Memory cells specific for myelin oligodendrocyte glycoprotein (MOG) govern the transfer of experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the CNS mediated by CD4(+) T cells directed against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin antigens like myelin oligodendrocyte glycoprotein (MOG). We have explored the transfer of EAE using MOG(35-55)-specific TCR transgenic (2D2) T cells. Unsorted 2D2 Th1 cells reliably transferred EAE. Further, we found that CD44(hi)CD62L(lo) effector/memory CD4(+) T cells are likely responsible for the disease transfer due to the up-regulation of CD44. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.

Figure 1

2D2 mice exhibit early onset EAE following immunization with MOG_35-55 compared to WT mice. A, 2D2 and WT mice were immunized with MOG_35-55 in CFA, given pertussis toxin, and monitored for clinical signs of EAE. B and D, Brains and; C and E, spinal cords were harvested 14 d following immunization and stained with H&E. Arrows designate focal areas of mononuclear cell infiltration (WT n = 3; 2D2 n = 5).

Figure 2

Following differentiating culture conditions, 2D2 cells exhibit Th1 and Th17 phenotypes. A, CD4⁺ splenocytes from male 2D2 mice were analyzed by flow cytometry for the presence of CD62L and CD44 prior to culture. Cells were then incubated in the presence of 20 μg/ml MOG_35-55 and 0.5 ng/ml rIL-12 or in Th17 differentiating conditions with 10 μg/ml MOG_35-55, 25 ng/ml IL-6, 2 mg/ml anti-IFN-γ, 1 μg/ml anti-IL-4, and 0.65 μg/ml anti-IL-12. Following a 48 h incubation, B, Th1 and Th17 cells were analyzed for the expression of IL-17 and IFN-γ and C, T-bet; cell populations were gated on CD4⁺ cells. D, Supernatants were also analyzed for the presence of secreted IFN-γ and IL-17; error bars represent SEM. Data are representative of 3 independent experiments.

Figure 3

Th1- but not Th17-differentiated 2D2 cells initiate CNS inflammation and cause EAE following initial stimulation. Male 2D2 splenocytes were cultured in Th1- or Th17-differentiating conditions. After 48 h, 10 × 10⁶ were injected i.p. into naïve male WT recipients. A, Th1 and Th17 cell recipients were monitored for the development of EAE; error bars represent SEM (***p < 0.0001). Brain and spinal cords were taken from transfer recipients 18 d post transfer and stained with H&E. B and E, Brain sections were visualized at 10x and spinal cord sections were visualized at 10x (for panels C and F), and 20x (for panels D and G). Data represent 3 combined experiments (Th1 cell recipients n = 19; Th17 cell recipients n = 11).

Figure 4

Intrinsic memory T cells are required for Th1-differentiated 2D2 cells to transfer EAE following initial stimulation. Unsorted 2D2 T cells, purified naïve (CD4⁺CD62L⁺) cells, and purified memory (CD4⁺CD44⁺) cells were cultured with WT APCs in 24-well plates in the presence of 20 μg/ml MOG_35-55 and 0.5 ng/ml IL-12 for 48 h. A, CD4⁺ cells were then analyzed for the expression of intracellular IL-17, IFN-γ, and B, T-bet. C, Unsorted and naïve T cells (5 × 10⁶) were injected i.p. into WT recipients and monitored for the development of EAE (*p < 0.05) (Unsorted T cell recipients n = 6; Naïve T cell recipients n = 5). D, Unsorted and purified memory cells were analyzed for the expression of intracellular IFN-γ and T-bet and E, cell surface-associated CD44 following culture.

Figure 5

Male 2D2 mice have a greater number of effector/memory T cells resulting in enhanced EAE following adoptive transfer. Unsorted splenocytes from male and female 2D2 mice were cultured in the presence of 20 μg/ml MOG_35-55 and 0.5 ng/ml IL-12 for 48 h. A, Male and B, female 2D2 CD4⁺ splenocytes were analyzed for effector/memory (CD44^hiCD62L^lo), central memory (CD44^hiCD62L^hi), and naïve (CD44^loCD62L^hi) cell populations. C, Central and effector memory populations from male and female 2D2 mice were quantified (*p < 0.05). Plots shown are representative of data collected from 4 2D2 males and 4 2D2 females, from which the quantification in panel C was derived. Cells were washed following culture and 10 × 10⁶ cells were injected i.p. into naïve WT sex-matched recipients. D, Transfer recipients were monitored for the development of EAE. Data represent 3 separate experiments (**p = 0.01) (Female recipients n = 15; Male recipients n = 9). E, Unsorted male and female splenocytes were normalized for the absolute number of memory T cells such that the number of memory cells transferred was the same between males and females. Following culture, 8-10 × 10⁶ 2D2 cells were injected i.p. into naïve WT sex-matched recipients and monitored for EAE (Female recipients n = 5; Male recipients n = 4).