ELISAs, respectively, and <1 U/ml for anti-dsDNA ELISA).

FIGURE 7.

Enhancement of B cell function by activated iNKT cells. B cells purified from (NZB × NZW)F1 splenocytes were cocultured with iNKT cells sorted from a line derived from the spleen of a 5-wk-old (NZB × NZW)F1 mouse. The sorted iNKT cells were previously irradiated then activated, or not, with anti-CD3. A, Proliferation of B cells was measured by [3H]thymidine incorporation after 5 days of incubation. B, Total IgM and IgG Ab production was measured in the culture supernatant by ELISA after 10 days in culture. C, Levels of IgM anti-dsDNA were also measured by ELISA in day 10 culture supernatants. Results are expressed as the mean ± SE from a representative of three independent experiments. BD, below detection (detection limit values were 2 and 4 ng/ml for the IgM and IgG ELISAs, respectively, and <1 U/ml for anti-dsDNA ELISA).

Close modal

A critical immunoregulatory role has been assigned to iNKT cells in a variety of autoimmune diseases in both mice and humans, such that their absence enhances autoimmunity, whereas their enrichment alleviates it (6, 8, 9, 11, 13, 17). Moreover, therapeutic targeting of iNKT cells in mice by administration of αGalCer has been proven to control diabetes and experimental allergic encephalomyelitis by enhancing their immunoregulatory properties (14, 15, 16, 26). The immunoregulatory role of iNKT cells in SLE, although suggested by previous studies, has not been clearly documented. Studies in various mouse disease models have yielded conflicting results in favor of either a protective function or a pathogenic role of iNKT cells (18, 19). The current study provides the first detailed assessment of the numbers and effector functions of iNKT cells in (NZB × NZW)F1 lupus-prone mice during the course of disease progression and explores their immunoregulatory role according to their functional characteristics and the age of the mice.

Several studies indicate the possibility of a selective reduction of circulating iNKT cells in patients with SLE and in various organs of lupus-prone mouse strains preceding the development of the disease (6, 9, 29, 30). In the mouse studies, iNKT cells were tracked using a combination of NK1.1 and TCR as phenotypic markers, which is now known to not be completely specific for iNKT cells (31). Moreover, in studies of lupus-prone mice, the age and strain were not taken carefully into consideration to properly analyze the status of iNKT cells during the progression of SLE. In human SLE, iNKT cells were quantified in the blood, which has been recently suggested to provide a poor representation of those cells in other organs (32). However, a defect of iNKT cells before the onset of disease was accurately reported by CD1d-αGalCer tetramer staining in both spontaneous (MRL/lpr mice) and induced (pristane-treated BALB/c mice) animal models of SLE, suggesting that lupus progression is a consequence of the deficiency in iNKT cells (18, 33). In some models, the disease was accelerated after depletion of NK1.1+ cells or deletion of CD1d genes (30, 33, 34). Conversely, lupus was delayed by transfer of enriched NK1.1+ T cells or by repeated injection of αGalCer (18, 30). All these data suggest an association between a systemic deficiency of NKT cells and the development of lupus, and hence favor a protective role for iNKT cells in this disease.

In contrast, other data attribute to iNKT cells a pathogenic role in lupus. Indeed, elevated numbers of iNKT cells have been previously reported in the liver and spleen of (NZB × NZW)F1 adult mice, as confirmed in the current study (19, 35). Chronic treatment of (NZB × NZW)F1 mice with anti-CD1d mAb delayed glomerulonephritis, whereas transfer of autoreactive CD1d-restricted T cells accelerated the disease (36, 37). More recently, αGalCer treatment of 20-wk-old adult (NZB × NZW)F1 mice has also been shown to accelerate the disease (19).

In the current study we demonstrated conclusively that, in contrast to diabetic NOD and lupus-prone MRL/lpr mice, the development of lupus in (NZB × NZW)F1 mice is not linked to a deficiency in the number of iNKT cells. By contrast, we found that as the mice aged and developed lupus, iNKT cells significantly expanded in various organs, such as spleen, kidney, liver, and thymus. This expansion was not observed in normal C57BL/6 mice at either young or more advanced ages. We used C57BL/6 as a prototypical normal mouse strain for these studies because of the wealth of background information available on this strain and the availability of these mice at a variety of different ages in our facility. In studies of six different normal strains of mice (C57BL/6, BALB/c, C3H/HeJ, CBA/J, DBA/2, and A/J), we did not find C57BL/6 to have unusually low or high levels of iNKT cells in any tissues; thus, we believe the frequencies of iNKT cells in this strain to be a reasonable representation of those in normal animals (J. S. Im and S. Porcelli, unpublished observations). It is well known that the severe SLE-like disease in (NZB × NZW)F1 animals is a consequence of multiple interacting genetic loci from both parental strains (20), and it is thus possible that a genetic locus or loci from one or both of the parental strains could be implicated in controlling the iNKT cell expansion. In fact, preliminary studies suggest that iNKT cell expansion may be a feature of the NZW and not the NZB parental strain, although we have not yet conducted genetic mapping studies to identify a specific locus (C. Forestier, unpublished observations).

Two reasons could account for the enrichment of iNKT cells in various tissues after the onset of the disease. First, resident iNKT cells may expand and proliferate in response to an activation stimulus in vivo. iNKT cells have been described as autoreactive by design, because they recognize endogenous ligands bound to CD1d (38). These endogenous ligands could be unleashed in target organs as a consequence of the progression of lupus. Indeed, lupus is a disease associated with inflammation and apoptosis, and it is conceivable that dying cells could serve as a potential reservoir of modified forms of autoantigens that may, in turn, activate iNKT cells (39, 40). In this regard, it will be of interest to assess the expression in tissues of SLE-prone mice of potential activating ligands of iNKT cells, such as the isoglobotrihexosylceramide iGb3 that has been recently identified as a putative major endogenous natural ligand (41, 42). A second possibility is that iNKT cells may be more actively recruited to and retained in various tissues of SLE-prone mice, leading to their accumulation in the kidney, spleen, and liver. Indeed, iNKT cells are known to display a variety of chemokine receptors on their surface that enable them to respond by migrating rapidly to sites of inflammation (43).

Interestingly, in the spleen and kidney of (NZB × NZW)F1 mice, the majority of tetramer-positive cells was CD44highCD69NK1.1, although in normal C57BL/6 mice, the majority was CD44highCD69+NK1.1+. It is noteworthy that in the spontaneous lupus of MRL/lpr mice, the NK1.1 subset was found to be especially deficient, suggesting that the alterations in iNKT cells may vary between genetically distinct models of SLE (18). The abundant CD44highCD69NK1.1 iNKT cell subset of (NZB × NZW)F1 mice could represent either immature iNKT cells that escaped the thymus before acquiring the NK1.1 marker or in vivo activated iNKT cells that have down-regulated this marker (44, 45). However, the phenotype that we observed in SLE-prone mice was atypical for immature NK1.1 iNKT cells, which express low levels of CD69, but are heterogeneous for CD44 expression (46). In addition, normal levels of NK1.1+ cells were observed in both the thymus and liver of (NZB × NZW)F1 mice, probably excluding a blockage in the maturation process. Taken together, these findings suggested that the unusual phenotype of iNKT cells in certain organs of these mice was due to spontaneous activation in vivo, possibly as a result of recognition of self-glycolipid ligands in tissues such as the spleen and kidney.

The expanded iNKT cells of (NZB × NZW)F1 mice were also unusual in their pattern of cytokine secretion compared with iNKT cells of normal mice (44, 47). After activation in vitro, iNKT cells of (NZB × NZW)F1 mice exhibited hyperactivity, characterized by a very high secretion of both IFN-γ and IL-4 compared with C57BL/6 mice. The overproduction of IFN-γ was particularly marked for iNKT cells from (NZB × NZW)F1 mice 20 wk of age or older and was also reflected by the serum cytokine levels resulting from in vivo stimulation of iNKT cells by a single injection of αGalCer. Our analysis of intracellular cytokine levels of CD1d-tetramer positive cells suggested that the increased IFN-γ levels after αGalCer stimulation in (NZB × NZW)F1 mice were in part associated with a direct enhancement of cytokine production by the iNKT cells themselves. However, it is clear that much of the IFN-γ generated after αGalCer stimulation of mixed cell populations such as splenocytes or in the intact animal after αGalCer injection is secreted by NK cells or possibly other cell types that are undergoing secondary bystander activation (21). Nevertheless, this secondary activation of downstream effectors is believed to be entirely dependent on the initial response of iNKT cells. Thus, the enhanced IFN-γ production observed in (NZB × NZW)F1 mice may be another reflection of the expansion and hyperactivity of the iNKT cells in these animals. Given that IFN-γ has been associated with the progression of SLE in a variety of murine models (48, 49, 50, 51), the overproduction of this cytokine may account in part for the harmful effect of αGalCer previously reported when administered to 20-wk-old adult (NZB × NZW)F1 mice (19).

Our data strongly suggest that the outcome of αGalCer injection will depend on the age-related changes in iNKT cells in (NZB × NZW)F1 mice. A beneficial effect could result from the artificial activation of iNKT cells in young (NZB × NZW)F1 mice as a result of the ability of the iNKT cells in those animals to secrete abnormally high amounts of IL-4, a cytokine known to prevent disease in some forms of lupus, combined with relatively normal IFN-γ production (52). The remarkable increase in older (NZB × NZW)F1 mice in the production of IFN-γ, a cytokine known to play an important role in the pathogenesis of tissue injury, might then account for the previously reported deleterious effect of αGalCer injection in adult animals (48).

Hereditary lupus in female (NZB × NZW)F1 mice is characterized by lethal immune complex glomerulonephritis attributed to pathogenic autoantibodies (53). Consistent with previously published results (19), we showed that artificial activation of iNKT strongly augmented autoantibody secretion by autoreactive B cells of (NZB × NZW)F1 mice in vitro, suggesting a helper function of iNKT cells. We do not yet know whether this helper function has a pathogenic role in SLE in (NZB × NZW)F1 mice. Given that the production of IgG anti-dsDNA, the autoantibodies most frequently linked to renal disease in this model of SLE (53), was not induced by activated iNKT cells, it seems unlikely these T cells alone are sufficient to drive the excessive autoantibody production that occurs in these animals. We also did not observe the production of increased levels of polyclonal IgG2a, an isotype that has been associated with severe renal disease in the (NZB × NZW)F1 model, when activated iNKT cells were cultured with purified B cells (53). These results, although limited to in vitro culture experiments at this point, suggest a model in which the expanded and hyperactivated iNKT cells synergize with other autoreactive T cell populations to drive pathogenic autoantibody production in SLE.

In conclusion, this study provides a clear example of an autoimmune mouse model in which iNKT cells are naturally expanded and activated in vivo, possibly as a result of recognition of a CD1d-presented autoantigen that has not yet been identified. Although the status of iNKT cells undergoes a dramatic evolution during aging and disease onset in (NZB × NZW)F1 mice, it is still not clear whether expansion and activation of iNKT cells are the consequences of SLE or two of the primary factors leading to its development. However, the features of iNKT cells in this model suggest that iNKT cells may have a complex and variable role in SLE, with a potential protective role before disease onset, followed by a potential pathogenic role once the disease is established. This study raises the possibility that a switch in the immunoregulatory function of iNKT cells occurs in SLE as a result of age, progression of disease, and immune activation in vivo and points to the need for careful assessment before introducing modulators of this T cell subset as a therapeutic approach.

We thank T. P. DiLorenzo for helpful comments. We appreciate the assistance of Karl Yu with the statistical analysis.

The authors have no financial conflict of interest.

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 AI51392 (program project grant awarded to B.D., A.D., and S.A.P.), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Grant AI45889 (awarded to S.A.P.), and Medical Research Council Grants G990177 and G0000895 (awarded to G.S.B.). S.A.P, was supported by the Irene Diamond Foundation and is the recipient of a career Scientist Award from the Irena T. Hirschl Trust. G.S.B. is a Lister Jenner Research Fellow. C.F. was supported by an award from the Human Frontier Science Program.

3

Abbreviations used in this paper: iNKT, invariant NK T cell; SLE, systemic lupus erythematosus; NZB, New Zealand Black; NZW, New Zealand White; αGalCer, α-galactosylceramide.

1
Kronenberg, M., L. Gapin.
2002
. The unconventional lifestyle of NKT cells.
Nat. Rev. Immunol.
2
:
557
-568.
2
Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, et al
1997
. CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides.
Science
278
:
1626
-1629.
3
Chen, H., W. E. Paul.
1997
. Cultured NK1.1+ CD4+ T cells produce large amounts of IL-4 and IFN-γ upon activation by anti-CD3 or CD1.
J. Immunol.
159
:
2240
-2249.
4
Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, A. Bendelac.
1999
. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells.
J. Immunol.
163
:
4647
-4650.
5
Mars, L. T., J. Novak, R. S. Liblau, A. Lehuen.
2004
. Therapeutic manipulation of iNKT cells in autoimmunity: modes of action and potential risks.
Trends Immunol.
25
:
471
-476.
6
van der Vliet, H. J., B. M. von Blomberg, N. Nishi, M. Reijm, A. E. Voskuyl, A. A. van Bodegraven, C. H. Polman, T. Rustemeyer, P. Lips, A. J. van den Eertwegh, et al
2001
. Circulating V(α24+) Vβ11+ NKT cell numbers are decreased in a wide variety of diseases that are characterized by autoreactive tissue damage.
Clin. Immunol.
100
:
144
-148.
7
Wilson, S. B., S. C. Kent, H. F. Horton, A. A. Hill, P. L. Bollyky, D. A. Hafler, J. L. Strominger, M. C. Byrne.
2000
. Multiple differences in gene expression in regulatory Vα 24Jα Q T cells from identical twins discordant for type I diabetes.
Proc. Natl. Acad. Sci. USA
97
:
7411
-7416.
8
Sumida, T., A. Sakamoto, H. Murata, Y. Makino, H. Takahashi, S. Yoshida, K. Nishioka, I. Iwamoto, M. Taniguchi.
1995
. Selective reduction of T cells bearing invariant Vα24 JαQ antigen receptor in patients with systemic sclerosis.
J. Exp. Med.
182
:
1163
-1168.
9
Kojo, S., Y. Adachi, H. Keino, M. Taniguchi, T. Sumida.
2001
. Dysfunction of T cell receptor AV24AJ18+, BV11+ double-negative regulatory natural killer T cells in autoimmune diseases.
Arthritis Rheum.
44
:
1127
-1138.
10
Illes, Z., T. Kondo, J. Newcombe, N. Oka, T. Tabira, T. Yamamura.
2000
. Differential expression of NK T cell Vα24 JαQ invariant TCR chain in the lesions of multiple sclerosis and chronic inflammatory demyelinating polyneuropathy.
J. Immunol.
164
:
4375
-4381.
11
Baxter, A. G., S. J. Kinder, K. J. Hammond, R. Scollay, D. I. Godfrey.
1997
. Association between αβTCR+CD4CD8 T-cell deficiency and IDDM in NOD/Lt mice.
Diabetes
46
:
572
-582.
12
Frey, A. B., T. D. Rao.
1999
. NKT cell cytokine imbalance in murine diabetes mellitus.
Autoimmunity
29
:
201
-214.
13
Yang, Y., M. Bao, J. W. Yoon.
2001
. Intrinsic defects in the T-cell lineage results in natural killer T-cell deficiency and the development of diabetes in the nonobese diabetic mouse.
Diabetes
50
:
2691
-2699.
14
Sharif, S., G. A. Arreaza, P. Zucker, Q. S. Mi, J. Sondhi, O. V. Naidenko, M. Kronenberg, Y. Koezuka, T. L. Delovitch, J. M. Gombert, et al
2001
. Activation of natural killer T cells by α-galactosylceramide treatment prevents the onset and recurrence of autoimmune type 1 diabetes.
Nat. Med.
7
:
1057
-1062.
15
Hong, S., M. T. Wilson, I. Serizawa, L. Wu, N. Singh, O. V. Naidenko, T. Miura, T. Haba, D. C. Scherer, J. Wei, et al
2001
. The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice.
Nat. Med.
7
:
1052
-1056.
16
Singh, A. K., M. T. Wilson, S. Hong, D. Olivares-Villagomez, C. Du, A. K. Stanic, S. Joyce, S. Sriram, Y. Koezuka, L. Van Kaer.
2001
. Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis.
J. Exp. Med.
194
:
1801
-1811.
17
Lehuen, A., O. Lantz, L. Beaudoin, V. Laloux, C. Carnaud, A. Bendelac, J. F. Bach, R. C. Monteiro.
1998
. Overexpression of natural killer T cells protects Vα14-Jα281 transgenic nonobese diabetic mice against diabetes.
J. Exp. Med.
188
:
1831
-1839.
18
Yang, J. Q., V. Saxena, H. Xu, L. Van Kaer, C. R. Wang, R. R. Singh.
2003
. Repeated α-galactosylceramide administration results in expansion of NK T cells and alleviates inflammatory dermatitis in MRL-lpr/lpr mice.
J. Immunol.
171
:
4439
-4446.
19
Zeng, D., Y. Liu, S. Sidobre, M. Kronenberg, S. Strober.
2003
. Activation of natural killer T cells in NZB/W mice induces Th1-type immune responses exacerbating lupus.
J. Clin. Invest.
112
:
1211
-1222.
20
Kono, D. H., A. N. Theofilopoulos.
2000
. Genetics of systemic autoimmunity in mouse models of lupus.
Int. Rev. Immunol.
19
:
367
-387.
21
Yu, K. O., J. S. Im, A. Molano, Y. Dutronc, P. A. Illarionov, C. Forestier, N. Fujiwara, I. Arias, S. Miyake, T. Yamamura, et al
2005
. Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of α-galactosylceramides.
Proc. Natl. Acad. Sci. USA
102
:
3383
-3388.
22
Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C. R. Wang, Y. Koezuka, M. Kronenberg.
2000
. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers.
J. Exp. Med.
192
:
741
-754.
23
Katz, J. B., W. Limpanasithikul, B. Diamond.
1994
. Mutational analysis of an autoantibody: differential binding and pathogenicity.
J. Exp. Med.
180
:
925
-932.
24
Marzio, R., J. Mauel, S. Betz-Corradin.
1999
. CD69 and regulation of the immune function.
Immunopharmacol. Immunotoxicol.
21
:
565
-582.
25
Bendelac, A., P. Matzinger, R. A. Seder, W. E. Paul, R. H. Schwartz.
1992
. Activation events during thymic selection.
J. Exp. Med.
175
:
731
-742.
26
Miyamoto, K., S. Miyake, T. Yamamura.
2001
. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells.
Nature
413
:
531
-534.
27
Hahn, B. H..
1998
. Antibodies to DNA.
N. Engl. J. Med.
338
:
1359
-1368.
28
Hammond, K. J., L. D. Poulton, L. J. Palmisano, P. A. Silveira, D. I. Godfrey, A. G. Baxter.
1998
. α/β-T cell receptor (TCR)+CD4CD8 (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10.
J. Exp. Med.
187
:
1047
-1056.
29
Mieza, M. A., T. Itoh, J. Q. Cui, Y. Makino, T. Kawano, K. Tsuchida, T. Koike, T. Shirai, H. Yagita, A. Matsuzawa, et al
1996
. Selective reduction of Vα14+ NK T cells associated with disease development in autoimmune-prone mice.
J. Immunol.
156
:
4035
-4040.
30
Takeda, K., G. Dennert.
1993
. The development of autoimmunity in C57BL/6 lpr mice correlates with the disappearance of natural killer type 1-positive cells: evidence for their suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune symptoms.
J. Exp. Med.
177
:
155
-164.
31
Benlagha, K., A. Weiss, A. Beavis, L. Teyton, A. Bendelac.
2000
. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers.
J. Exp. Med.
191
:
1895
-1903.
32
Berzins, S. P., K. Kyparissoudis, D. G. Pellicci, K. J. Hammond, S. Sidobre, A. Baxter, M. J. Smyth, M. Kronenberg, D. I. Godfrey.
2004
. Systemic NKT cell deficiency in NOD mice is not detected in peripheral blood: implications for human studies.
Immunol. Cell Biol.
82
:
247
-252.
33
Yang, J. Q., A. K. Singh, M. T. Wilson, M. Satoh, A. K. Stanic, J. J. Park, S. Hong, S. D. Gadola, A. Mizutani, S. R. Kakumanu, et al
2003
. Immunoregulatory role of CD1d in the hydrocarbon oil-induced model of lupus nephritis.
J. Immunol.
171
:
2142
-2153.
34
Yang, J. Q., T. Chun, H. Liu, S. Hong, H. Bui, L. Van Kaer, C. R. Wang, R. R. Singh.
2004
. CD1d deficiency exacerbates inflammatory dermatitis in MRL-lpr/lpr mice.
Eur. J. Immunol.
34
:
1723
-1732.
35
Morshed, S. R., K. Mannoor, R. C. Halder, H. Kawamura, M. Bannai, H. Sekikawa, H. Watanabe, T. Abo.
2002
. Tissue-specific expansion of NKT and CD5+ B cells at the onset of autoimmune disease in (NZB×NZW)F1 mice.
Eur. J. Immunol.
32
:
2551
-2561.
36
Zeng, D., M. K. Lee, J. Tung, A. Brendolan, S. Strober.
2000
. Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice.
J. Immunol.
164
:
5000
-5004.
37
Zeng, D., M. Dick, L. Cheng, M. Amano, S. Dejbakhsh-Jones, P. Huie, R. Sibley, S. Strober.
1998
. Subsets of transgenic T cells that recognize CD1 induce or prevent murine lupus: role of cytokines.
J. Exp. Med.
187
:
525
-536.
38
Bendelac, A., M. Bonneville, J. F. Kearney.
2001
. Autoreactivity by design: innate B and T lymphocytes.
Nat. Rev. Immunol.
1
:
177
-186.
39
Kaplan, M. J..
2004
. Apoptosis in systemic lupus erythematosus.
Clin. Immunol.
112
:
210
-218.
40
Mevorach, D..
2004
. The role of death-associated molecular patterns in the pathogenesis of systemic lupus erythematosus.
Rheum Dis. Clin. North Am.
30
:
487
-504.
41
Zhou, D., J. Mattner, C. Cantu, III, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. P. Wu, T. Yamashita, et al
2004
. Lysosomal glycosphingolipid recognition by NKT cells.
Science
306
:
1786
-1789.
42
Zhou, D., C. Cantu, III, Y. Sagiv, N. Schrantz, A. B. Kulkarni, X. Qi, D. J. Mahuran, C. R. Morales, G. A. Grabowski, K. Benlagha, et al
2004
. Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins.
Science
303
:
523
-527.
43
Brigl, M., M. B. Brenner.
2004
. CD1: antigen presentation and T cell function.
Annu. Rev. Immunol.
22
:
817
-890.
44
Benlagha, K., T. Kyin, A. Beavis, L. Teyton, A. Bendelac.
2002
. A thymic precursor to the NK T cell lineage.
Science
296
:
553
-555.
45
Chen, H., H. Huang, W. E. Paul.
1997
. NK1.1+ CD4+ T cells lose NK1.1 expression upon in vitro activation.
J. Immunol.
158
:
5112
-5119.
46
Gadue, P., P. L. Stein.
2002
. NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation.
J. Immunol.
169
:
2397
-2406.
47
Pellicci, D. G., K. J. Hammond, A. P. Uldrich, A. G. Baxter, M. J. Smyth, D. I. Godfrey.
2002
. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1CD4+ CD1d-dependent precursor stage.
J. Exp. Med.
195
:
835
-844.
48
Peng, S. L., J. Moslehi, J. Craft.
1997
. Roles of interferonγ and interleukin-4 in murine lupus.
J. Clin. Invest.
99
:
1936
-1946.
49
Hasegawa, K., T. Hayashi, K. Maeda.
2002
. Promotion of lupus in NZB × NZWF1 mice by plasmids encoding interferon (IFN)γ but not by those encoding interleukin (IL)-4.
J. Comp. Pathol.
127
:
1
-6.
50
Theofilopoulos, A. N., S. Koundouris, D. H. Kono, B. R. Lawson.
2001
. The role of IFN-γ in systemic lupus erythematosus: a challenge to the Th1/Th2 paradigm in autoimmunity.
Arthritis Res.
3
:
136
-141.
51
Jacob, C. O., P. H. van der Meide, H. O. McDevitt.
1987
. In vivo treatment of (NZB × NZW)F1 lupus-like nephritis with monoclonal antibody to γ interferon.
J. Exp. Med.
166
:
798
-803.
52
Santiago, M. L., L. Fossati, C. Jacquet, W. Muller, S. Izui, L. Reininger.
1997
. Interleukin-4 protects against a genetically linked lupus-like autoimmune syndrome.
J. Exp. Med.
185
:
65
-70.
53
Kewalramani, R., A. K. Singh.
2002
. Immunopathogenesis of lupus and lupus nephritis: recent insights.
Curr. Opin. Nephrol. Hypertens.
11
:
273
-277.