A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis

Abstract

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) characterized by plaques of infiltrating CD4(+) and CD8(+) T cells. Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4(+) myelin-specific T cells. The role of CD8(+) myelin-specific T cells in mediating EAE or MS has not been described previously. Here, we demonstrate that myelin-specific CD8(+) T cells induce severe CNS autoimmunity in mice. The pathology and clinical symptoms in CD8(+) T cell-mediated CNS autoimmunity demonstrate similarities to MS not seen in myelin-specific CD4(+) T cell-mediated EAE. These data suggest that myelin-specific CD8(+) T cells could function as effector cells in the pathogenesis of MS.

Figure 1

MBP-specific CD8⁺ cytotoxic T cells are present in wild-type mice. (A) T cell clones derived from C3H wild-type mice express an α/β⁺ TCR as well as CD8. All MBP79-87 T cell clones isolated from C3H and C3H.shi mice were of the CD8⁺ TCR α/β⁺ lineage. Data is shown for the T cell clone used for disease transfer experiments. (B) MBP-specific CD8⁺ T cells derived from wild-type mice lyse MBP-expressing target cells and (C) target cells coated with MBP79-87 peptide in a similar dose response as T cell clones derived from C3H.shi mice in a standard ⁵¹Cr release assay. An effector to target (E/T) ratio of 10:1 was used for the peptide dose response. Data shown is representative of six independent MBP-specific T cell clones isolated from C3H wild-type mice and 10 T cell clones isolated from C3H.shi mice.

Figure 3

Histologic analysis of brain from control (VAC–CTLs transferred) and clinically affected (MBP–CTLs transferred) mice. All sections were stained with H&E unless otherwise noted. (A) Section of cerebellum from a control mouse. The darkly stained nuclei of the granular layer outline the central white matter. (B) Multiple vessels within the cerebellar white matter of an affected mouse are prominent due to perivascular cuffing. Loss of staining intensity of the surrounding tissue is evident. The arrow designates a vessel shown at a higher magnification in C. (C) Venule with a perivascular cuff composed of lymphocytes and macrophages. The arrow indicates one of several inflammatory cells marginated to the endothelium. (D) Apoptotic granular cell nuclei (arrows) in a zone peripheral to an involved venule similar to those in B. (E) Section of cerebellum from a control mouse stained with LFB. (F) LFB-stained section of an affected mouse demonstrates myelin loss in an area similar to that in B. (G) LFB-stained section of white matter showing loss of myelin around an axon remnant (large arrow) and an apoptotic glial cell nucleus (small arrow). (H) Crystal Violet–stained section demonstrates fragments of nuclear debris (arrows) in a focus of cerebellar white matter adjacent to an affected vessel. (I) Section of normal gray matter from a control mouse. (J) Section of gray matter from an affected mouse showing perivascular cuffing and vacuolation. (K) Immunohistochemical detection of GFAP demonstrates a solitary astrocyte (arrow) adjacent to a vessel in the gray matter of a control mouse. (L) GFAP staining of a serial section to that shown in J demonstrates marked perivascular astrogliosis. Sections are shown at original magnification: 20× (A, B, E, F, I, J, K, and L) or original magnification: 100× (C, D, G, and H). The perivascular lesions shown are highly representative of lesions found in all anatomical compartments in all affected mice.

Figure 2

MBP-specific CTLs isolated from C3H wild-type mice transfer CNS autoimmunity. (A) Mean clinical score of recipient mice after transfer of MBP–CTL or Vaccinia-specific CTLs (VAC–CTL) into C3H, C3H.scid, or C3H.shi recipient mice. (B) MBP–CTLs induced severe weight loss in recipient mice expressing endogenous MBP. The mean clinical score and weight loss is the average of three independent experiments using the same T cell clone. Of two independent T cell clones studied, both induced similar CNS disease. Incidence of disease after transfer of MBP–CTLs into recipient mice was 17/19 in C3H, 12/13 in C3H.scid, 0/15 in C3H.shi, and 0/14 after transfer of VAC–CTLs into C3H.

Figure 2

MBP-specific CTLs isolated from C3H wild-type mice transfer CNS autoimmunity. (A) Mean clinical score of recipient mice after transfer of MBP–CTL or Vaccinia-specific CTLs (VAC–CTL) into C3H, C3H.scid, or C3H.shi recipient mice. (B) MBP–CTLs induced severe weight loss in recipient mice expressing endogenous MBP. The mean clinical score and weight loss is the average of three independent experiments using the same T cell clone. Of two independent T cell clones studied, both induced similar CNS disease. Incidence of disease after transfer of MBP–CTLs into recipient mice was 17/19 in C3H, 12/13 in C3H.scid, 0/15 in C3H.shi, and 0/14 after transfer of VAC–CTLs into C3H.

Figure 4

Mechanism of MBP-specific CTL-mediated autoimmunity. Mean clinical scores are shown for recipient mice after intrathecal transfer of MBP–CTLs. Recipient mice are (A) C3H, C3H.scid, or C3H.shi mice, (B) C3H mice coinjected with anti–IFN-γ or control Ab, (C) C3H recipients coinjected with TNFR:Fc fusion protein or control Ab, and (D) C3H or C3H.lpr mice. These experiments have been repeated twice. Incidence of disease was 35/43 for C3H mice; 14/19 for C3H.scid mice; 0/24 for C3H.shi; 14/18 for C3H plus anti-IFN-γ; 15/17 for C3H plus hamster IgG; 7/9 for C3H plus TNFR:Fc; 7/10 for C3H plus human IgG; and 9/10 for C3H.lpr mice.

Figure 4

Mechanism of MBP-specific CTL-mediated autoimmunity. Mean clinical scores are shown for recipient mice after intrathecal transfer of MBP–CTLs. Recipient mice are (A) C3H, C3H.scid, or C3H.shi mice, (B) C3H mice coinjected with anti–IFN-γ or control Ab, (C) C3H recipients coinjected with TNFR:Fc fusion protein or control Ab, and (D) C3H or C3H.lpr mice. These experiments have been repeated twice. Incidence of disease was 35/43 for C3H mice; 14/19 for C3H.scid mice; 0/24 for C3H.shi; 14/18 for C3H plus anti-IFN-γ; 15/17 for C3H plus hamster IgG; 7/9 for C3H plus TNFR:Fc; 7/10 for C3H plus human IgG; and 9/10 for C3H.lpr mice.