Supplementary Figure 2: T_reg and T_reg-conditioned media promote brain slice myelination and remyelination

(a) Schematic diagram and 3D image of Foxp3-eGFP⁺ T_reg in organotypic brain slice cultures representing z-stack images at 3 d.i.v. (scale bar = 100 μm, green = eGFP). (b,c) Analysis of (b) MBP⁺ cells per FOV and (c) myelination index (NF200⁺MBP⁺) of brain stem slices with or without T_reg cells at 3 d.i.v., Control: MBP⁺ counts n = 25 FOV and myelination index n = 24 FOV. T_reg: MBP⁺ counts n = 15 and myelination index n = 15 FOV. Fields of view were selected from 3-6 slices/group (MBP⁺ counts: t = 3.058, d.f. = 38, P = 0.0041; myelination index: t = 3.146, d.f. = 37, P = 0.0033, unpaired, two-tailed, Student’s t test). (d) Representative images of brain slices from (b, c) taken from a z-stack image (scale bar = 100 μm, green = NF200, red = MBP). (e) Analysis of myelination index (NF200⁺MBP⁺) of brain stem slices with T_reg or T_conv cells at 3 d.i.v. T_conv n = 15 FOV and T_reg n = 14 FOV from 3-6 slices per group (U = 26, P = 0.0006, two-tailed Mann-Whitney U test). (f) Representative flow cytometric analysis of non-polarized (NP) T cells and T_reg after reactivation for 72h, FSC = Forward Scatter. (g) Analysis of myelination index (NF200⁺MBP⁺) of brain stem slices treated with non-polarized (NP) or Treg-conditioned media, at 7 d.i.v. NP n= 14 FOV and T_reg n = 15 FOV from 3-6 slices per group (t = 4.559, d.f. = 27, P < 0.0001, unpaired two-tailed Student’s t test). (h-j) Electron microscopic analysis of control (non-demyelinated; M) and remyelinating (RM) brain slices with and without T_reg-conditioned medium, n = 3 slices/condition, scale bar = 2 μm (number of myelinated fibres: F(2,6) = 22.89, P = 0.0016; g-ratio: F(2,6) = 5.915, P = 0.0381, one-way ANOVA with Bonferroni post hoc test). Number of myelinated fibres: F = 22.89, Rsquare = 0.8841,C(M) v C(RM) t = 6.592, C(M) v Treg t = 1.978, C(RM) v Treg t = 4.614, P = 0.0016. g-ratio: F = 5.915, R square = 0.6635, C(M) v C(RM) t = 3.344, C(M) v Treg (RM) t = 2.370, p = 0.0381. (k) ELISA of IL-6 in brain slice culture-conditioned media up to day 6 in vitro. n = 4 wells, each from an independent experiment. Data shown are representative of at least 2 (a-e, g) 7 (f) or 1 (h-j) independent experiments. Data presented with mean values indicated, error bars = SEM, *P<0.05, ** P <0.01, *** P <0.001.