The Role of B Cells in the Early Onset of the First Demyelinating Event of Acute Optic Neuritis

Visual Neuroscience The Role of B Cells in the Early Onset of the First Demyelinating Event of Acute Optic Neuritis

Anna Feldman,1 Michael Gurevich,1 Ruth Huna-Baron,2,3 and Anat Achiron3,4

1Neurogenomics Unit, Sheba Medical Center, Tel-Hashomer, Israel 2Neuro-Ophthalmology Unit, Sheba Medical Center, Tel-Hashomer, Israel 3Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 4Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Israel

Correspondence: Anat Achiron, PURPOSE. We evaluated the molecular pathways that operate in the early phase of acute optic Multiple Sclerosis Center, Sheba neuritis (ON) by studying gene expression profiles of peripheral blood mononuclear cells Medical Center, Tel-Hashomer, Israel (PBMCs) subpopulations, including CD19þ B cells, CD14þ macrophages, and CD4þ and and Sackler School of Medicine, Tel- CD8þ T cells. Aviv University, Tel-Aviv, Israel; [email protected]. METHODS. Samples of PBMC subpopulations were obtained from 18 MS patients within 96 Submitted: August 6, 2014 hours of the acute onset of the first demyelinating event of ON, and from 14 age- and sex- Accepted: January 2, 2015 matched healthy subjects. High throughput gene expression analysis was performed on samples from six ON patients and nine healthy subjects using Affymetrix technology. Data Citation: Feldman A, Gurevich M, were analyzed using Partek software, and most informative genes (MIGs) were defined as Huna-Baron R, Achiron A. The role of B cells in the early onset of the first genes with P < 0.01 and fold change > 2.0. Molecular pathways were analyzed by Ingenuity demyelinating event of acute optic software. Verification of key MIGs was done on samples from five independent ON patients neuritis. Invest Ophthalmol Vis Sci. and five healthy subjects by quantitative (Q) RT-PCR and Western blot. Functional assay to test 2015;56:1349–1356. DOI:10.1167/ antigen presentation ability of sorted B cells was performed on blood samples from seven iovs.14-15408 additional ON patients.

RESULTS. Significantly differentiating gene expression signatures consisting of 467, 55, and 55 MIGs respective of CD19þ, CD14þ, and CD4þ cells, were identified between ON and healthy subjects. No MIGs were detected for CD8þ cells. The major involvement of CD19þ B cells in the early stage of ON was characterized by enrichment of genes involved in activation of immune mechanisms (P ¼ 3.2 3 1025 to 2.5 3 103), including cellular immune response (P ¼ 7.1 3 1012), B-cell cellular growth and proliferation (P ¼ 1.0 3 107), activation of immune cells trafficking pathways (P ¼ 5.4 3 1015 to 2.0 3 103), and stimulation of antigen presentation (P ¼ 8.9 3 1011). This massive B-cell–restricted initiation of the immune response in the early disease process of ON was followed by low CD14þ and CD4þ cells activity and CD8þ cells anergy.

CONCLUSIONS. Our findings demonstrate that CD19þ B cells have a significant role in the pathogenesis of the first demyelinating event of acute ON and suggest their role as a possible target for immunomodulation. Keywords: optic neuritis, B cells, multiple sclerosis

ptic neuritis (ON) is a common manifestation of multiple Thereafter, these cells migrate through the blood–brain barrier O sclerosis (MS); it appears as the presenting symptom in (BBB) in response to chemotactic signals, stimulate CD14þ approximately 25% of MS patients and occurs in 30% to 70% of macrophage by secretion of inflammatory cytokines, such as patients with MS during the course of their illness.1–4 Clinically, IFN-c and IL-1, to induce myelin damage.9 In addition to CD4þ ON is characterized by sudden painful onset of visual loss that T-cell–mediated demyelination, there is evidence implicating develops within hours to a few days and is accompanied by humoral immune involvement in ON with identification of decreased sensation of light brightness, decrease in color clonal populations of B lymphocytes and plasma cells in the vision, and central visual field defect.5,6 The pathological cerebrospinal fluid.10 changes in ON involve a complex interplay between active Studying gene expression patterns of immune cell subpop- de- and re-myelination associated with axonal loss.6 The ulations is of importance for revealing the role and influence of majority of patients have plaques in their optic nerves, each of them in the early ongoing ON pathological process.11 irrespective of a clinical history of ON.7 Although the In the current study, we aimed to characterize the molecular mechanism of injury in ON is inflammatory demyelination, mechanisms involved in the first event of acute ON that later the operating triggering components are unclear. develops to MS. Using gene expression microarrays technology, In MS it is suggested that autoreactive CD4þ T cells are we profiled CD19þ B lymphocytes, CD14þ macrophages, CD4þ activated in the peripheral blood by nonself antigens that T lymphocytes, and CD8þ T lymphocytes to identify the early resemble central nervous system (CNS) myelin proteins.8 pathogenic mechanisms involved. Understanding the nature of

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the molecular mechanisms that operate in the first event of ON ANOVA. Biological functional annotation and pathway analysis that later progress to MS is important to identify effective were performed by Ingenuity software (available in the public therapeutic targets. The involvement of B cells in early ON was domain at www.ingenuity.com). Significance of functional not previously described in the literature and it is one of the analyses were defined as P < 0.01 by The Benjamini and innovative findings of our work. Hochberg False Discovery Rate method.

Verification by Quantitative RT-PCR MATERIALS AND METHODS Key MIGs were verified by quantitative RT-PCR on a 7500 Subjects Realtime PCR Cycler (Applied Biosystems, Carlsbad, CA, USA) Patients with the first clinical presentation of acute unilateral using Taqman probes adjusted to specific genes. ON without previous neurological symptoms, and age- and sex- All reactions were run in triplicates and processed on an AB matched healthy subjects participated in the study. 7500 HT Detection System. Amplification data was analyzed Inclusion criteria were as follows: (1) age between 18 and with the ABI Prism SDS 2.1 software (Applied Biosystems). 40 years; (2) a diagnosis of ON based on unilateral visual loss of Relative quantification of the gene expression was performed >20/40 associated with dyschromatopsia, afferent pupillary by the DDCt method. HTRP1 was used as a housekeeping gene, defect, and central/paracentral visual field defect; (3) no prior as it is characterized by stable expression in our data. neurological symptomatology of any kind; (4) brain magnetic resonance imaging (MRI) suggestive of demyelinating disease Verification by Western Blot Analysis according to the criteria of Barkhof et al.12; (5) time from the onset of visual symptoms to blood sampling 96 hours; and Protein levels of significant key genes HLA-DQB1 and HLA- (6) development of definite MS within one year of clinical DOB were analyzed using 12% acrylamide SDS-PAGE gel. follow-up. Proteins were transferred into nitrocellulose membranes and Sheba Medical Center Internal Review Board (IRB) commit- stained by monoclonal HLA-DQ (TAL4.1) and HLA-DOB (V16) tee approved the study and all subjects signed written antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) informed consent. The study was conducted in compliance and by goat–anti-mouse HRP-conjugated antibody (Jackson with the Helsinki declaration guidelines for studies in humans. Laboratories, Bar Harbor, ME, USA). Samples from HLA-DQ– and HLA-DO–typed subjects were used as positive controls. Experiments were repeated four times. Cell Sorting Peripheral blood mononuclear cells (PBMC) were purified Functional Assay to Test Antigen Presenting using Ficoll Hypaque gradient (Invitrogen, Carlsbad, CA, USA). Function of Sorted B Cells Cell subsets of CD4þ, CD8þ T lymphocytes, CD19þ B cells, and CD14þ macrophages subpopulations were isolated using FITC- B cells were tested in vitro using stimulation with CD40L and IL-4 conjugated magnetic microbeads (purity > 95%; BD Pharmi- to study their ability to be efficient myelin-specific antigen- gen, San Jose, CA, USA) and analyzed by fluorescence activated presenting cells to autologous T cells as previously de- cell sorting (FACS; Beckton Dickinson, Franklin Lakes, NJ, scribed.13–15 Purified B cells (1 3 106 cells/mL) from 7 patients USA). with acute ON were cultured with or without the presence of human CD40L, irradiated with 10,000 rad to inhibit proliferation, Microarray Preparation and stimulated with 10 ng/mL recombinant human IL-4 (rhIL-4; R&D Systems, Minneapolis, MN, USA) for 5 days in human Microarray gene expression was performed separately for each culture medium (10% human AB serum, 100 lg/mL penicillin, cell subpopulation. Total RNA was purified using Trizol 100 IU streptomycin, 2 mM L-glutamine, 5 lg/mL insulin, 90% (Invitrogen), and RNA integrity was assessed by RNA Experion Iscove’s modified Dulbecco’s medium). Thereafter, 2 3 106 automated electrophoresis system (Bio-Rad, Hercules, CA, USA). CD40L-activated B cells were harvested and incubated with 10 Probe synthesis using 3 lg total RNA was performed using the lg biotin-labeled myelin basic protein (MBP; Millipore Corpora- two-cycle RNA amplification kit protocol and in vitro transcription, Darmstadt, Germany). The number of B cells specifically tion performed with the GeneChip Labeling Kit (both Affyme- binding MBP peptide was measured by FACS flow cytometer trix, Inc., Santa Clara, CA, USA). The biotin-labeled IVT–RNA using B cells incubated without CD40L as a negative control. hybridized to a Genechip array (HU133A-2, including well annotated 14,500 human genes), washed in a GeneChip Fluidics Station 450 (Hewlett Packard, Palo Alto, CA, USA), and scanned RESULTS (GeneArray-TM scanner G2500A) according to the manufactur- er’s protocol. Gene expression raw data were submitted to the Subjects public Gene Expression Omnibus (GEO) repository (available in the public domain at [email protected]), accession We included in the study 18 patients with acute onset of ON and number GSE62584. 14 healthy subjects. Samples from six patients (four females/ two males, average age 6 SD 35.0 6 7.1 years) and nine healthy subjects (five females/four males, age 36.0 6 5.4 years) were Data Analysis used for gene array experiments, and samples from five patients Data analysis was performed by Partek Genomics Solution with acute onset of ON (three females/two males, age 37.0 6 software (available in the public domain at www.partek.com). 4.2 years) and five healthy subjects (four females/one male, age Expression values were computed from raw CEL files by 35.5 6 4.6 years) were used for verification by QRT-PCR and applying the Robust Multi-Chip Average background correction Western blot. algorithm included: (1) values background correction, (2) Samples from seven patients with acute onset of ON (four quintile normalization, (3) log2 transformation, and (4) median females/three males, average age 6 SD 38.0 6 2.0 years) were polish summarization. Most informative genes (MIGs) were applied for functional assay to test antigen presenting function defined as those with a P < 0.01 and a change of ‡2.0-fold by of sorted B cells.

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ON Gene Expression Signature The characterizing ON gene expression signature included 467 MIGs in CD19þ B-lymphocytes, 55 MIGs in CD14þ macrophages, and 55 MIGs in CD4þ lymphocyte subpopulations. None of the genes in CD8þ T cells reached statistical signiﬁcance and, thus, this subpopulation was excluded from further analysis. The CD19þ, CD4þ, and CD14þ cells heat maps of the MIGs are presented in Figure 1, and the list of MIGs is in Supplementary Tables S1 through S3.

ON Related CD19þ Gene Expression Signature The functional analysis of 467 ON MIGs related to CD19þ Bcells demonstrated significant enrichment of genes related to activation of immune mechanisms (P ¼ 3.2 3 1025 to 2.5 3 103,125 genes), such as cellular immune response (P ¼ 7.1 3 1012,26 genes) and inflammation (P ¼ 3.6 3 1012, 42 genes); genes related to activation of cellular movement and immune cells trafficking pathways (P ¼ 5.4 3 1015 to 2.0 3 103, 104 genes); cellular growth and proliferation (P ¼ 6.3 3 1014 to 2.5 3 103, 140 genes), including proliferation of B lymphocytes (P ¼ 1.0 3 107, 22 genes) and stimulation of antigen presentation (P¼8.93 1011 to 2.5 3 103, 69 genes; Table 1). The most unexpected findings in B cells were activation of genes associated with MHC class II antigen presentation, like HLA-DQB1/HLA-DOB1 and their transcription factor RFX5, that influences HLA class II expression. The activation of B-cell proliferation and survival correlated with overexpression of the antiapoptotic BCL2 and BCL7C genes, as well as genes related to cell interaction and adhesion, like ITGB7 and BIN1, together with chemokine and chemoattractant family members, like CNTNAP2 and CCR7, known to be associated with memory B cells. Further evidence that implicate B-cell activation was the overexpression of genes related to restricted activation of B-cell–mediated immunity and antibody production, represented by CD79A/B, IL4R, FCER2, FCGRT, and immunoglobulin genes, like IGHG3 and IGHM.The activation of CD79A/B was followed by overexpression of their upstream transcriptional regulators TCF3 and FOXO1,aswellas transcriptional activators, like NFATC1 and RELA. Low level of apoptosis was an additional significantly enriched function demonstrated by B-cell–related genes. This was based on underexpression of the classical apoptosis- related genes, like CASP1, DAPK1, TNFRSF1A/1B/13, and TNFAIP6; apoptotic activating factors, like APAF1 and VDR; followed by suppression of signal transduction-related molecules, such as TLR-2 and TLR-5, IRAK3, and MYD88. Moreover, genes involved in cell-to-cell interaction, like complement component C1QR1 and DF, were found to be suppressed. Altogether, the specific B-cell gene expression profile in acute ON is comprised of evidence for B-cell involvement in antigen-presenting mechanism, B-cell memory cell generation, antibody production, and enhancement of B-cell proliferation and survival, followed by suppression of apoptotic-related mechanisms. Transcription levels of selected functionally relevant genes are presented in Supplementary Figures S1 through S5.

Reconstruction of CD19þ Related Gene Expression Networks The reconstructed regulatory network operating in B-cells is demonstrated in Figures 2A and 2B. This include overexpres- þ þ þ sion of CD79A/B and TCF3, which have a crucial role in the B- FIGURE 1. The CD19 ,CD14,andCD4 cells heat maps of the MIGs. cell proliferation,16,17 and activation of genes, like IGKC, The left dendrogram demonstrates unsupervised hierarchical clustering of ON patients (left vertical, green) and healthy subjects (HS; left vertical, IGHG3, IGHM, and IGL@, which are known to be involved in ) according to the expression of MIGs (upper dendrogram) 18–20 yellow the process of immunoglobulin variable region generation ; obtained in CD19þ,CD14þ,andCD4þ cells subpopulations. Overex- thus, conﬁrming the role of B-cell activation and antibody pressed genes are shown in red and down-expressed in blue.

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TABLE 1. The ON Related CD14þ, CD4þ, and CD19þ MIGs Functional Annotations

Functional annotation CD14þ* CD4þ* CD19þ* 55 MIGs*55MIGs 467 MIGs Overexpressed Overexpressed 25*/ Overexpressed 146*/ 22*/down-expressed 33 down-expressed 30 down-expressed 321 Antigen presentation NFYA, CD8A, CD8B1 HLA-DQA1, HLA-DQB1, HLA-DQB1,* HLA-DOB1,* HLA-DQB2, HLA-DPA1, RFX5, IRF4,* CD23,* CD83, BAT2 NFATC1,* IRS1* Apoptosis DDX3X, LCN TINP1, PDCD4, GZMB, DAPK1, DUSP2, CDKNC1, APAF1, TNFRSF1A/1B/13, TNFRSF25, DUSP2, LDHB, TNFRSF17, DEFCAP, COX4I1, TNFAIP6, TGFB, TP53I3, HNRPA1, SMYD2, CDC14A CARD4 VDR, CAMK, CARD15, CASP1, Calcineurin, MSR1, ETS2, DAPK1, HSPA6, HSP70, COX5B, MSR1,* ETS2* Cell cross-talk and Col2A1,* Col5A1,* Col9A3,* CD58,* CD84,* TCF8, LAMP3, IL16,* XCL1,* CCR7,* CXCR4,* adhesion CEACAM6/8,* NEBL,* LAMP, CCR1, CX3CR, CD9, NCAM1, ITGB7,* CNTNAP2,* BIN1,* SATB1, ITK, DGKL, ICOS, APBA2, ADAM28, KLRF1, KLRC3, CD1D, LST1, IL13RA1, LRRN3 KIR2DL5 C1QR1, DF Cell activation and AKT2,* GAP4,* LCN2,* IMP-3,* PDGFC,* CRIP,* MRP23,* RPL7/21/29/36A,* BCL2,* BCL7C,* BNIP3,* PBXIP,* proliferation/signal MMP9,* EGR1,* HOXA9,* TCEB2,* SKP1A,* SF3B5,* p38MAPK,* MAPK13,* IL2RG,* transduction HIST1H1C/1D,* HISTIH2B/ E1F4B,* HIST1 H1C/1D,* FCER2,* MDR1*/CFTR, RELA,* 1E/H2/H2BS,* CAMP,* TRPC1, HIST1H2B/1E/H2e/ TNFRSF17,* ADAM,* ABCB1,* PASK, PLXDC1, KANK, NYREN7 H2bf/H2BS/H1BF,* RPS5,* TLR2/5, IRAK3, MYD88 PSMD8,* C1 orf24,* ATP5G3,* EIF4B* Cell-mediated immunity/ KIR3DL2, KLRC3, KLRC4, KIR2DS3/ CD160, TRD@ TCF3,* FOXO1,* CD22,* antibody production 2TOB, HGFL, PDE3B, RGS1, LEF1 CD79A/B,* IL4R,* CD64,* TCL1,* TCF4,* LYB2,* IGLJ3,* IGL@,* FCER2,* CD72,* IGHM,* IGHG3,* IGKC,* BLR1,* IGKV1D-13* TLR2/5, IRAK3, MYD88 * Overexpressed genes. Remainder are down-expressed genes.

production. In addition to B-cell activation, we have demon- Verification of Key Genes by Quantitative RT-PCR strated B-cell–restricted antigen presentation as shown by and Western Blot upregulation of MHC class II genes, like HLA-DOB1, HLA- DQB1, and their transcription factor RFX5. This finding is The expression of eight key genes (RFX5, HLADQB1, FCER2, important in the context of anergy of APC-restricted genes in FCGRT, CNTNAP2, BIN1, ABCB1, and CCR7) within CD19þ B- the gene expression signature of CD14þ macrophages, where cell–restricted ON-induced signature by QRT-PCR correlated the HLA-related genes were not activated and HLA transcrip- with the microarray findings demonstrating corresponding tion factor NFYA was significantly suppressed. activation at the mRNA level in ON patients as compared to healthy subjects (Table 2). þ þ þ þ Expression of HLA type genes in CD14 and CD19 cells by ON Related CD14 and CD4 Gene Expression Western blot demonstrated activation of the MHC class II Signatures proteins HLA-DQB1, HLA-DQA1, and HLA-DOB in CD19þ B þ The CD14þ and CD4þ ON-related genes did not significantly cells and not in CD14 macrophages of ON patients during first fulfill any canonical pathway or biological function. Single gene 96 hours from onset (Fig. 3). analysis of the 55 CD14þ macrophage related genes (Table 1) showed that the overexpressed genes (n ¼ 22) were related to Functional Assay to Test Antigen Presenting adhesion (Col2A1, CEACAM6/8), cell activation, and prolifer- Function of Sorted B Cells ation (EGR1, AKT2). The group of down-expressed genes (n ¼ 33) included apoptotic-related genes (DDX3X, PDCD4, In vitro functional assay to test antigen-presenting ability of isolated B cells demonstrated that activation of B cells with TNFRSF25, DUSP2), genes involved in cell-to-cell interactions CD40L resulted in 4-fold increase (P 0.0001) in the number (ICOS, ITK), antigen presentation (NFYA), and cell-mediated ¼ of MBP binding B cells compared to B cells not activated with immunity (KLRC3/4). Within the ON-related 55 MIGs charac- CD40L (Fig. 4). terizing the CD4þ T cells (Table 1), the overexpressed genes (n ¼ 25) included genes involved in T- and B-cell interaction, like CD58 and CD84. The down-regulated genes included essential DISCUSSION apoptotic genes, like GZMB, DAPK1, and CARD4; MHC Class II genes, like HLA-DQA1/B1/B2 and BAT2; cell-mediated In the current study, we characterized the gene expression immunity related genes, like CD160; adhesion molecules, like pattern of peripheral blood immune cells subtypes in the very NCAM1, and chemokines, like CCR1 and CX3CR. early onset, within 96 hours, of the first occurrence of acute

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FIGURE 2. Optic neuritis–related regulatory networks. (A) B-cell restricted antigen presentation. (B) B-cell activation. Each node represents a gene. Red represents overexpressed genes, while green represents down-expressed genes.

TABLE 2. Verification of Key Genes ON in subjects who later had MS. To the best of our knowledge, our study is pioneering in assessing the molecular QRT-PCR Microarray Analysis processes early in ON using a large scale transcriptome Gene Fold Change Fold Change profiling technology in CD4þ, CD8þ, CD14þ, and CD19þ cells, Symbol ON vs. HS P Value ON vs. HS P Value and reconstructing the operating biological pathways. We RFX5 3.8 0.03 2.00 0.003 demonstrated major activation of CD19þ B cells in the early HLADQB1 4.02 0.04 3.25 0.006 þ þ stage of ON with a background of low CD14 and CD4 cells FCER2/CD23 15.3 0.01 3.16 0.003 activity and CD8þ cells anergy. FCGRT 3.5 0.04 2.19 0.007 The role of B cells in MS is suggested by their presence in CNTNAP2 3.2 0.01 3.50 0.003 acute and chronic demyelinating lesions in the brain,21 the BIN1 6.9 0.05 2.12 0.002 presence of cerebrospinal fluid oligoclonal IgG antibodies,22 ABCB1 1.9 0.03 4.45 0.009 CCR7 3.95 0.02 2.62 0.002 and by the efficacy of monoclonal antibody treatments, such as

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FIGURE 3. Veriﬁcation of MHC class genes by Western blot in CD19þ B cells and CD14þ macrophages. Upper: HLA-DQB detection. Lower: HLA-DOB detection. All experiments included genetically typed HLA-positive controls.

Rituximab, to reduce disease activity in relapsing–remitting MS resulting in IL4 production and upregulation of MHC class II patients.23 genes through STAT6. Additionally, activated transcription Immunologically, there are emerging evidences that mam- factor RFX5 also activate MHC class II genes and initiate malian B lymphocytes operate to activate T cells as antigen- antigen presentation. On the other hand, the inhibition of presenting cells (APC), as well as precursors of antibody- repressor genes, like MSR1 and ETS2, contribute to B-cell secreting plasma cells24; however, their contribution to ON survival.36 pathogenesis and their relationship with other immune- The upregulation of the molecules related to cell migration competent cells were demonstrated in our study for the first across endothelial barriers in CD19þ B cells, like ITGB7, BIN1, time. The possible explanation for this very early restricted B- CNTNAP2, and chemokine receptors CCR7, CXCR4,35,37,38 cell antigen presentation could be related to the amount of the suggests that B cells may penetrate the BBB and induce myelin antigen. The B cells can serve as efficient APCs for T-cell damage.39 activation when low amount of antigen is applied, but are not Despite the negative expression of most specific CD4þ required for T cells priming when mice are immunized with related genes, like GZMB, the activation of CD58 and CD84 high dose of an antigen.25 These dose-dependent B-cell antigen- that optimize recognition in immune CD4þ T lymphocytes/ presentation properties are well corroborated with the early APC interactions40,41 allows us to assume that cells participate disease process of ON. Later on increased structural damage is in antigen presentation, but possibly join at a later stage. caused by epitope spreading leading to activation of the In conclusion, our results demonstrate B-cell–induced classical APC. immune mechanisms in early acute ON, including cell Alternatively, the trigger for stimulation of B cells to APC activation, antigen uptake, antigen presentation, and immuno- could be related to the nature of the antigen. For instance, viral antigens more probably stimulate B-cell–dependent antigen presentation.26 The involvement of B cells in antigen presentation is either T-cell–dependent or independent.18,24 In the current study, we demonstrated that groups of genes known to be involved in the T-cell–independent antigen presentation by B cells, as TLR-2/5, IRAK3, and MYD88,27–29 were down-expressed in the CD19þ signature. Thus, the process of antigen presentation by CD19þ B cells during the first 96 hours of ON is T-cell–dependent. In support of the inflammatory T-cell–dependent type of B- cell–restricted antigen presentation in early ON, overexpression of the chemoattractant factor IL-16–soluble ligand for CD4þ, known to be involved in selective recruitment of T helper cells by B lymphocytes,30 and overexpression of XCL1- lymphotactin, known to attract T cells and natural killer cells,31 were evident in the ON CD19þ signature. Specifically the B-cell activation in early ON is demonstrated by overexpression of CD72 and CD79A/B genes followed by antibody production that occurs via IL-4 receptor (IL4R).32 The

IL4R receptor, a key molecule for B-cell activation and FIGURE 4. Myelin basic protein–peptide binding to CD40L-activated B 33 immunoglobulin production, was highly expressed in the cells. Flow cytometry analysis demonstrated 4-fold increase in the signature. The IL4R signaling mediated by CD23 overexpres- number of MBP-binding CD40L-activated B cells. The y-axis shows the sion34 leads to downstream activation of IRF4 and NFAT,35 absolute number of MBP-biotin–streptavidin-PE–positive B cells.

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