Figure 6. Opn-deficiency in non-T cells delays EAE onset.

(A) EAE development in Rag2^−/− hosts. Naive CD4⁺ 2D2 Tg Opn-deficient T cells (1.4×10⁵ cells/mouse) were adoptively transferred into Opn wt or Opn-deficient B6.Rag2^−/− hosts. Disease development and incidence are shown for each group. (Opn wt, ●, n=9; Opn^−/−, ○, n=8; error bars denote mean + SEM. Data are representative of 3 experiments. (B–E) Analysis of mice at different time-points in separate experiments. (B) CD4⁺ 2D2 T cells from B6 2D2 Tg mice were adoptively transferred to either Opn wt or Opn-deficient B6 Rag2^−/− mice, as described in (A). Twenty days later, the total number of spinal cord-infiltrated cells, spinal cord-infiltrated macrophages (%) (F4/80⁺CD45^hi), and total CD4⁺ T cells were enumerated. Increased lymphocyte infiltration was observed in Opn wt hosts (●) compared with Opn-deficient (○) hosts. Negative control (✳) mice are Rag2^−/− mice without T cell transfer. Average disease scores of the Opn wt and Opn-deficient (Spp1^−/−) hosts used for these experiments (at day 20) were 3.8 and 1.2 for Opn wt and Opn-deficient hosts, respectively. Horizontal lines denote mean values. (C) Serum IL-27 analysis. Rag2^−/− hosts were analyzed when EAE scores were either 0 (day 5) or 3~3.5 (day 12 and day 17 for Opn wt and Opn-deficient hosts, respectively). Error bars represent mean ± S.D. from values of 3 mice (score 0) or 4 mice (score 3~3.5) each of Opn wt and Opn-deficient Rag2^−/− hosts. (D) Th17 development. Cells from DLN were harvested from mice on day 5 (score =0), treated immediately with PMA and ionomycin and incubated with IL-17 or IFNγ Abs and subject to FACS analysis; dot plots were gated on CD4⁺ cells. (E) Th subsets in CNS. Cells from spinal cord or brain were harvested from score 3~3.5 mice on day 12 and 17 from Opn wt and Opn-deficient Rag2^−/− hosts, respectively, treated immediately with PMA and ionomycin and incubated with IL-17 or IFNγ Ab. Panels represent results of 3 mice (score 0) or 4 mice (score 3~3.5) each from Opn wt and Opn-deficient Rag2^−/− hosts.