Myelin oligodendrocyte glycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice

Abstract

Multiple sclerosis (MS) is a clinically and pathologically heterogeneous inflammatory/demyelinating disease of the CNS. In the MS variant Devic disease, lesions are predominantly found in the optic nerves and spinal cord but not the brain. The immunological bases of the different forms of MS are unknown. We previously generated myelin oligodendrocyte glycoprotein-specific (MOG-specific) TCR transgenic mice (TCRMOG mice; also referred to as 2D2 mice) and reported that a large proportion of these mice develop spontaneous isolated optic neuritis. We have now crossed the TCRMOG mice with MOG-specific Ig heavy-chain knock-in mice (IgHMOG mice; also referred to as Th mice), in which one-third of the B cells are specific for MOG. In these mice, MOG-specific B cells are very efficient in presenting MOG to the transgenic T cells and undergo class switching to IgG1 in the presence of the transgenic T cells. Sixty percent of TCRMOG x IgHMOG mice spontaneously developed a severe form of experimental autoimmune encephalomyelitis (EAE). Histological examination of the CNS revealed a selective distribution of meningeal and parenchymal inflammatory lesions in the spinal cord and optic nerves. Thus, CNS antigen-specific T and B cells cooperate to induce a distinct clinicopathologic EAE pattern that closely replicates human Devic disease.

Figure 1. MOG-specific B cells enhance antigen presentation to MOG-specific T cells.

(A) Proliferation of splenocytes from TCR^MOG×IgH^MOG, TCR^MOG×IgH^NP, IgH^MOG, IgH^NP, and TCR^OVA×IgH^MOG mice incubated with different doses of MOG aa 35–55 peptide or rMOG protein as antigen. (B) Proliferation of TCR^MOG T cells cultured in the presence of purified MOG (IgH^MOG) or NP-specific (IgH^NP) B cells and increasing doses of antigen. (C and D) Splenocytes from TCR^MOG mice were stimulated in vitro with increasing doses of MOG aa 35–55 peptide or rMOG protein, either alone or in the presence of MOG-specific B lymphocytes (IgH^MOG) or NP-specific B lymphocytes (IgH^NP) as control. (C) Proliferation of MOG-specific T lymphocytes was measured by CFSE labeling of CD4⁺ T cells after 3 days of culture. (D) Cytokine secretion in the culture supernatants was measured by ELISA after 4 days of culture. Data are representative of at least 3 independent experiments.

Figure 2. Proliferation of MOG-specific B lymphocytes upon stimulation with activated T lymphocytes.

(A) IgH^MOG B lymphocytes were cultured in the presence of T cells from TCR^MOG mice. (B) IgH^MOG and IgH^NP B lymphocytes were cultured in the presence of T cells from TCR^MOG or TCR^OVA mice. Cultures were stimulated with increasing doses (0–40 μg/ml) of rMOG protein, MOG aa 35–55 peptide, or OVA aa 323–339 peptide as indicated. Proliferation of B lymphocytes was measured by CFSE labeling of B220⁺ cells after 4 days of culture.

Figure 3. Histopathological analysis of CNS tissues of TCR^MOG ×IgH^MOG mice with Devic-like disease.

(A) Paraffin sections show extensive mononuclear cell infiltrates in meninges and spinal cord (upper panel, Luxol fast blue–H&E stain; original magnification, ×16) and optic neuritis (lower panel, H&E stain; original magnification, ×40) of TCR^MOG×IgH^MOG mice. (B) Cryosections of TCR^MOG×IgH^MOG spinal cord showing T cell (anti-CD4 and anti-Vβ11 staining) and prominent B cell infiltrates in the subarachnoid space. Asterisks indicate the same vessel in each section. Original magnification, ×160. (C) Paraffin sections showing a spinal cord leptomeningeal infiltrate composed primarily of lymphocytes (H&E stain). The adjacent section shows a follicle-like organization pattern (reticulin stain). Original magnification, ×160.

Figure 4. Phenotypic analysis of T and B cells and MOG-specific antibodies in TCR^MOG ×IgH^MOG mice.

(A) Surface staining of CD69 on CD4⁺ T cells in the spleens and lymph nodes of TCR^MOG mice as well as healthy and sick TCR^MOG×IgH^MOG mice. The percentage of CD69⁺ cells in CD4⁺ T cells is indicated. (B) Expression of B7.1 and B7.2 on splenic B cells (B220⁺) from WT and IgH^MOG mice as well as healthy and sick TCR^MOG×IgH^MOG mice. The percentage of B220⁺B7.1⁺ or B220⁺B7.2⁺ cells is indicated. Dot plots in A and B are representative of 3 experiments. (C) MOG-specific antibodies in serum samples from individual mice were measured by ELISA for the different isotypes: IgM, IgG1, IgG2a, IgG2b, and IgG3. Isotype IgG3 was below the detection level in all groups (not shown). Results are expressed as OD; horizontal bars indicate mean OD for each group.

Figure 5. IL-17– and IFN-γ–producing CD4⁺ T cells infiltrate the CNS of TCR^MOG ×IgH^MOG mice with Devic disease.

(A) Mononuclear cells from the brains and spinal cords of WT and IgH^MOG mice as well as healthy and sick TCR^MOG×IgH^MOG mice were isolated by percoll gradient and stained with anti-CD4 antibody. Dot plots show CD4 expression versus side scatter (SSC). The percentage of CD4⁺ T cells present in the gate is indicated. (B) Mononuclear cells isolated from the brains and spinal cords of TCR^MOG×IgH^MOG mice with Devic disease were stimulated with PMA and ionomycin, and the presence of IL-17– and IFN-γ–secreting CD4⁺ T cells was determined by intracellular cytokine staining. The percentage of IL-17– and IFN-γ–positive and –negative cells in CD4⁺ T cells is indicated in each dot plot. (C) RNA was prepared from the spinal cords of TCR^MOG mice with EAE (n = 2) and TCR^MOG×IgH^MOG mice with Devic-like disease (n = 2). Expression of IL-17 and IFN-γ cDNAs was determined by real-time PCR, and results are expressed as relative to β-actin.

Figure 6. MOG mRNA is more abundant in the spinal cord than in the brain.

Brains, spinal cords, and optic nerves were isolated from C57BL/6 mice (n = 18) and pooled to prepare mRNA. Expression of MOG mRNA was determined by real-time PCR and expressed as relative to β-actin.