Characterization of myelin oligodendrocyte glycoprotein (MOG)35-55-specific CD8+ T cells in experimental autoimmune encephalomyelitis

Abstract

Background: The pathogenesis of multiple sclerosis (MS) is mediated primarily by T cells, but most studies of MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have focused on CD4 T cells. The aims of the current study were to determine the pathological interrelationship between CD4 and CD8 autoreactive T cells in MS/EAE.

Methods: Female C57BL/6 mice (n = 20) were induced by myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. At 14 days after immunization, T cells were isolated from the spleen and purified as CD4 and CD8 T cells by using CD4 and CD8 isolation kits, and then the purity was determined by flow cytometric analysis. These cells were stimulated by MOG35-55 peptide and applied to proliferation assays. The interferon-gamma (IFN-γ) and interleukin (IL)-4 secretion of supernatant of cultured CD4 and CD8 T cells were measured by enzyme-linked immunosorbent assays (ELISA). For adoptive transfer, recipient mice were injected with MOG35-55-specific CD8 or CD4 T cells. EAE clinical course was measured by EAE score at 0-5 scale and spinal cord was examined by staining with hematoxylin and eosin and Luxol fast blue staining.

Results: CD8CD3 and CD4CD3 cells were 86% and 94% pure of total CD3 cells after CD8/CD4 bead enrichment, respectively. These cells were stimulated by MOG35-55 peptide and applied to proliferation assays. Although the CD8 T cells had a generally lower response to MOG35-55 than CD4 T cells, the response of CD8 T cells was not always dependent on CD4. CD8 T cell secreted less IFN-γ and IL-4 compared with CD4 T cells. EAE was induced in wildtype B6 naïve mice by adoptive transfer of MOG35-55-specific T cells from B6 active-induced EAE (aEAE) mice. A similar EAE score and slight inflammation and demyelination were found in naive B6 mice after transferring of CD8 T cells from immunized B6 mice compared with transfer of CD4 T cells.

Conclusion: Our data suggest that CD8 autoreactive T cells in EAE have a lower encephalitogenic function but are unique and independent on pathogenic of EAE rather than their CD4 counterparts.

Figure 1

Beads-enriched CD8 T Cells are high purified. T cells were isolated from spleen cells of aEAE mice by passage through a nylon wool column, then purified by CD4 and CD8 isolation kits, finally the purity of the isolated cell fraction was determined by flow cytometric analysis with APC-conjugated anti-CD3 antibody, FITC-conjugated anti-CD4 antibody, and PE-conjugated antibody anti-CD8. aEAE: Activated EAE; APC: Allophycocyanin; FSC: Forward scatter; SSC: Side scatter; EAE: Experimental autoimmune encephalomyelitis; PE: Phycoerythroprotein; FITC: Fluorescein isothiocyanate.

Figure 2

Purified CD8⁺ T Cells are MOG_35–55-specific. To compare of the response of the purified CD4 and CD8 T cell populations to MOG_35–55, CD8 or CD4 enriched T cells from MOG_35–55-immunized wild-type B6 mice were prepared and seeded at 4 × 10⁵ cells/well in 96-well plates and cultured at 37°C for 48 h in a total volume of 200 μL of medium, with or without MOG_35–55, in the presence of mytomycin C-treated syngeneic spleen APCs (1 × 10⁵), and [³H] thymidine incorporation during the last 8 h was assessed. The proliferative response is expressed as the mean counts per minute (cpm) ± SD of triplicate determinations. ^∗P < 0.01. APC: antigen presenting cell; CPM: Counts per minute; MOG: Myelin oligodendrocyte glycoprotein; SD: Standard deviation.

Figure 3

Cytokine profiles of MOG_35–55-specific CD8⁺ T Cells are similar to those of MOG_35–55-specific CD4⁺ T Cells. Cytokine levels in the culture medium of activated CD4⁺ and CD⁺8 T cells at 24 to 48 h poststimulation in vitro are measured by ELISA. Stimulation means that CD4 enriched T cells from MOG_35–55-immunized wild-type B6 mice were prepared and seeded at 8 × 10⁵ cells/well in 24-well plates and cultured at 37°C for 24 to 48 h in a total volume of 500 μL of medium, with or without MOG_35–55, in the presence of mytomycin C-treated syngeneic spleen APCs (2 × 10⁵). The data are the mean ± SD from three separate experiments. ^∗P < 0.01, †P < 0.05. APC: Antigen presenting cell; IFN-γ: Interferon-gamma; IL-4: Interleukin-4; MOG: Myelin oligodendrocyte glycoprotein; SD: Standard deviation.

Figure 4

Adoptive transfer of MOG_35–55-specific CD8 T Cells or CD4 T cells to naïve mice is able to induce tEAE. Attempts were made to induce disease in wild-type B6 naïve mice by adoptive transfer of CD8 MOG_35–55-specific T cells or CD8 MOG_35–55-specific T cells from B6 aEAE mice, and measurement of clinical signs of EAE score was assessed by daily check (A) and histopathology (B-G; H&E, original magnification ×50 [B and E]; LFB, original magnification ×50 [C and F]; LFB, original magnification ×200 [D and G]). The data are the mean±SD from three separate experiments. aEAE: Activated EAE; H&E: Hematoxylin and eosin; LFB: Luxol fast blue; MOG: Myelin oligodendrocyte glycoprotein; SD: Standard deviation; LFB: Luxol fast blue.