Invariant NKT Cells Regulate Experimental Autoimmune Encephalomyelitis and Infiltrate the Central Nervous System in a CD1d-Independent Manner

Mice

The transgenic Vα14-Jα18 nonobese diabetic (NOD) mice have already been described (35). Transgenic NOD mice that express CD1d exclusively on DP thymocytes were generated by expressing CD1d under the control of the proximal Lck (pLck) promoter and crossing these mice with CD1d^−/− NOD mice obtained by >15 backcrosses with NOD (6, 36, 37). Five transgenic pLck-CD1d lines were selected and crossed with Vα14-Jα18 transgenic (Tg) NOD mice to generate Vα14-Jα18 Tg pLck-CD1d CD1d^−/− NOD mice (named Vα14 pLck). Two lines, Vα14 pLck436 and Vα14 pLck473, were selected for the present study. All mice used in this study were raised and housed under specific pathogen-free conditions. All experimental protocols were approved by the local ethics committee on animal experimentation and are in compliance with European Union guidelines.

Induction and clinical evaluation of EAE

EAE was induced by the immunization of 6- to 12-wk-old mice s.c. with 100 μg of MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) (Mimotopes) emulsified in CFA (Difco) and supplemented with 5 mg/ml Mycobacterium tuberculosis (strain H37RA) (Difco). Pertussis toxin (List Biological Laboratories) was injected i.v. at day 0 (200 ng) and day 2 (400 ng) postimmunization. Disease severity was scored daily on a 5 point scale: 1, tail atony; 2, hind limb weakness; 3, hind limb paralysis; 4, quadriplegia; 5, moribund or death.

CNS-infiltrating mononuclear cell purification

Brain and spinal cord were dissected from mice perfused transcardially with PBS. Brain tissue was dissociated by passages over three consecutive cell strainers of 150, 100, and 40 μm. Single-cell suspensions were resuspended in 30% Percoll and deposited on 70% Percoll. The interface was collected after a 20 min centrifugation at 3000 rpm and washed twice in medium.

RNA isolation

Isolated cells were resuspended in RNAprotect cell reagent (Qiagen) and kept at −80°C until use. RNA was extracted using a RNeasy Plus mini kit and QIAshredders according to the manufacturer’s instructions (Qiagen). The quality of the RNA was assessed by capillary electrophoresis (Agilent 2100 bioanalyzer, Agilent Technologies) and by spectrometry (λ260/λ280) before analysis. Then, 0.1 microgram of total RNA was converted into a representative cDNA pool using the SuperScript III first-strand synthesis system and random hexamer primers, according to the manufacturer’s instructions (Invitrogen). cDNAs were stored at −80°C until use.

Quantitative PCR primers and gene expression analysis

The following published primer pairs were used: granzyme B (GrB), forward 5′-CGATCAAGGATCAGCAGCCT-3′, reverse 5′-CTTGCTGGGTCTTCTCCTGTTCT-3′; hypoxanthine phosphoribosyltransferase (HPRT), forward 5′-TGACACTGGTAAAACAATGCAAACT-3′, reverse 5′-AACAAAGTCTGGCCTGTATCCAA-3′; IFN-γμ, forward 5′-TCAAGTGGCATAGATGTGGAAGAA-3′, reverse 5′-TGGCTCTGCAGGATTTTCATG-3′; IL-4, forward 5′-ACAGGAGAAGGGACGCCAT-3′, reverse 5′-GAAGCCCTACAGACGAGCTCA-3′; IL-10, forward 5′-GGTTGCCAAGCCTTATCGGA-3′, reverse 5′-CTGTCATCGATTTCTCCC-3′; IL-17, forward 5′-CCACGTCACCCTGGAACTCTC-3′, reverse 5′-CTCCGCATTGACACAGCG-3′ (38, 39, 40). The housekeeping gene HPRT was used as an endogenous control for each sample to normalize for differences in cDNA content or quality. Quantitative PCR was performed using the ABI Prism 7000 sequence detection system (Applied Biosystems). Each reaction was performed in 25 μl volume in a 96-well plate (Applied Biosystems) containing 1 μl of cDNA, 12.5 μl of SYBR Green I master mix 2× (Eurogentec), 30 nM of yeast tRNA (Sigma-Aldrich), and 300 nM each of the forward and reverse primer (Invitrogen). Conditions for the PCR were 2 min at 95°C, and then 40 cycles each consisting of 15 s at 95°C and 1 min at 60–62°C.

The ΔΔCt calculation for the relative transcript quantification was applied as follows: ΔΔCt = (Ct_{target gene} − Ct_HPRT)_x − (Ct_{target gene} − Ct_HPRT)_y, where x is the sample to analyze and y is an arbitrarily chosen control sample. Results for each sample were expressed as fold changes in target gene copies, with the amount of target transcripts = 2^−ΔΔCt. Two independent experiments were conducted for each gene and sample. In each experiment, each sample was tested in duplicate wells.

Flow cytometry

After Fcγ receptor blockade with the 2.4G2 mAb, surface staining was performed with the following mAbs: anti-αβ TCR (H57-597), anti-CD4 (RM4-5), anti-CD11c (HL3), anti-CD11b (Mac 1), anti-CD19 (1D3), anti-CD1d (1B1), Thy1.2 (53-2.1), MHC-II (39-10.8, crossreacts with I-A^g7), and CD45 (30-F11). Biotinylated CD1d:α-GalCer tetramers were prepared as previously described (41). Second-step stainings were performed with streptavidin-allophycocyanin, streptavidin-PerCP, and streptavidin-PE-Cy7. Cells were acquired with a FACSCalibur or FACSAria flow cytometer (BD Biosciences) and analyzed using CellQuest or FlowJo software. For cell sorting, cells were passed through a FACSAria flow cytometer (BD Biosciences) using FACSDiva software.

Analysis of NKT cell function in transgenic lines

To analyze NKT cell function in Vα14 pLck lines (Vα14-Jα18 Tg pLck-CD1d CD1d^−/− NOD mice), splenocytes were cultured in 96-well plates (5 × 10⁵cells/well) in RPMI 1640 containing 10% FCS, 2-ME, and penicillin-streptomycin. Cells were stimulated with Con A (4 μg/ml) (ICN Immunobiologicals) or with α-GalCer (100 ng/ml). When indicated, irradiated splenocytes (10⁵/well) from CD1d^+/+Cα^−/− NOD mice were added. After 48 h of culture, supernatants were harvested and IL-4 and IFN-γ were measured by sandwich ELISA as previously described (35). Recombinant mouse IL-4 and IFN-γ were from R&D Systems.

Assessment of the MOG_35–55 recall response of CD4⁺ T cells in vitro

To analyze the anti-MOG response, CD4⁺ T cells were purified in three steps. First, splenocytes were depleted of macrophages, granulocytes, and CD8⁺ T cells by incubation with the following Abs: anti-CD11b (Mac-1), anti-Gr1 (8C5), and anti-CD8 (Lyt-2) and subsequently with magnetic beads coupled to anti-rat Ig (Dynal Biotech, Invitrogen). Second, B cells were depleted using anti-mouse Ig (Dynal Biotech). Finally, the suspensions were incubated with anti-CD8 (Lyt-2) mAb and anti-CD5 mAb (53-7.3) and sorted with a FACSVantage cell sorter (BD Biosciences) as CD8⁻CD5⁺ cells. The suspension contained 97–99% of CD4⁺ T cells. Purified CD4⁺ splenocytes (1.5 × 10⁵/well) plus irradiated (3000 rad) syngeneic splenocytes (7.5 × 10⁵/well) were stimulated with Con A (4 μg/ml) or MOG_35–55 (100 μg/ml). After 48 h of culture, supernatants were harvested and IFN-γ and IL-4 were measured by ELISA (35). Proliferation was assessed after 48 h of the culture by incorporation of [³H]thymidine (1 μCi/well). In a second set of experiments, the supernatants were tested for 10 independent cytokines (IFN-γ, TNF-α, CXCL10, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17) using LINCOplex multiplex assays acquired on Luminex instrumentation (Millipore).

Statistical analysis

Data were analyzed using the Mann-Whitney U test with the exception of cytokine production, which was compared using the unpaired Student t test, and the kinetics of EAE development, which were compared using the log-rank test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

NKT cells accumulate in the CNS during CNS inflammation

MOG_35–55-immunized NOD mice develop a relapsing-remitting EAE driven by an I-A^g7-restricted CD4⁺ T cell response. We have previously shown that the enrichment of NKT cells in Vα14 Tg mice renders NOD mice resistant to the induction of EAE, despite generating a limited inflammatory infiltrate in the CNS (33). To further characterize this infiltrate, we purified the CNS-infiltrating mononuclear cells 15 days after immunization (Fig. 1⇓), corresponding to the first peak of clinical disability in wild-type NOD mice. At this time point, the Vα14 Tg mice exhibited occasionally a very mild clinical deficit (Fig. 1⇓A). In NOD mice, we observed a CD4⁺ T cell infiltrate (Fig. 1⇓B), representing on average 8.9% of infiltrating mononuclear cells, amounting to 1.1 × 10⁵ cells per inflamed CNS (Fig. 1⇓C). In the protected Vα14 Tg mice, the frequency of infiltrating CD4⁺ T cells was significantly reduced (4.8%), but it still represented on average 0.3 × 10⁵ CD4⁺ T cells per CNS. When assessing the infiltrating NKT cell compartment, we observed a significant increase in the frequency of CD1d:α-GalCer tetramer-positive NKT cells in Vα14 Tg vs NOD mice (3.7 vs 0.4%, respectively, Fig. 1⇓B), corresponding to 12 × 10³ NKT cells in the protected Vα14 Tg CNS and 2 × 10³ NKT cells in the diseased wild-type CNS (Fig. 1⇓, B and D). These data indicate that the composition of the inflammatory infiltrate in the protected CNS of Vα14 Tg mice comprises CD4⁺ T cells and is enriched in NKT cells. This is of interest, as CNS-resident APC populations are known to express CD1d under inflammatory conditions. CD1d has been identified on microglia during inflammatory demyelination in the rat, while in human MS lesions, CD1d expression was found on microglia and reactive astrocytes (24, 42).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

CNS accumulation of CD4⁺ T cells and enrichment in NKT cells in protected Vα14 Tg mice. A, Clinical score of NOD (n = 22) and Vα14 Tg mice (n = 21) 15 days after EAE induction. B, Flow cytometry analysis of CNS-infiltrating mononuclear cells 15 days after immunization. NKT cell frequency (top) was assessed using CD1d:α-GalCer tetramers combined with a TCRβ-specific mAb on Thy1.2-gated T cells. The mean frequency of NKT cells ± SD was calculated from 10 individual experiments containing 2–7 pooled mice/observation. The frequency of infiltrating conventional CD4⁺ T cells (bottom) was defined as the percentage of CD4⁺ CD1d:α-GalCer tetramer-negative cells among total mononuclear cells. The mean frequency of CD4⁺ cells ± SD was calculated from 10 individual experiments containing 2–7 pooled mice/observation. C, Absolute number of CNS-infiltrating CD4⁺ T cells (CD1d:α-GalCer tetramer-negative) per CNS. The average number of CD4⁺ cells ± SEM was calculated from eight individual experiments containing two to seven pooled mice/observation. D, Absolute number of NKT cells (CD1d:α-GalCer tetramer-positive) per CNS. The average number of NKT cells ± SEM was calculated from seven individual experiments containing two to seven pooled mice/observation.

CD1d expression and NKT development in Vα14 pLck mice

To address the role of lipid-Ag presentation in the activation of regulatory NKT cells in EAE, we used transgenic mouse lines that express CD1d under the control of the proximal Lck promoter, which targets gene expression selectively to DP thymocytes. These transgenic pLck-CD1d mice were then crossed twice onto CD1d^−/− mice to eliminate endogenous CD1d alleles, thereby restricting the expression of CD1d on DP thymocytes. This was sufficient to ensure the development and persistence of NKT cells (6). Introduction of the TCR Vα14-Jα18 transgene in these mice lacking extrathymic CD1d allowed the enrichment in NKT cells. Flow cytometric analysis illustrated that both selected transgenic lines (Vα14 pLck436 and 473) express CD1d in the thymus at levels similar to, or higher than, wild-type NOD mice (Fig. 2⇓A). In the Vα14 pLck473 line, the CD1d expression profiles on splenic αβ TCR⁺, CD19⁺, CD11b⁺, and CD11c⁺ cells were identical to those of CD1d^−/− mice (Fig. 2⇓B). In the Vα14 pLck 436 line, the staining for CD1d was similar to that in CD1d^−/− mice on splenocytes of CD19⁺, CD11b⁺, or CD11c⁺ cells, whereas minute CD1d expression was detected on αβ TCR⁺ (Fig. 2⇓B). Next, we analyzed the frequency and functional status of NKT cells in the Vα14 pLck mouse lines. Fig. 2⇓C shows that both lines exhibit a similar enrichment in α-GalCer:CD1d tetramer⁺ αβ TCR⁺ cells in the spleen as do Vα14 Tg NOD mice (7–9.3%), compared with 0.6% in wild-type NOD mice. We next assessed whether NKT cells from Vα14 pLck mice also exhibit the unique IL-4 and IFN-γ cytokine profile upon TCR triggering. To this end, splenocytes from Vα14 pLck mice and CD1d-sufficient Vα14 control mice were restimulated in vitro with α-GalCer. As shown in Fig. 2⇓D, α-GalCer alone failed to stimulate the NKT cells from Vα14 pLck mice, confirming their extrathymic CD1d deficiency. However, upon addition of CD1d^+/+ APCs together with α-GalCer, NKT cells in the Vα14 pLck mice produced IL-4 and IFN-γ in similar quantities as those of CD1d-sufficient Vα14 Tg mice. Consequently, NKT cells from the pLck lines have retained their capacity to recognize and respond to a specific agonist if properly presented. Collectively, these experiments support the observation that thymic CD1d expression is sufficient for the generation of functionally mature NKT cells without affecting their long-term survival in peripheral lymphoid tissues (6).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Thymic CD1d expression suffices for the maturation and persistence of NKT cells in Vα14 pLck mice. Lymphoid organs of 6- to 8-wk-old mice were analyzed by flow cytometry. CD1d expression on (A) thymocytes (top) and splenocytes (bottom) and (B) splenic CD11b⁺ (myeloid cells), CD11c⁺ (DCs), TCRαβ⁺ (T cells), and CD19⁺ (B cells) of the different mouse lines (open histograms) in comparison to NOD mice (filled histograms). Columns from left to right (open histograms): CD1d^−/−, Vα14 Tg, Vα14 pLck436, and Vα14 pLck473; the MFI of each histogram is indicated in the top right-hand corner. Data are representative of three mice per group. To determine NKT cell frequency and function, the spleens of the indicated mice were analyzed at the age of 6–8 wk. C, Flow cytometry analysis depicting the frequency of NKT cells among splenocytes using TCRβ-specific mAbs and CD1d:α-GalCer tetramers. D, Ag-specific activation of NKT cells. Splenocytes (5 × 10⁵/well) were incubated in medium alone (striped bars), Con A (open bars), α-GalCer (gray bars), or α-GalCer plus irradiated CD1d^+/+ APCs (black bars). After 48 h of culture, IFN-γ (left panel) and IL-4 (right panel) release was measured in the culture supernatant by ELISA. Indicated values represent the mean concentration ± SD from three individual mice. Similar data were obtained in two independent experiments.

CD1d expression in the inflamed CNS

To assess the potential for local glycolipid presentation in the CNS, we analyzed the expression of CD1d on CNS-resident APC populations at the first peak of disease, 15 days after immunization. Using a flow cytometry approach, we identified CD45^highCD11c^high DCs, CD45^highCD11b^highCD11c^low macrophages, and CD45^lowCD11b^high microglial cells (Fig. 3⇓A) and measured their expression of CD1d and MHC-II. In wild-type mice, a clear MHC-II expression was found only on DCs (Fig. 3⇓B). The expression pattern of CD1d was much broader, including DCs, macrophages, and some microglial cells (Fig. 3⇓B). APC populations in the protected CNS of Vα14 Tg mice revealed a very similar expression pattern with strong CD1d expression on DCs and macrophages (Mφ) although at a slightly lower intensity (NOD: DCs mean fluorescence intensity (MFI)^CD1d = 285, Mφ MFI^CD1d = 190; Vα14 Tg DCs MFI^CD1d = 195, Mφ MFI^CD1d = 156). To confirm the absence of CD1d expression in the mice lacking extrathymic CD1d expression, we used pLck mice that did not express the Vα14-Jα18 transgene to ensure local CNS inflammation. In the diseased pLck mice, CD1d staining was identical to that seen in CD1d^−/− mice on CNS DCs, Mφ, and T cells (Fig. 3⇓B and data not shown). Therefore, even strong local inflammatory conditions cannot unveil CD1d expression in the pLck mice, emphasizing the validity of this model.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

CNS DCs and macrophages from wild-type NOD and Vα14 Tg mice express CD1d during inflammation, while no CD1d expression is detectable in the pLck mice. CNS mononuclear cells were purified 15 days after EAE induction in 6- to 12-wk-old NOD (n = 3), Vα14 Tg (n = 3), CD1d^−/− (n = 3), and pLck473 mice (n = 2). A flow cytometry analysis was performed using mAbs directed against CD45, CD11c, CD11b, CD1d, MHC-II, and CD4. A, Microglia were identified as CD45^lowCD11b⁺ cells. CD45^high cells comprise multiple subpopulations (CD11b⁺CD11c⁻ macrophages (Mφ), CD11c⁺ DCs), while the CD11b⁻CD11c⁻ subpopulation contains mostly T and B cells. B, MHC-II and CD1d expression was analyzed on DCs, macrophages, and microglia (top to bottom) from CD1d^−/−, NOD, Vα14 Tg, and pLck473 mice (columns from left to right). MHC-II expression is observed on activated DCs in all four genotypes, while CD1d expression is observed on DCs, macrophages, and some microglia only in NOD and Vα14 Tg mice.

NKT cell enrichment prevents EAE even in the absence of extrathymic CD1d

The Vα14 pLck mice permitted us to assess the necessity for Ag presentation in the activation of regulatory NKT cells in the context of EAE. Based on the analogy with conventional T cells, one would expect that the peripheral absence of CD1d would abrogate the regulatory function of NKT cells by preventing their activation. Alternatively, if CD1d is dispensable for the regulatory function of NKT cells, the pLck mice would prove resistant to EAE. To test these hypotheses, we immunized the three groups of mice (NOD, Vα14 Tg, and Vα14 pLck) with MOG_35–55. All immunized NOD mice developed a typical relapsing-remitting disease (Fig. 4⇓A and Table I⇓), whereas Vα14 Tg mice were mostly protected. Vα14 pLck mice also developed a significantly delayed and milder EAE than did NOD mice. To assess whether this inhibition of EAE could be merely ascribed to the expression of the Vα14-Jα18 transgene by conventional T cells and thereby perturb their encephalitogenic potential, we performed control experiments using Vα14 Tg mice on a CD1d-deficient background. These Vα14 Tg CD1d^−/− mice, which express the Vα14-Jα18 TCR transgene but lack NKT cells, developed an EAE indistinguishable from wild-type NOD mice, while their CD1d-expressing Vα14 Tg counterparts were resistant (Fig. 4⇓B). These data demonstrate that the encephalitogenic potential of the αβ T cells in the CD1d-expressing Vα14 Tg mice is unaltered by the transgene expression. These data reinforce the implication of NKT cells in the observed alleviation of EAE and indicate that this regulatory function of NKT cells can be induced in the absence of CD1d-dependent TCR triggering. In fact, given the very similar disease evolution in the Vα14 Tg and Vα14 pLck mice, it appears that NKT cell regulation is as efficacious in a CD1d-sufficient as in a CD1d-deficient environment.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Enrichment of NKT cells prevents EAE irrespective of extrathymic CD1d expression. A, EAE was induced in 6- to 12-wk-old NOD (open circles, n = 13), Vα14 Tg (black circles, n = 9), and Vα14 pLck mice (gray circles, n = 11: for line 436, n = 5; for line 473, n = 6) in two independent experiments. B, EAE was induced in 6- to 12-wk-old NOD (open circles, n = 10), Vα14 Tg CD1d^−/− (filled diamonds, n = 9), and Vα14 Tg CD1d^+/+ mice (filled circles, n = 5) in two independent or a single (Vα14 Tg CD1d^+/+) experiment. The clinical severity of EAE is presented as the mean clinical score over time.

NKT cells suppress the encephalitogenic T cell response in a CD1d-independent manner

We have previously shown that, in the Vα14 Tg mice, the priming of the MOG_35–55-specific T cell response is unaffected in draining lymph nodes but is reduced in the spleen (30), where NKT cells are more frequent (1). To assess the role of CD1d in the NKT cell-mediated regulation, we analyzed the intensity and cytokine profile of the MOG_35–55-specific CD4⁺ T cells in the different mice. CD4⁺ T cells were isolated from the spleen 12 days after immunization and restimulated in vitro in the presence of irradiated I-A^g7+CD1d^−/−TCRCα^−/− splenocytes (Fig. 5⇓). CD4⁺ T cells from the NOD mice proliferated readily to MOG_35–55 restimulation, whereas CD4⁺ T cells from either the Vα14 Tg or Vα14 pLck mice failed to expand in response to MOG_35–55 stimulation, suggesting that the encephalitogenic T cells are either absent or anergized. This process was indiscriminate between Th1 and Th17 responses, as the MOG_35–55-specific production of both IFN-γ (Fig. 5⇓B) and IL-17 (Fig. 5⇓C) was severely impaired. Collectively, these results show a similar inhibition of the encephalitogenic T cell response in Vα14 Tg mice and Vα14 pLck mice, and they argue that CD1d is dispensable in this regulatory process.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

NKT cell enrichment similarly inhibits the encephalitogenic T cell response in the presence or absence of CD1d. The MOG_35–55-specific CD4⁺ T cell response was assessed in the spleens of NOD (n = 3), Vα14 Tg (n = 3), and Vα14 pLck 473 mice (n = 3) 12 days after immunization. Purified CD4⁺ T cells were cocultured with irradiated splenocytes and stimulated with MOG_35–55 (100 μg/ml) or Con A (4 μg/ml). After 48 h of culture (A) the proliferation was measured by thymidine incorporation, or the supernatants were harvested and (B) IFN-γ was measured by ELISA. Data represent the mean proliferation or cytokine concentration ± SD and are representative of two independent experiments. C, IL-17 production was assessed under similar culture conditions by 10-plex Luminex analysis. The pooled data of two independent experiments are presented as the mean cytokine concentration ± SD of NOD (n = 8), Vα14 Tg (n = 7), and Vα14 pLck mice (n = 7). In the same experiments, the drastic reduction in IFN-γ production in the Vα14 Tg and Vα14 pLck mice was confirmed by Luminex analysis.

CNS-infiltrating NKT cells are predominantly double negative and exhibit a cytotoxic profile generated independently of CD1d expression

The Ag specificity of conventional αβ T cells and the presence of their restriction element are essential for their recruitment to or retention within the CNS, as well as their local reactivation (43, 44, 45, 46). To determine whether CD1d Ag presentation exerts a similar selective pressure on CNS-infiltrating NKT cells, we assessed their frequency, subset composition, and effector phenotype in the different mouse lines 15 days after immunization (Fig. 6⇓). Interestingly, the absence of CD1d had no effect on the accumulation of NKT cells in the CNS, as Vα14 pLck mice exhibited a similar NKT cell infiltration (5–6% of αβ T cells) as did CD1d-sufficient Vα14 Tg mice (Fig. 6⇓B). Since previous studies have suggested that various NKT cell subsets (DN vs CD4⁺, or NK1.1⁺ vs NK.1⁻) could be localized in different tissues and exert specialized functions, we have analyzed the phenotype and cytokine production of NKT cells present in the CNS and the spleen (47, 48). In all genotypes, the CNS-infiltrating NKT cells were predominantly of the DN rather than the CD4⁺ subset (Fig. 6⇓A), excluding a role for CD1d in subset selection. To analyze whether the local CNS expression of CD1d influenced the differentiation of CNS-infiltrating NKT cells, we FACS purified NKT cells from the CNS and the spleen (Fig. 6⇓C) to permit mRNA content analysis by real-time PCR. Splenic NKT cells purified from NOD mice produced predominantly IFN-γ, but no detectable IL-4 or IL-10. By contrast, splenic NKT cells from both Vα14 Tg and Vα14 pLck mice produced a similar level of IFN-γ transcripts but also IL-4 and IL-10 (Fig. 6⇓D). In the CNS, rather than a regulatory cytokine profile, the infiltrating NKT cells expressed cytotoxic and inflammatory molecules such as IFN-γ, IL-17, and granzyme B, while IL-4 and IL-10 were undetectable (Fig. 6⇓E). Again, no significant differences were observed between NKT cells from CD1d-sufficient Vα14 Tg mice and CD1d-deficient Vα14 pLck mice. This implies that in the spleen of NKT cell-enriched mice, the attenuation of the encephalitogenic T cell response coincides with the presence of antiinflammatory NKT cells producing IL-4, IL-10, and IFN-γ. In the same mice the protected CNS is enriched in NKT cells producing IFN-γ, IL-17, and granzyme B, indicative of a proinflammatory, cytotoxic phenotype. Lastly, the generation of the splenic antiinflammatory NKT cells, the infiltration and accumulation of NKT cells in the CNS, as well as their phenotype are all independent of the expression of CD1d in these organs.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Cell-surface phenotype and cytokine profile of splenic and CNS-infiltrating NKT cells 15 days after immunization. A, Cell-surface phenotype gated on CD1d:α-GalCer⁺TCRβ⁺ NKT cells, assessing the frequency of CD4⁺ vs DN NKT cells in the spleen (top) and CNS (bottom) in wild-type NOD (n = 8), Vα14 Tg (n = 6), and Vα14 pLck 473 mice (n = 4) 15 days after immunization. The mean percentages of CD4⁺ NKT cells ± SD from three independent experiments are added. B, Frequency of CD1d:α-GalCer⁺TCRβ⁺ NKT cells among CNS (left) and splenic (right) T cells of NOD (open bars), Vα14 Tg (black bars), or Vα14 pLck 473 mice (gray bars) 15 days after immunization. C, Flow cytometry analysis to illustrate the purification procedure of CD1d:α-GalCer⁺TCRβ⁺ NKT cells. D, Real-time PCR analysis of mRNA from purified NKT cells from the spleen of NOD (open bars), Vα14 Tg (black bars), or Vα14 pLck 473 mice (gray bars) 15 days after immunization. E, Real-time PCR analysis of mRNA transcripts from purified CNS-infiltrating NKT cells from NOD (open bars), Vα14 Tg (black bars), or Vα14 pLck 473 mice (gray bars) 15 days after immunization. For D, Three to five individual spleens per group; for E, two groups of three pooled CNS were analyzed for each genotype. Data represent the mean fold increase ± SEM. ND, not detected.