NeuroImmunology

We used human brain microvascular ECs (hECs; TY10), hASTs with AQP4 expression, and hPCTs, each of which was conditionally immortalized by transfection with ts-SV40-LT (Figure 1A). These transfected cell lines proliferate at 33°C due to ts-SV40 LT's temperature sensitive inhibition activity of p53 and Rb. On the other hand, cells could differentiate into mature cells at 37°C because Ts-SV40 LT is inactivated and exhibit growth arrest28,33,34 (Figure 1B). In these previous articles, we confirmed the expression markers of hECs, hASTs, and hPCTs (hECs; occludin, Claudin-5, Claudin-12, ZO-1, ZO-2, JAM-A, GLUT-1, CAT-1, LAT-1, 4F2hc, MCT-1, calreticulin, MDR1, ABCG2, MRP1, MRP2, MRP4, and MRP5, and hASTs; glial fibrillary acidic protein, EAAT2, and AQP4, hPCTs; platelet-derived growth factor receptor beta, Desmin, Kir 6.1, osteopontin, alfa smooth muscle actin, and NG2). To construct triple coculture systems of these human BBB cell lines, multiple steps were organized by including cell culture on Upcell dish with coated temperature-responsive polymer (eFigure 1, A and B, links.lww.com/NXI/A591). First, hASTs were cultured on abluminal side of insert membranes having 3-μm pores and incubated for 24 hours so that some astrocytic endfeet with AQP4 could protrude through the membrane pores (eFigure 1C and links.lww.com/NXI/A591). Second, after culturing of hPCTs on the luminal side of membranes (eFigure 1E, links.lww.com/NXI/A591), hASTs and hPCTs were cocultured at 33°C for 24 hours. Third, hECs were cultured on Upcell dish with coated temperature-responsive polymer, which can achieve sheet-like detachment of confluent cells and extracellular matrix by temperature shifting to 20°C (eFigure 1, F and G, links.lww.com/NXI/A591). Then, sheet-like detachment of confluent hECs was transferred onto the hPCTs. After cocultuting of these cell lines at 33°C for 24 hours, they differentiated into mature cells under the condition of 37°C. Confocal 3D analysis with living staining of each cell line showed that multicultured insert constituted the five-layer structures, which is consisted of hECs, hPCTs, astrocytic endfeet, membrane, and hAST (eFigure 1, H, links.lww.com/NXI/A591). Some endfeets protruded through the membrane pores. hPCTs and endfeet of hASTs were close to the hEC layer.

The study was supported by an unrestricted research grant from Merck-Serono.

For many many years we have been fed a diet of MS is an autoimmune disease of T cell reactivity to myelin basic protein.This has been lapped up by researchers and with any dissenting voices their …...

The complement cascade is a key component of the innate immune system that is rapidly
recruited through a cascade of enzymatic reactions to enable the recognition and clearance
of pathogens and promote tissue repair.

Abstract SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the new coronavirus responsible for the pandemic disease in the last year, is able to affect the central nervous system (CNS)....

COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear.Here, we interrogated the brain stems a…...

The immune system is the brain's best frenemy. It protects the brain from infection and helps injured tissues heal, but it also causes autoimmune diseases and creates inflammation that drives neurodegeneration.

New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers.Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometr...

Researchers were first to demonstrate direct evidence of autoreactive immune cells in narcolepsy patients, providing evidence of autoimmune basis...

Extremely invasive, GBMs cannot be completely resected by surgery, and are resistant to radiation and chemotherapy. A subpopulation of cells called glioma stem cells (GSCs), which exhibit NSC properties, are responsible for drug resistance and tumour recurrence.

Abstract Background and Objectives To describe the marked clinical and biological responses of a targeted treatment with anti–interleukin-6 (IL-6)–receptor antibody and Janus kinase (JAK) inhibitors in a patient with a severe, corticoresistant CNS toxicity of immune-checkpoint inhibitor (ICI) therapy. Methods A 58-year-old man was admitted for subacute paraparesis, urinary retention, and ascending paresthesia. He was under treatment with ipilimumab and nivolumab for metastatic melanoma. Spine MRI disclosed multiple T2-hyperintense, contrast-enhancing longitudinally extensive lesions. A diagnosis of ICI-related acute transverse myelitis was made. Results ICIs were immediately discontinued, and the patient received high-dose glucocorticoids plus 1 session of plasma exchange, but he did not improve. Based on the marked elevation of CSF IL-6 (505 pg/mL), a second-line targeted therapy with anti-IL-6-receptor tocilizumab (8 mg/kg/mo for 3 infusions) plus JAK inhibitor ruxolitinib (50 mg/d) was administered. Patient neurologic status started to improve shortly after, with corresponding radiologic resolution. At 9 months, the patient was able to walk independently, presenting only slight residual disability while remaining in oncologic partial response. Discussion Our case suggests that some patients with severe, corticoresistant CNS immune-related toxicities of ICIs may benefit from cytokine blockade. Cytokine measurement in serum and CSF might help in selecting patients for personalized treatment strategies. Neurologic immune-related adverse events (irAEs) of immune-checkpoint inhibitors (ICIs) are uncommon but often severe and potentially life-threatening. High-dose glucocorticoids may be insufficient,1 but optimal treatment in severe and refractory cases remains to be defined. In this study, we report the case of a 58-year-old man who started combined treatment with ipilimumab and nivolumab for BRAF–wild-type metastatic melanoma. After the fourth ICI cycle, the patient presented with rapidly progressing paraparesis, urinary retention, and ascending paresthesia, which peaked in 10 days from onset. Neurologic examination showed severe flaccid paraparesis, a dorsal sensory level (T10) with impaired proprioception, and absent deep tendon reflexes. Spine MRI disclosed multiple T2 hyperintensities at C2, C3, C7-T2, T4-T7, and T8-conus levels with associated patchy contrast enhancement (Figure 1, B and C). CSF analysis displayed increased proteins (2.57 g/L) and cell count (112/mm3) with no malignant cells. Screening for anti-aquaporin-4, anti-myelin oligodendrocyte glycoprotein, anti-glutamic acid decarboxylase, and onconeural antibodies (including anti-Hu, anti-Ri, anti-CV2, and anti-amphiphysin) was negative. Cytokine assessment showed a marked elevation of interleukin-6 (IL-6) in the CSF (505 pg/mL, upper normal limit 4 pg/mL) (Figure 1A). After excluding alternative infectious, vascular, and neoplastic causes, a diagnosis of ICI-related acute transverse myelitis was made. Figure 1 Imaging, Treatment, CSF Monitoring, and Functional Outcome in a Patient With Severe ICI-Related Myelitis (A) Timeline of patient functional status (as expressed by the modified Rankin Scale [mRS], gray curve), IL-6 levels in CSF (orange dots) and serum (blue dots), and administered treatments. The first CSF analysis at day 10 from symptom onset displayed an inflammatory CSF with markedly increased IL-6 level (515 pg/mL) that rapidly decreased after the first administration of tocilizumab and introduction of oral ruxolitinib (8 pg/mL at day 29 and indetectable at day 50). Follow-up CSF analysis at 3 months (day 107) disclosed a slight rise in CSF inflammatory markers (CSF IL-6: 6 pg/mL; CSF proteins: 1.8 g/L; and CSF cells: 17/mm3) that justified a third administration of IV tocilizumab despite the sustained clinical and radiological improvement. (B and C) Lumbosacral spine MRI at diagnosis displayed a longitudinally extensive lesion with spinal swelling from T8 to the conus in T2-weighted sequences (T2w) (B), with an associated faint parenchymal and leptomeningeal enhancement in postgadolinium T1-weighted images (T1w-Gd) (C). (D and E) Lumbosacral spine MRI 1 week after the introduction of tocilizumab and ruxolitinib, showing a marked reduction of the spinal hyperintensities in T2-weighted images (D) and resolution of the contrast enhancement in postgadolinium T1-weighted images (E). ICI = immune-checkpoint inhibitor; IL-6 = interleukin-6; IVMP = intravenous methylprednisolone; PLEX = plasma exchange; Toci = tocilizumab. ICIs were immediately discontinued, and the patient received IV methylprednisolone (3.5 g over 4 days), followed by oral prednisone tapering (from 100 mg/d) and 1 session of plasma exchange to remove residual circulating ICIs, but the patient did not improve. Based on our recent experience of poor responses to high-dose glucocorticoids and plasma exchanges in ICI-related myelitis1 and on the marked IL-6 elevation in the CSF, a second-line treatment with the anti-IL-6-receptor tocilizumab (8 mg/kg IV every 4 weeks) was started, 14 days after symptom onset. To broaden the inhibition of cytokine signaling, the Janus kinase (JAK) inhibitor ruxolitinib (25 mg orally, twice daily) was initiated (Figure 1A). JAKs are crucial enzymes in downstream signaling of type I and type II cytokine receptors including IL-6R, being required for the activation of mitogen-activated protein kinase, phosphatidylinositol-4,5-bisphosphate-3-kinase, and signal transducer and activator of transcription pathways. Ruxolitinib was also added to prevent a possible initial worsening of inflammation due to the expected transient tocilizumab-induced elevation of IL-6 and consequent signal transduction activation.2 Five days after starting second-line treatment, the patient started improving (Video 1), showing a parallel radiologic amelioration (Figure 1, D and E), a resolution of CSF inflammation, and a dramatic decrease of CSF IL-6 levels (Figure 1A). Measurement of drug levels, 2 weeks after starting second-line treatment, confirmed that both compounds were present in the CSF at therapeutic concentrations (tocilizumab: serum 21 μg/mL, CSF 6 μg/mL and ruxolitinib: serum 138 ng/mL, CSF 12.6 ng/mL). At that time, protein levels in the CSF were 0.55 g/dL, reflecting a very mild blood-brain barrier damage. Tocilizumab was continued for a total of 3 infusions, until a steady decrease of IL-6 in the CSF was observed, whereas ruxolitinib was discontinued after 3 weeks because of grade 3 anemia. Video 1 The patient five days after the start of the second-line therapy with tocilizumab plus ruxolitinib, regaining legs movement.Download Supplementary Video 1 via http://dx.doi.org/10.1212/001073_Video_1 At 3 months from myelitis onset, the patient was able to walk with support (Video 2) and constantly improve with physical therapy. At the last follow-up, 9 months from onset, he could walk independently without limitations of the walking perimeter (Video 3), and he was able to resume his work, although he still had mild sensory ataxia and a bladder and bowel dysfunction. Spinal MRI showed a substantial regression of previously reported abnormalities. Oncologic re-evaluation showed persistent partial response with no progression of his metastatic melanoma, and he remained on simple surveillance. Video 2 The patient three months after the start of the second-line therapy, able to walk with aid.Download Supplementary Video 2 via http://dx.doi.org/10.1212/001073_Video_2 Video 3 The patient at last follow up, nine months after the start of the second-line therapy, able to walk independently.Download Supplementary Video 3 via http://dx.doi.org/10.1212/001073_Video_3 The present case, recently included in a series of longitudinally extensive myelitis,1 underscores the value of personalized treatment approaches in severe neurologic irAEs, as recently proposed for other irAEs.3,4 In this case, the marked increase of CSF IL-6 suggested the potential value of IL-6-receptor inhibition. Tocilizumab has recently demonstrated its efficacy in patients with ICI-related cerebritis5 and other non-ICI-related CNS inflammatory conditions, such as neuromyelitis optica spectrum disorder (NMOSD).6 Similarly, ruxolitinib was also effective in a patient with severe NMOSD.7 In this study, we demonstrated that both agents reach therapeutic concentrations in the CNS and might be effectively used to treat severe, corticoresistant neurologic irAEs. Nevertheless, we acknowledge the limitations of single-case reporting; we cannot exclude that glucocorticoids and plasma exchange, or a naturally improving course of the disease itself, could have played a role in clinical and biological improvement. Our report is hypothesis generating, and further studies are needed. Cytokine measurement in serum and CSF might help to select patients that would benefit from cytokine blockade. Data Availability Further anonymized data will be made available upon reasonable request. Study Funding The authors report no targeted funding. Disclosure The authors report no disclosures relevant to the manuscript. Go to Neurology.org/NN for full disclosures. Appendix Authors Footnotes Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. ↵* These authors contributed equally to this work. The Article Processing Charge was funded by the authors. Received February 28, 2021. Accepted in final form June 15, 2021. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. References 1.↵Picca A, Berzero G, Bihan K, et al. Longitudinally extensive myelitis associated with immune checkpoint inhibitors. Neurol Neuroimmunol Neuroinflamm. 2021;8(3):e967. 2.↵Teachey DT, Lacey SF, Shaw PA, et al. Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Cancer Discov. 2016;6(6):664-679. 3.↵Martins F, Sykiotis GP, Maillard M, et al. New therapeutic perspectives to manage refractory immune checkpoint-related toxicities. Lancet Oncol. 2019;20(1):e54-e64.OpenUrlPubMed 4.↵Esfahani K, Elkrief A, Calabrese C, et al. Moving towards personalized treatments of immune-related adverse events. Nat Rev Clin Oncol. 2020;17(8):504-515.OpenUrlPubMed 5.↵Stroud CR, Hegde A, Cherry C, et al. Tocilizumab for the management of immune mediated adverse events secondary to PD-1 blockade. J Oncol Pharm Pract. 2019;25(3):551-557.OpenUrlCrossRefPubMed 6.↵Zhang C, Zhang M, Qiu W, et al. Safety and efficacy of tocilizumab versus azathioprine in highly relapsing neuromyelitis optica spectrum disorder (TANGO): an open-label, multicentre, randomised, phase 2 trial. Lancet Neurol. 2020;19(5):391-401.OpenUrlCrossRef 7.↵Hodecker SC, Stellmann JP, Rosenkranz SC, et al. Ruxolitinib treatment in a patient with neuromyelitis optica: a case report. Neurol Neuroimmunol Neuroinflamm. 2017;4(2):e328.

KEY POINTS Transcription factor TCF4 in DCs protects against autoimmune neuroinflammation. TCF4 regulates p38 MAPK activation and proinflammatory cytokine expression in DCs. TCF4 activation in DCs limits pathological effector T cell differentiation. Abstract Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1β, TNF-α, and IL-12p40. Consistent with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology in TCF4ΔDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation. This article is featured in Top Reads, p.1223 Footnotes This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Awards (DK097271 and DK123360) and Augusta University Awards (IGPB0003 and ESA00041) (to S.M.). The online version of this article contains supplemental material. Abbreviations used in this article DC dendritic cell DLN draining lymph node EAE experimental autoimmune encephalomyelitis ICS intracellular cytokine staining LRP5/6 low density lipoprotein receptor-related protein 5 and 6 MΦ macrophage MHC II MHC class II MOG myelin oligodendrocyte glycoprotein MS multiple sclerosis pDC plasmacytoid DC pi postimmunization TCF/LEF T cell factor/lymphoid enhancer–binding factor Tr1 type 1 Treg Treg regulatory T cell Wnt wingless/integrated WT wild-type Received January 7, 2021. Accepted June 28, 2021. Copyright © 2021 by The American Association of Immunologists, Inc.

18 Jul 2021 Evidence that the brain harbors its own cadre of immune cells continues to grow. In the July 12 Nature Neuroscience, researchers led by Gerd Meyer zu Hörste, University Hospital Münster, Germany, describe using single-cell transcriptomics to profile lymphocytes in the meninges, the membranes that envelope the central nervous system. The scientists report that both mature and immature B cells populate the dura, the outer meningeal layer that adjoins the skull. The paper comes hot on the heels of recent reports from the labs of Jonathan Kipnis and Marco Colonna at Washington University, St. Louis. These groups reported that a reservoir of progenitors contained in a thin layer of bone marrow inside the skull supplies the brain's meninges with their own private stock of B cells. Perhaps unlike those that mature in the spleen or blood, those better recognize antigens in the brain as self (Jun 2021 news).  In contrast, zu Hörste concludes that the skull bone marrow is an unlikely source of dural B cells, at least during times of inflammation. Instead, he believes, the dura has its own progenitors that might be capable of maintaining the dural B cell population independently of bone marrow. That would be a first. Meningeal Layers. The meninges that protect the brain comprise the dura close to the skull, the subarachnoid space bathed in cerebrospinal fluid, and the pia, which covers the gray and white matter of the brain. Shafflick separated all three and tested them for lymphoid cells. [Courtesy of Shafflick et al., Nature Neuroscience, 2021.] Zu Hörste set out to better characterize the lymphocytes in the brain meninges. First author David Shafflick and colleagues used single-cell RNA-Seq to profile cells of the rat brain's membranes. He looked separately in the three meningeal layers: the outer dura, the middle arachnoid layer that is bathed with cerebrospinal fluid, and the inner pia that covers the brain tissue (see image above). From 26,301 total cell transcriptomes, he identified 12 major cell types among six tissues: CNS, subdural meninges, the dura, choroid plexus, CSF, and blood. Focusing specifically on the dura, Shafflick found that it had the most diverse population of all the tissues, sporting 17 transcriptional clusters. Four subclusters of B cells (Bc) reside there. Interestingly, the Bc cluster expressed genes involved in proliferation and Nf-κB signaling, processes usually associated with B cell mobilization in the bone marrow or spleen. “This unique phenotype and expression pattern suggested dura as a potential site of homeostatic Bc residence and proliferation,” the authors wrote. Almost all the transcription patterns seen in rat dura were found in mouse dura as well, albeit less prominently. Do these dural B cells respond to immune challenge? In mice that had experimental autoimmune encephalitis (EAE), a model of multiple sclerosis, dural B cells increased expression of MHC II, which is involved in antigen presentation, and they also upregulated ribosomal and transcription factor genes. They shut off expression of Ki67, a gene involved in proliferation. All told, it appeared that these B cells had left a homeostatic proliferation state to adopt a mature, antigen-presenting gait. Where do these B cells come from? Unexpectedly, Shafflick found two transcriptional clusters in rat dura indicative of B cell progenitors. Dubbed pre-Bc and late pro-Bc, these clusters resembled progenitor cells that previously had been detected only in the bone marrow (see image below). Mouse dura contained similar clusters. Using flow cytometry, the authors also detected cells expressing immature B cell markers in the mouse dura. These data raise the tantalizing idea that the dura contains its own supply of B cell progenitors and does not rely on obtaining them from the bone marrow. Dural Progenitors. Single cell RNA-Seq of the dura (left) identified transcriptional profiles in common with immature (brown) and mature (turquoise) B cells, but also with pre-B and late pro-B cell progenitors. In situ hybridization (right) identified dural cells expressing the progenitor genes dntt (red), Igll1 (white), and the common B cell marker CD19 (green). [Image courtesy Shafflick et al., Nature Neuroscience, 2021.] Further evidence for this came from chimeric mice. The authors introduced bone marrow from a mouse that harbors the CD45.1 isoform of the cell surface protein tyrosine phosphatase into a mouse that expresses the CD45.2 isoform. All lymphocytes express CD45, and both these isoforms are equally functional, hence CD45.1 served merely to mark cells as originating from the graft. Injecting the CD45.1 bone marrow into the tail veins of CD45.2 mice after they had received a sublethal dose of radiation ensured that the host cells would be replenished with CD45.1-positive cells. Indeed, after eight weeks, about 95 percent of B cells in the lymph and skull bone marrow were replaced with donor cells; alas, in the dura, 15 percent of the B cells were still from the host. This implies either that these cells were more resistant to irradiation, or that they were replenished from within the dura, not from the injected bone marrow. To avoid the vagaries of radiation, the authors next turned to parabiosis. They hooked up mice to a partner that ubiquitously expresses a red fluorescent protein. After seven to 10 weeks, B cells in blood, spleen, and lung were at a 1:1 ratio between host and parabiosis partner, i.e., 50 percent of the cells were read in each animal. In contrast, B cells in the host's dura were still 78 percent from the host; only 22 were red. Skull bone marrow was similarly discerning, having 34 percent red B cells. The findings are in keeping with the idea that both the dura and the skull bone marrow are replenished more slowly, or that they have their own source of B cell progenitors. Could the skull bone marrow be supplying B cells to the dura then, as Kipnis and Colonna had previously reported? Shafflick and colleagues do not think so. They labelled B cells in the bone marrow with a dye and watched through the skull to see where these cells ended up. Even 17 days after induction of EAE, no labelled cells were found in the dura. “The skull BM thus did not likely provide large amounts of leukocytes to the dura in neuroinflammation,” conclude the authors. That’s likely to be a topic of debate and more research. —Tom Fagan REFERENCES News Citations Private Stock—Brain Taps Skull Bone Marrow for Immune Cells 11 Jun 2021 FURTHER READING News As Mice Age, T Cells Traipse Around Their Meninges. Mayhem Ensues 26 May 2021 Do Immune Cells in the Meninges Help with … Memory? 11 Oct 2019

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. Glossary ɑCD20 mAb=anti-CD20 monoclonal antibody; BCR=B-cell receptor; EAE=experimental autoimmune encephalomyelitis; mELT=meningeal ectopic lymphoid tissue; MOG=myelin oligodendrocyte glycoprotein; MPO=myeloperoxidase; MS=multiple sclerosis; RRMS=relapsing-remitting multiple sclerosis; PMS=progressive MS; SLO=secondary lymphoid organ; LFB-PAS=Luxol fast blue and periodic acid-Schiff reaction; TCR=T-cell receptor B-cell-depleting strategies with anti-CD20 monoclonal antibodies (ɑCD20 mAbs), such as rituximab, ocrelizumab, or ofatumumab, are among the most potent therapies currently available in the treatment of relapsing-remitting multiple sclerosis (RRMS). Modest efficacy has also been shown in progressive MS (PMS), specifically active primary PMS, and early, relapse-independent progression in RRMS.1,-,4 Their success highlights the importance of B cells for MS pathogenesis. Among other factors, the therapeutic effect of B-cell depletion may be mediated by interrupting antigen presentation by B cells to T cells in the CNS.5,-,9 Leukocytic infiltrates, forming meningeal ectopic lymphoid tissue (mELT), are found in patients with PMS and to a lesser extent in RRMS. They correlate with an earlier disease onset, a more rapid disease progression, and subpial cortical damage.10,-,15 In general, ectopic lymphoid tissue (ELT) is formed by aggregates of B and T cells, which range from tight-cell clusters to highly organized structures and resemble secondary lymphoid organs (SLOs).16,-,20 In a model of MS, it was demonstrated that B-cell repertoire expansion occurs in mELT, suggesting germinal center activity in the CNS.21 Hereby, mELT may facilitate smoldering autoimmunity in the CNS parenchyma sequestered beyond the blood-brain barrier. Yet, it remains unclear whether and how mELT contributes to early or progressive MS. Because B cells are an integral part of mELT and ɑCD20 mAb that targets B cells not only in the periphery but also in the meninges,22 it seems possible that ɑCD20 mAb inhibits the formation or retention of mELT. Assuming that mELT plays a role in MS pathogenesis, preventing or suppressing mELT may contribute to the efficacy of B-cell depletion in MS. In this study, we investigated the effects of ɑCD20 mAb on mELT in a B-cell-dependent and T-cell-dependent experimental autoimmune encephalomyelitis (EAE) model of MS. Approximately, 50% of 2D2 (TCRMOG) × Th (IgHMOG) mice develop spontaneous chronic EAE and exhibit meningeal clusters of T and B cells in the spinal cord, reminiscent of those found in MS.23,-,25 In this model, we depleted B cells to investigate whether ɑCD20 mAb prevents mELT formation. Surprisingly, our results showed that, despite depleting virtually all B cells from the meninges, mELT still formed or persisted. Materials and Methods Mice 2D2xTh mice, developing spontaneous chronic EAE with mELT, were generated as previously described by crossing myelin oligodendrocyte glycoprotein (MOG)-specific T-cell receptor (TCR) transgenic 2D2 (TCRMOG) mice with MOG-B-cell receptor (BCR) knockin Th mice (IgHMOG).23,24 Mice were bred and maintained in-house at the Center of Preclinical Research (CPR) at Klinikum rechts der Isar (Technical University of Munich) under specific pathogen-free conditions, according to FELASA recommendations with free access to water and standard rodent chow. EAE and Clinical Assessment 2D2xTh mice develop spontaneous chronic EAE with an incidence of approximately 30–50% in our facility. Clinical scores of mice were evaluated on a daily basis and assessed as follows: 0 = no clinical signs, 1 = tail weakness, 1.5 = additional impaired tail erection, 2 = ataxia and hind limb weakness, 2.5 = hind limb paresis, 3 = high-grade hind limb paresis, 3.5 = additional minor weakness of the fore limbs, 4 = additional fore limb paresis, 4.5 = tetraplegia, and 5 = moribund. ɑCD20 mAb Treatment 100 µg of antimouse CD20 mAb (clone 18B12, provided by F. Hoffmann-La Roche, Switzerland) in a murine IgG2a Fc-format or antihen egg lysozyme isotype control (Crown bio, Cat: C0006) in 100 µL PBS was injected intraperitoneally (i.p.) at day 0, 7, 14, and 21 for a total of 4 doses. Mice were grouped for prevention or treatment of EAE. In an attempt to prevent EAE, mice received the first injection at the age of 26 ± 3 days (n = 18 in both groups). For the evaluation of the therapeutic potential, mice were treated when reaching a clinical score of ≥3 (n = 11 ɑCD20 mAb-treated, n = 14 isotype-treated). Mice were killed 28 days after the first injection. Reference mice at the onset of disease (figure 3C) were dissected immediately after reaching an EAE score of ≥3 without treatment. Cell Isolation and Flow Cytometric Analysis Blood was freshly obtained by terminal cardiac puncture and mixed with 500 µL 2 mM EDTA. Cervical lymph nodes were carefully dissected and passed through a 30-µm cell strainer. Samples were washed with RPMI medium, and erythrocytes were lysed using BD Pharm Lyse Buffer (BD Biosciences). Cells were washed again, and the number of living cells was determined using a hemocytometer (Cellometer Auto 2000, Nexcelom Bioscience). Single-cell suspensions were stained with antibodies in PBS containing 2% fetal calf serum for 20 minutes at 4°C in the dark. Fc-receptors were blocked to prevent nonspecific antibody binding. Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen). Cells were washed twice and acquired on a CytoFLEX flow cytometer (Beckman Coulter). The following antimouse antibodies were used: CD16/CD32 (2.4G2), CD45R/B220 (RA3-6B2, FITC), and CD3e (145-2C11, PE) (BD Bioscience). Data were analyzed using FlowJo version 10 (TreeStar). Histology, Immunohistochemistry, and Imaging Spinal cord, spleen, and inguinal lymph nodes were perfused before dissection, fixed with 4% paraformaldehyde, and paraffin embedded. The spinal cord was additionally decalcified with Osteosoft (Sigma-Aldrich) 72 hours before embedding. To determine the degree of meningeal infiltrates, the spinal cord was sectioned horizontally into 13–16 tissue blocks (thickness: 2 × 5 mm cervical, subsequently 2.5 mm) per mouse and stained with hematoxylin and eosin according to standard protocols. The stained tissues were digitized (Leica AT2 scanner) and examined using Image Scope (Pathology Slide Viewing Software, version 12.4.3, Leica Biosystems). Infiltrates with a size of ≥1 × 104 µm2 per section were considered as mELT. The cell density was analyzed using a nuclear algorithm (Nuclear v9, Image Scope; Leica). To characterize the cellular composition of mELT, immunohistochemistry was performed on representative sections with antimouse CD45R/B220 (BD, 550286, 1:50), CD3 (DCS, CI597R0, 1:50), and myeloperoxidase (MPO; Thermo Fisher, RB-373-A, 1:50). Mouse spleens served as positive controls. Numbers of B220+ and CD3+ positive cells were determined using a membrane algorithm (Membrane v9, Image Scope; Leica). In the CNS, MPO+ cells were counted manually. In the periphery, MPO+ cells were quantified by applying a nuclear algorithm (Membrane v9, Image Scope; Leica). Two cross-sections of the cervicothoracic, the thoracolumbar, and the lower lumbar part were stained with Luxol fast blue and periodic acid-Schiff reaction (LFB-PAS) according to standard protocols. LFB-PAS staining was analyzed semiquantitatively by dividing spinal cord sections into quadrants and counting quadrants with demyelination. All histologic evaluations were performed in a blinded manner. Detection of Anti-MOG Antibodies Antirecombinant mouse MOG (1–125) (Eurogentec, Cat: AS-55150-100) IgG was measured with a noncommercial ELISA in duplicates as described,26 using MOG at a final concentration of 10 µg/mL and horseradish peroxidase-labeled goat antimouse IgG (Thermo Fisher Scientific). The optical density was measured at a wavelength of 450 nm with 540 nm as a reference wavelength using the ELISA reader (infinite M200 PRO, TECAN). Statistical Analysis Data are shown as mean ± SEM or 95% CI. Statistical significance between groups was analyzed using the unpaired Student t test. For correlation analysis, simple linear regression was calculated for each group. A value of p ≤ 0.05 was considered significant with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Standard Protocol Approvals, Registrations, and Patient Consents All animal experiments were approved by the competent authority, Regierung von Oberbayern, Munich, Germany (ROB-55.2-2532.Vet_02-16-100). Data Availability Data not published within this article will be made available by request from any qualified investigator. Results ɑCD20 mAb Efficiently Depletes B Cells in the Periphery but Does Not Affect the Clinical Course of Spontaneous Chronic EAE To analyze the effects of ɑCD20 mAb on mELT in the context of EAE, we crossed MOG Ig-VH knock-in (IgHMOG, Th) mice, in which B cells express a MOG-specific BCR heavy chain, with MOG-specific TCR transgenic mice (TCRMOG, 2D2). As previously described, 2D2xTh mice develop spontaneous chronic EAE with B-cell-rich and T-cell-rich mELT along the spinal cord.23,24 2D2xTh mice were treated weekly for a total of 4 intraperitoneal (i.p.) doses with antimurine CD20 or isotype control mAb. Two treatment paradigms were pursued in parallel: (1) to prevent EAE and mELT formation, mice received the first dose as soon as they were weaned about 26 days after birth (figure 1A) and (2) to treat fully established EAE, ɑCD20 mAb was first administered after mice developed an EAE score of ≥3 (figure 3A). ɑCD20 mAb efficiently depleted B cells in the peripheral blood (figure 1B.a, upper panel) and lymph nodes (figure 1B.b, lower panel). However, its application neither attenuated the rather severe and chronic course of EAE compared with the control groups in either condition (figures 1C and 3B) nor changed the incidence of EAE. In total, 44% of preventatively treated mice developed spontaneous chronic EAE, which was similar to 39% of control treated mice (figure 1D). 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. mELT Formation and Retention Occurs in the Absence of B Cells in Preventatively Treated Mice To investigate whether mELT forms and persists under B-cell-depleting conditions, we analyzed cross-sections along the spinal cord of 2D2xTh mice, which developed clinical signs of EAE during the observation/treatment period. With a mean EAE duration of 19 days (range 13–24 days) in the ɑCD20 mAb-treated group and 16 days (range 3–21 days) in the isotype-treated group, all mice showed mELT in at least 5 of 12–16 segments with a maximum along the thoracolumbar part of the spinal cord (figure 2A). Infiltrates were considered as mELT when their size was ≥1 × 104 µm2 per section. Surprisingly, the mean area of mELT per section, including sections in which no mELT was detected, was similar in both groups with a mean of 81,152 µm2 per section in ɑCD20 mAb and 103,968 µm2 in isotype-treated controls (figure 2B, upper panel). Hence, despite the absence of B cells in circulation and cervical lymph nodes, structures of mELT formed. With B cells being a main component of mELT,21,23,24 we performed B220 immunohistochemistry on sections of the thoracolumbar part of the spinal cord. We found that, although mELT was present to a similar extend, mELT of ɑCD20 mAb-treated mice only exhibited 0.59% of B220+ B cells observed in controls (figure 2B, lower panel). 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. Interestingly, in both groups, mice without clinically apparent EAE over the treatment period of 28 days also showed histologic signs of disease in approximately 60% of cases, ranging from scattered cellular infiltrates in the meninges in some cases to mELT in 30% of the ɑCD20 mAb-treated mice and 36% of the isotype controls (figure 2C). In these clinically unremarkable animals, mELT was also mainly found in the thoracolumbar part of the spinal cord and was comparable in size in both groups (figure 2D). Our data indicate that the absence of B cells in preventatively treated 2D2xTh mice is not sufficient to prevent mELT formation and that not only mELT formation but also its retention may be independent of B cells. Moreover, mELT formation in our model occurs before developing clinical signs of EAE. ɑCD20 mAb Treatment Does Not Influence the Size of mELT In a second experiment, more closely resembling a clinical scenario, mice were randomized into groups treated with ɑCD20 or control mAbs after disease onset, i.e., when having developed an EAE score of ≥3 (aged on average 47.6 days in the ɑCD20 mAb and 48.4 days in the isotype-treated group). As a baseline, we used mice which were euthanized without further treatment immediately after having developed a score of ≥3 (figure 3A). At that stage of disease (onset), all mice already exhibited extensive mELT with a distinct fraction of B220+ B cells (figure 3C), confirming that mELT was sufficiently present before we started our intervention. After 4 weeks of treatment, distribution of mELT throughout the spinal cord was almost identical in-between groups, with all mice exhibiting mELT in at least 1 of 14–16 cross-sections analyzed (figure 3D). Again, we did not observe a difference in the mean area of mELT in both groups (figure 3E). Similar to what was observed in autopsy cases of MS, the extent of mELT correlated with disease severity in control mice. However, this was not statistically significant in ɑCD20 mAb-treated animals (figure 3F). 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. Persisting High Titers of Anti-MOG Antibodies and Extensive Demyelination in B-Cell-Depleted Mice Reflects the Lasting Severe and Chronic Course of EAE Although B cells were effectively reduced not only in the periphery but also in the meningeal compartment, MOG-specific total IgG titers remained high even after 4 weeks of B-cell depletion (figure 4A). Correlating with the clinical disease course, the spinal cord was similarly affected by demyelination in ɑCD20 mAb-treated mice compared with controls (figure 4B). Together with persisting B-cell-free mELT, these observations may contribute to the lack of a clinical benefit of B-cell depletion in our model. 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. ɑCD20 mAb Treatment Changes the Density and Composition of the Cellular Content of mELT When we analyzed the cellular composition of mELT more closely, we found that, within the treatment paradigm, mELT in ɑCD20 mAb-treated mice was populated with 24.2% less cells (figure 5A). The count of B220+ B cells, which was the largest immune cell fraction in control mice, was drastically reduced on ɑCD20 mAb treatment, suggesting that the absence of B cells led to this reduced cellular density in mELT (figure 5B, upper panel). We did not notice a relevant change in CD3+ cells (figure 5B, middle panel), whereas the number of MPO positive neutrophil granulocytes was significantly increased under ɑCD20 mAb therapy (figure 5B, lower panel). Comparing these changes in mELT with SLOs, we observed that in the spleen and inguinal lymph nodes, B220+ B cells were also largely eliminated. MPO+ cells seemed to be increased, but this did not reach statistical significance. In contrast to mELT, however, the amount of CD3+ T cells was slightly increased in B-cell-depleted lymph nodes (figure 6). 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. Together, our findings show that ɑCD20 mAb, to some degree, depletes B cells in mELT in mice with spontaneous chronic EAE, yet mELT persists. The role and function of mELT devoid of B cells remains unclear. Discussion Although its exact role in MS pathogenesis remains unclear, meningeal inflammation has gained widespread attention in recent years. Because B cells are a major component of mELT and B-cell-depleting mAbs are among the most successful MS therapeutics today, the question how B-cell-depleting therapies affect mELT is obvious. In this study, we systematically addressed this question in a murine model of MS. Not unexpectedly, B-cell depletion did not change the disease course in our spontaneous chronic EAE model. It has previously been reported that ɑCD20 mAb did not significantly change the course of EAE in the same model because high titers of MOG-binding antibodies in conjunction with MOGTCR 2D2 T cells could support clinical disease in the absence of B cells.26 Importantly, ɑCD20 mAb does not target antibody-producing plasma cells. It could be debated that spontaneous chronic EAE in 2D2xTh mice, because of the MOG specificity of both T and B cells, is not dynamic enough to allow being modified by pharmaceutical interventions. However, laquinimod ameliorated spontaneous chronic EAE and reduced meningeal B-cell aggregates and thereby demonstrated its plasticity.27 Thus, other factors are likely to contribute. Although clinical effects were neglectable, we found that ɑCD20 mAb depleted virtually all B cells not only from SLOs and blood but also from mELT. It has been shown before that B cells infiltrating the meninges in EAE could be efficiently depleted by ɑCD20 mAb administered either intraperitoneally or intrathecally.22 This study used a model, however, which does not feature large meningeal B-cell aggregates such as mELT. A case report of a patient with gastrointestinal mantle cell lymphoma, who developed progressive multifocal leukoencephalopathy after rituximab therapy, showed that 8 months after the last dose, no B cells were found in perivascular spaces.28 By contrast, a case report of a young man with aggressive MS treated with rituximab and with no detectable peripheral B cells found some B cells in the parenchyma and in perivascular cuffs on CNS autopsy.29 Regarding B-cell-depleting antibodies used to treat MS, it is an encouraging finding that ɑCD20 mAb could deplete B cells present in mELT and prospectively prevent colonization of the meningeal compartment by B cells. Yet, open questions remain. The most unexpected finding in our study was that mELT formed or remained in the absence of B cells when mice were B-cell-depleted after EAE was fully established and even when treated before EAE onset. The size and extent of mELT was even comparable whether they contained B cells or not. This shows that, in our model, B cells are not essential for the induction or maintenance of mELT development. Yet, in the absence of B cells, mELT was less densely packed with cells and the cellular composition was altered. ɑCD20 mAb-treated mice had drastically fewer B220+ B cells, comparable amounts of CD3+ T cells, and significantly more MPO+ neutrophil granulocytes in mELT. A recent study showed that neutrophil granulocytes pioneer mELT formation and precede B cells in the meningeal compartment.30 In addition, several other cell types have been proposed as candidates to induce ELT formation, including IL-22 or IL-17 secreting immune cells, dendritic cells, and CD3−CD4+CD45+ lymphoid tissue inducer cells.16,31,-,35 Our own data showed that shortly after EAE onset, B220+ B cells were already present in mELT, but after 4 weeks of isotype control treatment, their amount had roughly doubled. Thus, B cells seem to fully populate mELT at later stages. This raises the question, which role B cells play in (later stage) mELT and whether mELT without B cells still has a pathogenic role. Generally, our mice would be expected to benefit from B-cell depletion, like it has been shown in another B-cell-dependent model without mELT or in MS itself.1,-,3,36 If mELT lacking B cells still contribute to autoimmunity, this could explain why the EAE disease course is unchanged in ɑCD20 mAb-treated 2D2xTh mice. As discussed above, other inherent properties of the model could contribute, such as the presence of high titers of MOG-binding antibodies and MOGTCR T cells with a potentially altered activation status under ɑCD20 mAb therapy. However, the notion that mELT without B cells persist in EAE and its persistence correlates with sustained severe disease, may give rise to interesting considerations: B-cell-depleting therapies are disproportionately more effective in RRMS than PMS. It could be speculated that future therapeutic approaches targeting mELT directly, instead of B cells, may be more effective in treating PMS. We observed that approximately one-third of 2D2xTh mice without clinically manifest EAE develop a minor degree of mELT both in the presence or absence of B cells. What does this imply regarding the role of mELT? Naturally, it remains unknown whether for these mice disease was imminent and the occurrence of mELT only preceding clinical signs. Alternatively, it is possible that 2D2xTh mice live with mELT without developing a phenotype. The latter would suggest that mELT may be a rather irrelevant epiphenomenon of encephalitogenic B and T cells being present in this model or that mELT may even have a regulatory role. One limitation of our study is that we depleted B cells relatively soon, i.e., before the onset of EAE or at an early stage of disease, where B cells are already present in mELT but to a lesser extent. We believe that it is reasonable to conclude from our results that B cells already present in mELT were depleted by ɑCD20 mAb. However, we could not formally assess the respective contributions of preventing B-cell influx from the periphery or direct depletion in the CNS itself to our finding that mELT was devoid of B cells after several weeks of treatment with ɑCD20 mAb. It is very challenging to study meningeal pathologies in patients in vivo or even postmortem. Thus, animal models are instrumental, and yet, the transferability of any findings in such models to MS must be questioned continuously. Our model of spontaneous chronic EAE has several characteristics that make it a suitable tool. EAE develops without an external stimulus, such as immunization or an adjuvant, and mice form meningeal inflammatory aggregates. Furthermore, mELT is readily available in high quality and quantity. However, it is a highly artificial model which is genetically determined through B-cell-mediated and T-cell-mediated autoimmunity against MOG. MS, by contrast, is not known to have a single autoantigen and certainly does not feature such a highly polarized adaptive immune system. Therefore, studies in human subjects treated with ɑCD20 mAb are required to confirm our results. In conclusion, we provide evidence that systemically applied ɑCD20 mAbs efficiently deplete B cells in meningeal inflammatory aggregates, and yet, mELT still form and persist with an altered cellular composition. The unchanged severe course of EAE, despite near complete B-cell depletion, both in the periphery and the meningeal compartment, may point to a yet unknown function of mELT, which is independent of B cells. Future studies are required, which aim to better understand the role and pathogenic relevance of mELT with and without B cells. Study Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) SFB TRR 130 TP17 to H. Radbruch. K. Lehmann-Horn received research support from the Deutsche Forschungsgemeinschaft (SFB-TR-128 and LE 3079/3-1), the Hertie Foundation (MyLab program), and the US National Multiple Sclerosis Society (NMSS) (G-1508-07064). Disclosure R.M. Brand, V. Friedrich, J. Diddens, M. Pfaller, F.R. de Franchis, H. Radbruch, and K. Steiger report no disclosures. B. Hemmer has served on scientific advisory boards for Novartis; he has served as DMSC member for AllergyCare, Polpharma, and TG therapeutics; he or his institution has received speaker honoraria from Desitin; his institution received research grants from Regeneron for MS research. He holds part of two patents: one for the detection of antibodies against KIR4.1 in a subpopulation of patients with MS and the other for genetic determinants of neutralizing antibodies to interferon. All conflicts are not relevant to the topic of this study. K. Lehmann-Horn has received research support (to TUM) from Novartis, honoraria, and compensation for travel expenses from Novartis, F. Hoffmann-La Roche, and Merck Serono. Go to Neurology.org/NN for full disclosures. Acknowledgment The authors would like to thank Melina Pekic Hajdarbasic, Beatrix Lunk, Olga Seelbach, Marion Mielke, Ulrike Mühlthaler (all TUM), and Vera Wolf and Sandra Meier (both Charité) for excellent technical assistance. The authors acknowledge support from F. Hoffmann-La Roche for providing the antimouse CD20 antibody 18B12. R.M. Brand is a fellow of the Hertie Foundation (medMS Doctoral Program). Appendix Authors Footnotes Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. Editorial, page e1000 Received January 25, 2021. Accepted in final form March 8, 2021. Copyright © 2021 The Author(s). 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