Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis

Abstract

Natural killer (NK) T cells recognize lipid antigens in the context of the major histocompatibility complex (MHC) class 1-like molecule CD1 and rapidly secrete large amounts of the cytokines interferon (IFN)-gamma and interleukin (IL)-4 upon T cell receptor (TCR) engagement. We have asked whether NK T cell activation influences adaptive T cell responses to myelin antigens and their ability to cause experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. While simultaneous activation of NK T cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) and myelin-reactive T cells potentiates EAE in B10.PL mice, prior activation of NK T cells protects against disease. Exacerbation of EAE is mediated by an enhanced T helper type 1 (Th1) response to myelin basic protein and is lost in mice deficient in IFN-gamma. Protection is mediated by immune deviation of the anti-myelin basic protein (MBP) response and is dependent upon the secretion of IL-4. The modulatory effect of alpha-GalCer requires the CD1d antigen presentation pathway and is dependent upon the nature of the NK T cell response in B10.PL or C57BL/6 mice. Because CD1 molecules are nonpolymorphic and remarkably conserved among different species, modulation of NK T cell activation represents a target for intervention in T cell-mediated autoimmune diseases.

Figure 1.

Flow cytometry profiles of the NK T cell population in B10.PL mice immunized either with PBS or α-GalCer. Mononuclear cells from the livers and spleens of mice immunized with PBS or with α-GalCer were stained with PE-labeled mCD1d/α-GalCer tetramers or with PE-labeled anti-NK1.1 antibodies in combination with FITC-labeled pan anti-TCR Vβ mAb. Numbers indicate the percentage of NK.1.1⁺TCR Vβ⁺ or tetramer⁺TCR Vβ⁺ cells in individual mice. These data are representative of two independent experiments.

Figure 2.

In vitro response of splenocytes to α-GalCer. (A) Splenocytes from naive B10.PL, C57BL/6, and C57BL/6.CD1^−/− mice were cultured with graded concentrations of α-GalCer for measurement of proliferation and cytokine secretion. Proliferative response and IFN-γ secretion is shown in individual mice. A representative of three experiments is shown. (B) Groups of B10.PL mice (two in each group) were immunized either with α-GalCer or with vehicle, and proliferation in response to an in vitro recall with 100 ng of α-GalCer was determined 3, 5, or 10 d later. Proliferative responses in individual mice are shown. These data are representative of two independent experiments.

Figure 2.

In vitro response of splenocytes to α-GalCer. (A) Splenocytes from naive B10.PL, C57BL/6, and C57BL/6.CD1^−/− mice were cultured with graded concentrations of α-GalCer for measurement of proliferation and cytokine secretion. Proliferative response and IFN-γ secretion is shown in individual mice. A representative of three experiments is shown. (B) Groups of B10.PL mice (two in each group) were immunized either with α-GalCer or with vehicle, and proliferation in response to an in vitro recall with 100 ng of α-GalCer was determined 3, 5, or 10 d later. Proliferative responses in individual mice are shown. These data are representative of two independent experiments.

Figure 3.

Coimmunization of α-GalCer with MBP promotes antigen-specific Th1 responses in B10.PL mice. Groups of B10.PL mice (two in each) were immunized either with α-GalCer or with PBS/vehicle at the time of challenge with MBP emulsified in CFA. 9 d later lymph node cells were cultured with graded concentrations of MBPAc1–9. Proliferation (A) and IFN-γ secretion (B) are shown. IL-4 production was below the level of detection, and response to PPD was similar in both groups of mice. These data are representative of two independent experiments.

Figure 4.

Coimmunization of α-GalCer with MBP exacerbates EAE in B10.PL mice. EAE was induced following the coimmunization protocol as described in Materials and Methods. The mean clinical disease course of mice (four in each group) in both vehicle and α-GalCer–immunized groups is shown. These data are representative of two independent experiments. A summary of all independent experiments is provided in Table II.

Figure 5.

Preimmunization with α-GalCer protects B10.PL mice from EAE. EAE was induced following the preimmunization protocol as described in Materials and Methods. Groups of mice (four in each) were immunized either once (A) or three times (B) with α-GalCer, and 1 wk after the last immunization, they were immunized with MBP/CFA/PT for the induction of EAE. A summary of all independent experiments is given in Table II.

Figure 6.

Preimmunization with α-GalCer decreases IFN-γ production and increases IL-4 production by MBPAc1–9–reactive T cells in B10.PL mice. Groups of B10.PL mice (two in each) were immunized either with α-GalCer or with PBS three times, 1 wk apart, 1 wk before challenge with MBPAc1–9 emulsified in CFA. 9 d later lymph node cells were cultured with Ac1–9 and supernatants were collected 48 h later for cytokine secretion. These data are representative of three experiments.

Figure 7.

IFN-γ is required for the α-GalCer-mediated potentiation of EAE in B10.PL mice. Groups of B10.PL.IFN-γ^2/− (four in each) were coimmunized for EAE with MBPAc1–9 and α-GalCer as described in Materials and Methods. Mean clinical disease scores in both vehicle and α-GalCer immunized groups are shown. These data are representative of two independent experiments.

Figure 8.

Potentiation and protection from EAE by α-GalCer in B10.PL mice are dependent upon the CD1d antigen presentation pathway. Groups of B10.PL.CD^−/− mice (six in each) were injected with α-GalCer and MBP following the coimmunization (A) or prior-immunization (B) protocol as described in Materials and Methods. The mean clinical disease scores in each group are shown.

Figure 9.

Coimmunization of α-GalCer with MOG35–55 prevents EAE in C57BL/6 mice. Groups of BL/6 mice (four in each) were immunized with MOG_35–55 peptide for EAE induction following the coimmunization protocol with α-GalCer. The mean clinical disease scores for both vehicle and α-GalCer immunized groups are shown. Summary of all experiments is provided in Table III. Monocytes from the liver and spleens of each PBS or α-GalCer immunized mouse were stained and analyzed by flow cytometry as described in Materials and Methods. These data are representative of two independent experiments.