Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide

Abstract

Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Valpha14+ NK T cells, and a panel of Valpha3/Valpha8+ CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d+ antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-gamma and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases.

Figure 1.

A distinct CD1d-restricted sulfatide-reactive T cell population in naive mice. (A) Proliferative response of splenocytes from naive BL/6 or BL/6.CD1d^−/− mice in response to an in vitro stimulation with indicated self-lipids (10 μg/ml) or α-GalCer (10 ng/ml) suspended in PBS/vehicle. Each symbol represents response of an individual animal in each group (n = 4–11), with the mean proliferative response indicated by a horizontal bar. *P = 0.004. (B) In vitro cytokine and proliferative response of splenocytes from naive BL/6 (▪) or BL/6.CD1d^−/− (○) mice to a titrated dose of sulfatide. One of three representative experiments is shown. (C) FACS^® analysis of lymphocytes from the liver, spleen, and thymus from BL/6 (CD1d^+/+) or BL/6.CD1d^−/− (CD1d^−/−) mice after staining with fluoresceinated tetramers or anti-NK1.1 mAbs in conjunction with the anti-TCRβ chain antibody. One of three representative experiments is shown. (D) Flow cytometric profile of splenocytes from BL/6 mice stained simultaneously with the CyChrome-labeled α-GalCer/CD1d tetramers and with the PE-labeled sulfatide/CD1d tetramers. (E) Flow cytometric profile of splenocytes from BL/6 and BL/6.Jα18^−/− mice stained with α-GalCer/CD1d tetramers or sulfatide/CD1d tetramers.

Figure 2.

Sulfatide-reactive T cell hyridomas stain with sulfatide/CD1d tetramer and secrete IL-2 in response to sulfatide in a CD1d-dependent manner. A representative T cell hybridoma reactive to sulfatide (Hy19.3) (B and D) and a α-GalCer–reactive hybridoma (Hy1.2) (A and C) were stained with sulfatide/CD1d or α-GalCer/CD1d tetramers and analyzed by FACS^®. Unloaded tetramer or GM1-filled tetramer staining is shown by dotted lines. For IL-2 secretion assay, hybridomas (50,000 cells/well) were incubated with irradiated (3000R) splenocytes (400,000 cells/well) from wild-type C57BL/6 (BL/6) or CD1d^−/− BL/6 (CD1KO) mice in the absence or presence of graded concentrations of α-GalCer or sulfatide, as shown. For blocking of CD1d-dependent presentation, 10 μg/ml of anti-CD1d mAb (1B1 [47]) or an isotype-matched mAb (IgG2b; BD Biosciences) was added during the incubation period. IL-2 secretion levels by Hy19.3 in response to plate-bound anti-CD3 were 1/8–1/10 that of Hy1.2. Concentration of IL-2 in the supernatants is shown. (E) The CD1d-restricted Vα3⁺ (A11, 24, IF4), Vα8⁺ (B11, 49), and Vα4⁺ (68) T cell hybridomas were incubated with irradiated CD1d⁺ splenocytes in the presence of graded concentrations of sulfatide, as above. Concentration of IL-2 in the supernatants is shown. All of the hybridomas responded well to plate-bound anti-CD3.

Figure 3.

Sulfatide/CD1d tetramer⁺ cells are increased severalfold in mice lacking the CGT. Thymocytes pooled from three age-matched, naive BL/6 or BL/6.CGT^−/− mice were stained with α-GalCer/CD1d or sulfatide/CD1d tetramers in conjunction with anti-NK1.1, anti-TCRβ, anti-CD4, or anti-CD8 mAb and analyzed by FACS^®. The CD4 and CD8 staining profiles of thymocytes were similar in both sets of mice. This is representative of two independent experiments.

Figure 4.

Sulfatide-reactive T cells infiltrate the CNS during the course of EAE. (A) Mononuclear cells from the CNS of diseased BL/6 mice (n = 7) were pooled and stained with the indicated lipid/CD1d tetramers and analyzed by FACS^®. A representative of two experiments is shown. (B) To analyze the TCR Vβ gene usage, sulfatide/CD1d tetramer⁺ cells were stained with various anti-Vβ mAbs. A typical positive and negative staining profile is shown. (C) Ex vivo intracytoplasmic staining of the CNS-infiltrating cells for IFN-γ and IL-4. (D) The percentages of α-GalCer– or sulfatide-reactive T cells in spleen, liver, and the CNS of naive animals or mice with EAE were determined by FACS^® analysis. The plot represents the averaged data of three independent experiments (a total of 19 mice in each group). *P < 0.0001.

Figure 5.

Increased frequency of cytokine-secreting, sulfatide-reactive lymphocytes after immunization with sulfatide. Groups of mice (two in each) were immunized in complete Freund's adjuvant with 20 μg of sulfatide or with 20 μg of mono-GM1 or with PBS/vehicle alone. 10 d later, splenocytes were isolated, recalled in vitro with sulfatide, and subjected to ELISPOT analysis as described in Materials and Methods. The average number of spots/10⁶ spleen cells for both IFN-γ and IL-4 in each group is shown.

Figure 6.

Immunization of mice with sulfatide protects mice from EAE. In a representative experiment, groups of female BL/6 or BL6.CD1^−/− mice were immunized i.p. with 20 μg of sulfatide (•) (n = 4) or with PBS/vehicle alone (□) (n = 4) at the time of disease induction with MOG_35–55/CFA/PT. The mean clinical disease course of mice in each group is shown. The summary of all independent experiments is provided in Table I. *P < 0.0001.

Figure 7.

Inhibition of cytokine secretion by MOG-reactive T cells after coinjection with sulfatide. Groups of female BL/6 mice were coinjected i.p. with either 20 μg of sulfatide in PBS/vehicle or PBS/vehicle alone at the time of s.c. challenge with 100 μg MOG_35–55 in CFA. 10 d later, LN cells were cultured with graded concentrations of MOG_35–55 or protein-purified derivative of Mycobacterium (PPD) for the measurement of proliferation and cytokine secretion by ELISA and ELISPOT analysis. Averaged responses within the group are plotted. A representative of three independent experiments is shown. *P-value < 0.004.