Anti-CD20 Depletes Meningeal B Cells but Does Not Halt the Formation of Meningeal Ectopic Lymphoid Tissue

After weaning, 2D2xTh mice received a weekly dose of 100 µg ɑCD20 mAb (n = 18) or isotype control mAb (n = 18) and were observed for 28 days (A + B) 8 of 18 aCD20 mAb-treated mice and 7 of 18 isotype mAb-treated mice developed EAE. One aCD20 mAb-treated mouse died prematurely and had to be excluded from the histologic analysis. (A) Quantitative distribution of mELT along the spinal cord with 12–16 cross-sections per animal analyzed. (B) Representative HE-stained (upper left panel) and B220-stained (lower left panel) sections of the thoracolumbar part of the spinal cord and corresponding close-up views of mELT. Quantification of the mean area of mELT including all sections (upper right panel) and the number of B220⁺ cells in mELT, analyzed in 2 randomly selected sections per animal, for all mice (lower right panel). (C) Analysis of histologic signs of disease in mice without clinically manifest EAE (11 of 18 isotype mAb-treated mice and 10 of 18 aCD20 mAb-treated mice). Representative HE sections given in (a) and (b) for mice with no pathologies, in (c) and (d) for mice with minor cellular infiltrates in the meninges, and in (e) and (f) for mice with mELT. (D) Quantitative analysis of the size and distribution of mELT along the spine in mice without clinically manifest EAE. In total, 12–16 sections analyzed per animal. Scale bars: 250 µm (overviews) and 100 µm (close-ups). When not stated differently, data shown as mean ±95% CI. *p < 0.05; ****p < 0.0001; statistical significance between groups was analyzed using the Student t test. ɑCD20 mAb = anti-CD20 monoclonal antibody; EAE = experimental autoimmune encephalomyelitis; HE = hematoxylin & eosin; mELT = meningeal ectopic lymphoid tissue; n/N = mice showing mELT or noted feature/total number of mice analyzed; ns = not significant.

(A) Experimental setup. As soon as 2D2xTh mice developed a score of ≥3, they received a weekly dose of 100 µg of ɑCD20 (n = 11) or isotype control mAb (n = 14) over a period of 28 days. Another 5 mice were euthanized and dissected directly after developing a clinical score of ≥3, without receiving treatment. They served as histologic reference at disease onset for the remainder of the mice for which treatment commenced at that stage. (B) Clinical course of EAE during application of ɑCD20 or isotype mAb. Data expressed as mean ± SEM. (C) Representative HE (upper left panel) and B220 (lower left panel) staining of the spinal cord mELT of mice at EAE onset and respective quantification in all mice (right panels). (D) Representative HE-stained cross-sections of the thoracolumbar part of the spinal cord of ɑCD20 mAb-treated and isotype-treated mice (left) and representation of the distribution of mELT along the spine with 14–16 sections analyzed per animal (right). (E) Quantification of the mean area of mELT per section. (F) Correlation of the mean area of mELT per section with the mean EAE score over the observation period. Simple linear regression was calculated for each group. Scale bars, 250 µm. When not indicated differently, data shown as mean ± 95% CI. Statistical significance between groups was analyzed using the Student t test. ɑCD20 mAb = anti-CD20 monoclonal antibody; EAE = experimental autoimmune encephalomyelitis; HE = hematoxylin & eosin; i.p. = intraperitoneally; mELT = meningeal ectopic lymphoid tissue; n/N = mice showing mELT/total number of mice analyzed; ns = not significant.

2D2xTh mice received 4 doses of ɑCD20 mAb (n = 11) or isotype control (n = 10) as soon as they reached an EAE score of ≥3. (A) Antimurine MOG IgG serum levels determined by ELISA (dilution 1:300). Data are shown as individual data points and mean ± 95% CI. (B) LFB-PAS staining of the spinal cord, showing representative thoracolumbar cross-sections and corresponding close-ups. For each mouse, 2 randomly chosen cross-sections per spinal cord segment—cervicothoracic (n = 5 for ɑCD20 mAb and n = 6 for isotype), thoracolumbar (n = 8 per group), and lumbar (n = 7 for ɑCD20 mAb and n = 6 for isotype)—were examined. Data are presented as minimum and maximum values and mean. Scale bars: 250 µm and 100 µm (close-ups). Statistical significance between groups was analyzed using the Student t test. ɑCD20 mAb = anti-CD20 monoclonal antibody; EAE = experimental autoimmune encephalomyelitis; LFB-PAS = Luxol fast blue and periodic acid-Schiff reaction; MOG = myelin oligodendrocyte glycoprotein; ns = not significant; OD = optical density.

2D2xTh mice received 4 doses of ɑCD20 mAb (n = 11) or isotype control (n = 10) as soon as they reached an EAE score of ≥3. (A) HE staining of representative spinal cord sections of (a) isotype-treated and (b) ɑCD20 mAb-treated mice visualizing the cellular density of mELT (c) and (d) show representative samples of the computer-aided quantification of nuclei in mELT with the corresponding graphs. (B) Quantification of B cells (B220), T cells (CD3), and neutrophil granulocytes (MPO) in spinal cord mELT. mELT of 1–4 randomly chosen cross-sections per mouse were analyzed. Scale bars: 50 µm (A) and 100 µm (B). Data are presented as individual data points and mean ± 95% CI. *p ≤ 0.05; **p < 0.01; ****p < 0.0001; statistical significance between groups was analyzed using the Student t test. ɑCD20 mAb = anti-CD20 monoclonal antibody; EAE = experimental autoimmune encephalomyelitis; HE = hematoxylin & eosin; mELT = meningeal ectopic lymphoid tissue; MPO = myeloperoxidase; ns = not significant.