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

Abstract

Objective: To investigate whether anti-CD20 B-cell-depleting monoclonal antibodies (ɑCD20 mAbs) inhibit the formation or retention of meningeal ectopic lymphoid tissue (mELT) in a murine model of multiple sclerosis (MS).

Methods: We used a spontaneous chronic experimental autoimmune encephalomyelitis (EAE) model of mice with mutant T-cell and B-cell receptors specific for myelin oligodendrocyte glycoprotein (MOG), which develop meningeal inflammatory infiltrates resembling those described in MS. ɑCD20 mAbs were administered in either a preventive or a treatment regimen. The extent and cellular composition of mELT was assessed by histology and immunohistochemistry.

Results: ɑCD20 mAb, applied in a paradigm to either prevent or treat EAE, did not alter the disease course in either condition. However, ɑCD20 mAb depleted virtually all B cells from the meningeal compartment but failed to prevent the formation of mELT altogether. Because of the absence of B cells, mELT was less densely populated with immune cells and the cellular composition was changed, with increased neutrophil granulocytes.

Conclusions: These results demonstrate that, in CNS autoimmune disease, meningeal inflammatory infiltrates may form and persist in the absence of B cells. Together with the finding that ɑCD20 mAb does not ameliorate spontaneous chronic EAE with mELT, our data suggest that mELT may have yet unknown capacities that are independent of B cells and contribute to CNS autoimmunity.

Figure 1. Spontaneous EAE Occurs in the Absence of Peripheral B Cells in Mice Preventatively Treated With ɑCD20 mAb

(A) Experimental setup. Mice were treated with a weekly dose of 100 μg of ɑCD20 mAb (n = 18) or isotype control (n = 18) after weaning. (B) Flow cytometric analysis of B220+ and CD3⁺ cells in blood (B.a) and lymph nodes (B.b) after 4 weeks of treatment. The representative flow cytometry contour plot (left) and quantification (right). (C) Daily evaluated EAE scores over the experimental period. Data are shown as mean ± SEM. (D) Incidence of mice developing clinical signs of disease. 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; i.p. = intraperitoneally; n/N = mice with EAE/all mice observed.

Figure 2. mELT Formation Occurs in the Absence of B Cells in Mice Preventatively Treated With ɑCD20 mAb

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.

Figure 3. ɑCD20 mAb Applied After EAE Onset Does Not Change the Clinical Course of Spontaneous EAE or the Size of mELT

(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.

Figure 4. B-Cell-Depleted Spontaneous Chronic EAE Mice Exhibit High Titers of Anti-MOG Antibodies and Extensive Demyelination, Reflecting Their Severe Disease Course

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.

Figure 5. ɑCD20 mAb B-Cell Depletion in Spontaneous Chronic EAE Alters Cellular Density and Composition of mELT

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.

Figure 6. ɑCD20 mAb Efficiently Depletes B Cells in SLOs

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. Quantification of B cells (B220), T cells (CD3), and neutrophil granulocytes (MPO) in spleens and inguinal lymph nodes. One randomly chosen cross-section per organ (n = 5 mice per group) was evaluated. Scale bars: 250 µm. Data are presented as individual data points and 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; LN = lymph node; MPO = myeloperoxidase; ns = not significant; SLO = secondary lymphoid organ; SPL = spleen.