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. 2017 May;20(5):674-680.
doi: 10.1038/nn.4528. Epub 2017 Mar 13.

Regulatory T Cells Promote Myelin Regeneration in the Central Nervous System

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Free PMC article

Regulatory T Cells Promote Myelin Regeneration in the Central Nervous System

Yvonne Dombrowski et al. Nat Neurosci. .
Free PMC article

Abstract

Regeneration of CNS myelin involves differentiation of oligodendrocytes from oligodendrocyte progenitor cells. In multiple sclerosis, remyelination can fail despite abundant oligodendrocyte progenitor cells, suggesting impairment of oligodendrocyte differentiation. T cells infiltrate the CNS in multiple sclerosis, yet little is known about T cell functions in remyelination. We report that regulatory T cells (Treg) promote oligodendrocyte differentiation and (re)myelination. Treg-deficient mice exhibited substantially impaired remyelination and oligodendrocyte differentiation, which was rescued by adoptive transfer of Treg. In brain slice cultures, Treg accelerated developmental myelination and remyelination, even in the absence of overt inflammation. Treg directly promoted oligodendrocyte progenitor cell differentiation and myelination in vitro. We identified CCN3 as a Treg-derived mediator of oligodendrocyte differentiation and myelination in vitro. These findings reveal a new regenerative function of Treg in the CNS, distinct from immunomodulation. Although the cells were originally named 'Treg' to reflect immunoregulatory roles, this also captures emerging, regenerative Treg functions.

Conflict of interest statement

Competing Financial Interest Statement

The authors have no competing interests as defined by Springer Nature, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Figures

Figure 1
Figure 1. Treg are required for efficient OPC differentiation and remyelination in vivo
(a) Immunohistochemical analysis of CC1+Olig2+ cells per lesion area in spinal cords of Foxp3-DTR and C57BL/6 mice at 14 days post lesion (d.p.l.). n = 6 mice in control and n = 5 mice in Treg-depleted groups (t = 2.703, d.f. = 9, *P = 0.0243; t = 5.624, d.f. = 9, ***P = 0.0003). (b) Representative images of (a) showing demyelination by luxol fast blue staining (scale bar = 200 µm) and CC1+Olig2+ cells in lesions (scale bar = 100 µm, green = Olig2+ cells, red = CC1+ cells, blue = DAPI, right panels = merged images). (c) Lesion size of Foxp3-DTR mice +/- DT at 5 d.p.l. n = 5 mice per group. (t = 1.773, d.f. = 8, P = 0.1142). (d) Olig2+Ki67+ cells per lesion area in spinal cords of Foxp3-DTR mice at 5 d.p.l. n = 5 mice per group. (t = 0.7789, d.f. = 8, P = 0.4584). (e) Electron micrographs showing distribution of remyelinated axons versus unmyelinated axons in spinal cord lesions of control or Treg-depleted mice at 17 d.p.l. Scale bar = 5 µm (top) and 1 µm (bottom). Three mice per group were analyzed (middle panel). Data (right panel) represent mean ± SEM from 109 micrographs from 3 mice per group. Two-tailed Mann-Whitney test. (U = 2, P < 0.0001) (f) CC1+Olig2+ cells per lesion area in spinal cords of DT-treated Foxp3-DTR mice with or without adoptively transferred Treg at 14 d.p.l. n = 15 mice in Treg-depleted, n = 8 mice in Treg-depleted/adoptively transferred Treg group pooled from 2 independent experiments. (t = 2.353, d.f. = 21, P = 0.0285). (g) Representative flow cytometric identification of adoptively transferred Treg in lymph nodes of Treg-injected mice from (f) and controls, gated on CD4+ cells. (h) Immunohistochemical analysis of CC1+Olig2+ cells per area of the corpus callosum at 2 weeks post-cuprizone withdrawal. n = 5 mice/group, data represent analysis of 1-2 regions of corpus callosum per mouse (t = 2.693, d.f. = 8, P = 0.0274). (i) Representative images of (h). Top: Black Gold II myelin stain. Bottom: Olig2+CC1+ cell staining (green = Olig2+ cells, red = CC1+ cells, scale bars = 100 µm). Data shown are representative of 4 (a,b), 2 (c,d,f,g) and 1 (e, h, i) independent biological experiments. Data presented with mean values indicated, error bars = SEM, unpaired two-tailed Student’s t test, unless otherwise indicated above. *p<0.05, ***p<0.001.
Figure 2
Figure 2. Treg directly promote brain tissue myelination and remyelination ex vivo
(a-c) Analysis of (a) MBP+ cells and (b) myelination index per field of view (FOV) in brain stem slices treated with control media or Treg-conditioned media. (c) Representative images taken from z-stacks at 7 d.i.v (scale bar = 100 µm, green = NF200, red = MBP). Control: MBP+ counts n = 17 FOV and myelination index n = 8 FOV. Treg: MBP+ counts n = 8 and myelination index n = 12 FOV. Fields of view were selected from 3-4 slices/group (MBP+ counts: U = 28.5, P = 0.0196, two-tailed Mann-Whitney U test; myelination index: t = 2.886, d.f. = 18, P = 0.0098, unpaired two-tailed Student’s t test). (d-f) Analysis of (d) MBP+ cells and (e) myelination index per FOV in control, demyelinated and remyelinating brain stem slices. (f) Representative images taken from z-stacks (scale bar = 100 µm, green = NF200, red = MBP). Control: MBP+ counts n = 15 FOV and myelination index n = 10 FOV. Demyelination: MBP+ counts n = 6 FOV and myelination index n = 6 FOV. Remyelination control: MBP+ counts n = 5 FOV and myelination index n = 20 FOV. Remyelination+Treg: MBP+ counts n = 26 and myelination index n = 32 FOV. Fields of view were selected from 3-6 slices/group (MBP+ counts: demyelination, U = 0, P < 0.0001, remyelination, U = 0, P < 0.0001, two-tailed Mann Whitney U test; myelination index: demyelination, U = 9, P = 0.0225, two-tailed Mann Whitney U test, remyelination, t = 5.845, d.f. = 50, P = 0.001, unpaired two-tailed Student’s t test). (g-i) Analysis of (g) MBP+ cells and (h) myelination index per FOV in brain stem slices. (i) Representative images taken from z-stacks (scale bar = 100 µm, green = NF200, red = MBP). Control: MBP+ counts n = 11 FOV and myelination index n = 10 FOV. Treg: MBP+ counts n = 13 and myelination index n = 12 FOV. Fields of view were selected from 3-6 slices/group (MBP+ counts: t = 7.537, d.f. = 22, P < 0.0001; myelination index: t = 2.334, d.f. = 20, P = 0.0301, unpaired two-tailed Student’s t tests). Data shown are representative of 6 (a-c) and 2 (d-f, g-i) independent experiments. Data presented with mean values indicated, error bars = SEM, *p<0.05, **p<0.01, ***p<0.001.
Figure 3
Figure 3. Treg directly enhance oligodendrocyte differentiation and myelination in vitro
(a-d) Immunofluorescence analysis of (a) MBP+ cell numbers (t = 8.200, d.f. = 10, P < 0.0001) (b) percentage area (U = 0, P = 0.0022), (c) total Olig2+ cell numbers (t = 0.8294, d.f. = 10, P = 0.4263) and (d) representative images of mixed glial cultures analyzed (scale bar = 100 µm, green = Olig2, red = MBP), n = 6 wells. (e,f) Immunofluorescence analysis of (e) Olig2+Ki67+ cell numbers (t = 1.299, d.f. = 10, P = 0.2230) with (f) representative images of mixed glial cultures (scale bar = 100 µm, green = Olig2, red = Ki67), n = 6 wells. (g,h) Immunofluorescence analysis of (g) MBP+ cell numbers (t = 6.431, d.f. = 22, P < 0.0001) and (h) representative images of pure OPC cultures analyzed (scale bar = 50 µm, red = MBP, blue = DAPI), n = 12 fields of view. (i,j) Immunofluorescence analysis of (i) MBP+CC1+cell numbers (n.d. = not detectable) and (j) representative images of DRG neuron-OPC co-cultures in the presence or absence of Treg-conditioned medium, n = 20 fields of view. Image scale bars = 50 µm and 20 µm (enlarged image), red = MBP, green = CC1, blue = DAPI. Data shown are representative of at least 12 (a,b,d), 3 (c,g-j) and 2 (e,f) independent experiments. Data presented with mean values indicated, error bars = SEM, Unpaired, two-tailed, Student’s t test (cell counts) and Mann-Whitney U tests (percentage area), ** p<0.01, *** p<0.001.
Figure 4
Figure 4. CCN3 is produced by Treg and promotes oligodendrocyte differentiation and myelination
(a) ELISA quantification of CCN3 in T cell-conditioned media, n = 4 independently generated supernatants using different mice (U = 0, P = 0.0294, two-tailed Mann-Whitney U test). (b) Western blot analysis of CCN3 in Treg-conditioned medium. Recombinant CCN3 (rCCN3, 25 ng, R&D Systems) was used as a positive control. (c,d) Immunofluorescence analysis of (c) MBP+ cell numbers (Control vs Treg: t = 3.648, d.f. = 10, P = 0.0045; Treg vs Treg+aCCN3: t = 6.104, d.f. = 10, P = 0.0001, unpaired two-tailed Student t test; Treg vs Treg+IgG: U = 10, P = 0.2207, Mann-Whitney U test) and (d) MBP+ percentage area in mixed glial cultures, n = 6 wells (Control vs Treg: U = 0, P = 0.0022; Treg vs Treg+aCCN3: U = 0, P = 0.0022, Treg vs Treg+IgG: U = 18, P = 1, two-tailed Mann-Whitney U test). (e) Myelination index (MBP+NF200+) of brain stem slices at 7 d.i.v. n = 15 FOV for control, Treg and Treg+anti-CCN3 conditions. n = 14 FOV for Treg+IgG condition. Fields of view were from 3-6 slices/group (control vs Treg: t = 2.739, d.f. = 28, P = 0.0106; Treg vs Treg+anti-CCN3: t = 4.998, d.f. = 28, P < 0.0001, unpaired two-tailed Student’s t test). (f) Immunofluorescence analysis of MBP+ percentage area in mixed glial cultures, n = 6 wells, (Control vs Treg: U = 0, P = 0.0022; Treg vs Treg CCN3-depleted: U = 3, P = 0.0152; Treg CCN3-depleted vs Treg IgG-depleted: U = 5, P = 0.0411, two-tailed Mann-Whitney U test). (g,h) Immunofluorescence analysis of (g) myelination index (MBP+NF200+) and (h) representative images of brain stem slices at 7 d.i.v., n = 20 FOV for Control, Treg, CCN3-depleted and IgG-depleted conditions; n = 15 FOV for CCN3-eluted condition; n = 13 FOV for IgG-eluted condition. Fields of view were from 3-4 slices/group (control vs Treg: t = 5.378, d.f. = 38, P < 0.0001; Treg vs CCN3-depleted: t = 4.159, d.f. = 38, P = 0.0002; CCN3-depleted vs IgG-depleted: t = 4.446, d.f. = 38, P < 0.0001, unpaired two-tailed Student’s t test; CCN3-eluted vs IgG-eluted: U = 43, P = 0.0129, two-tailed Mann-Whitney test). Scale bar = 100 µm, green = NF200, red = MBP. Data shown are representative of at least 4 (a), 2 (b, f-h) and 3 (c-e) independent experiments. Data presented with mean values indicated, error bars = SEM, *p<0.05, **p<0.01, ***p<0.001.

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