Article Figures & Data
Figures
- Figure 1.
Adoptive transfer of Bregs or B cells in EAE mice and spinal cord myelination. A, Disease progression and clinical score in three cohorts of EAE C57BL/6 mice: untreated, treated with Bregs, or B cells (n = 6). Arrows indicate the adoptive transfer of 2 × 106 Bregs or B cells. B, Spinal cord analysis with toluidine blue-stained sections (thoracic and lumbar region) in WT, EAE-untreated, EAE Bregs-treated, and EAE B-cell-treated mice. Arrows indicate regions of demyelination in the spinal cord of EAE-untreated and EAE B-cell-treated animals. C, Quantitative comparison of relative lesions in EAE Bregs or B-cell-treated mice 3.5 weeks after adoptive transfer, EAE mice 13 dpi, and WT animals. From each of the cross-sections in Figure 1A, the total of white matter was outlined. Lesion areas, defined as regions of white matter with demyelination or remyelination, were then traced in the same cross-sections. Data are representative of three independent experiments in vivo with n ≥ 6. Data are mean ± SEM. *p <0.05; ***p ≤ 0.001; ****p ≤ 0.0001, ns, not significant or p > 0.05; compared with the corresponding control, in 3 representative mice (Student's t test). ns, not significant.
- Figure 2.
TEM on spinal cords of WT, EAE-untreated mice, and Bregs or B-cell-treated EAE mice. A, Electron micrograph of spinal cords (3400×), thoracic and lumbar regions of WT mice, untreated EAE mice, Bregs, or B-cell-treated EAE mice in cross-sections. B, Box plots represent g-ratio (diameter of the inner axon:outer diameter of myelinated fiber). Data are representative of two independent experiments in vivo with n ≥ 6. Data are presented as box and whiskers (Min to Max). *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****P ≤ 0.0001; compared with the corresponding control, in 4 representative mice (Student's t test). ns, not significant.
- Figure 3.
Flow cytometry analysis of freshly isolated CNS-infiltrating monocytes/macrophages and resident microglia after Bregs adoptive transfer in EAE mice compared with WT animals. Content and phenotype of (A,B) monocytes/macrophages (CD11b+CD45highLy6c+) and (C,D) microglia (CD11b+CD45intLy6C–) were determined. Data are representative of two independent experiments in vivo with n ≥ 6. Data are mean ± SEM. *p <0.05; **p ≤ 0.01; ***p ≤ 0.001; compared with the corresponding control (Student's t test).
- Figure 4.
Oligodendroglial lineage after adoptive transfer with B cells or Bregs in EAE mice. Mice were killed starting 2 weeks after adoptive cell transfer, and spinal cords were collected for analysis by flow cytometry and Western blotting. Naive mice and untreated EAE mice were used as control. A, Gating strategy: single cells in side and forward scatter, cells, live cells followed by CD45–/low gate. B, Expression of oligodendroglial markers 2 (and 3.5 weeks for Bregs** treated animals) after adoptive transfer with Bregs or B cells compared with WT and EAE-untreated mice: A2B5, CD140, GalC, and O1 in WT mice and EAE C57BL/6 treated either with B cells or Bregs. C, Statistical analysis of the oligodendroglial marker expression in the spinal cords and brains 2 weeks after adoptive transfer. D, Oligodendroglial phenotyping from spinal cords in WT, Bregs, and B-cell-treated mice: A2B5+CD140+, early OPCs, and O1+GALC+ mature oligodendrocytes. E, Western blotting analysis on the expression of MOG, MBP, and paired-related homeobox protein 1 (PRXX1) by spinal cord oligodendrocytes from EAE C57BL/6 mice untreated, treated with Bregs or B cells, and WT mice 2 weeks after adoptive transfer, load control GADPH. Data are representative of three independent experiments in vivo with n ≥ 6. Data are mean ± SEM. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; compared with the corresponding control (Student's t test).
- Figure 5.
Effect of adoptive transfer of Bregs in IL-10−/− EAE mice. A, Disease progression and clinical score in two cohorts of EAE IL-10−/− mice treated with Bregs or B cells. Arrows indicate the adoptive transfer of 2 × 106 Bregs or B cells generated/obtained from IL-10-sufficient C57BL/6 mice. B, Survival curve of IL-10−/− mice following adoptive transfer with Bregs or B cells. Data are representative of two independent experiments in vivo with n = 6. Data are mean ± SEM.
- Figure 6.
Model of repair process induced by Bregs and effect in lymphoid organs and CNS. EAE manifested with an increase in autoreactive T cells and inflammation in lymphoid organs and the CNS causing demyelination and axon loss occur. In lymphoid organs, autoreactive T cells (Th1 and Th17) are induced after MOG immunization, and they then migrate in the CNS. The inflammatory microenvironment of the CNS induced demyelination and loss of oligodendrocytes. Myeloid-derived cells, both resident microglia and blood-derived monocytes/macrophages, showed a proinflammatory phenotype in B-cell-treated mice compared with WT or Bregs-treated animals (left). Adoptive transfer of Bregs can decrease the frequency of autoreactive T cell in the periphery and the CNS with concomitant increased of IL-10-producing T cells, both Tr-1 and canonical Tregs. Moreover, in mice that received Bregs, myeloid-derived cells, both monocytes/macrophages and microglia, displayed a more immunosuppressive phenotype, and their frequency is lowered respect to EAE mice (right). Concomitantly, a maturation/differentiation of OPCs was evident in the spinal cords of mice that received Bregs concurrent with remyelination.
