Abstract
Epidemiological data have identified Epstein–Barr virus (EBV) infection as the main environmental risk factor for multiple sclerosis, the predominant autoimmune disease of the central nervous system (CNS)1. However, how EBV infection initiates multiple sclerosis pathogenesis remains unclear. Here we demonstrate that EBV expands oligoclonal T-bet+CXCR3+ B cells that home to the CNS in humanized mice. Effector memory CD8+ T cells and CD4+ TH1 cells as well as CD4+ TH17 cells co-migrate to the brain of EBV-infected humanized mice. T-bet+CXCR3+ B cells can colonize submeningeal brain regions in the absence of other lymphocytes and attract T cells. Depletion of B cells with rituximab or blocking of CXCR3 significantly decreases lymphocyte infiltration into the CNS. Thus, we suggest that symptomatic primary EBV infection generates B cell subsets that gain access to the CNS, attract T cells and thereby initiate multiple sclerosis.
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Data availability
The scRNA-seq datasets analysed in this study are available from the Gene Expression Omnibus (Fig. 3; GSE299939) and Zenodo50 (Extended Data Fig. 5; https://doi.org/10.5281/zenodo.15602185). The multiplex immunofluorescence images are also available to download from Zenodo51 (https://doi.org/10.5281/zenodo.15599580).
Code availability
The code used to analyse scRNA-seq data and multiplex immunofluorescence images can be found via GitHub (https://github.com/EannaFennell/EBV-induces-CNS-homing-of-B-cells-attracting-inflammatory-T-cells). The package used for multiplex immunofluorescence analysis is also available from GitHub (https://github.com/EannaFennell/MISSILe).
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Acknowledgements
We thank the Functional Genomics Center Zürich of the University of Zürich and ETH Zurich, and in particular Q. Zhang, for support on the genomics analyses; K. Gehrmann for technical support with the humanized mice; J. Fullin for creating the graphical illustrations featured in the figures; and F.L. thanks L. Yuka for ongoing support. This research was in part financially supported by the Swiss National Science Foundation (310030_204470/1, 310030L_197952/1 and CRSII_222718_10000065), Cancer Research Switzerland (KFS-5896-08-2023-R), the Swiss MS Society (2023-17), the Swiss State Secretariat for Education, Research and Innovation for EU Horizon BEHIND-MS, CRPP-ImmunoCure of the University of Zürich, the Sobek Foundation, the Swiss Vaccine Research Institute, the Vontobel Foundation, Roche, Novartis and Viracta. L.R. was supported by career advancement grants from the University of Zurich (Forschungskredit, FK-21-034). É.F. is funded by HORIZON-TMA-MSCA-PF-GF (no. 101110227). G.F. is funded by Next Generation EU — NRRP M6C2 — Investment 2.1 Enhancement and strengthening of biomedical research in the NHS — PNRR-MCNT2-2023-12378122; CUP: C43C23000880006.
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F.L., I.P., É.F., S.S., L.R. and J. Mietz performed the in vivo experiments. F.L., I.P., E.B. and C.C. performed the in vitro EBV infection and transwell migration experiments. E.B. and A.M. performed the ChipCytometry experiments. É.F. and P.G. performed the bioinformatics analysis. I.L.-P., L.B., A.G., K.E. and J.S. performed light-sheet microscopy. M.L. and J. Mautner performed serological assays for viral and autoantigen recognition by IgM. G.P. and G.F. performed HLA typing. C.M. supervised the study. F.L., E.B. and C.M. wrote the first draft of the manuscript. All authors edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 T cell infiltration into the CNS and associated NfL release.
a, Weekly blood viral loads (IU/ml blood) of EBV-infected humanized NSG and BRGS-A2DR2 mice (p-value = 0.010256; NSG = 8, BRGS-A2DR2 = 8). b, IHC staining of EBV-infected BRGS-A2DR2 tongue and spleen sections, scale bar=100μm. c, Weekly blood viral loads (IU/ml) of EBV-infected humanized BRGS-A2DR2 mice (n = 28). d, Correlation between brain EBV viral load and bioluminescent signal (n = 23). e, UMAP of marker expression by T cell populations. f, Percentage of the indicated T cell differentiation subpopulations. g, Frequency of CD19+ B cells in brains of PBS or EBV-infected mice (PBS n = 20, EBV n = 23). h-j, Total numbers of CD3+, activated HLA-DR+CD4+ (p-value = 0.0435), and activated HLA-DR+CD8+ T cells (p-value = 0.0002) in brains of PBS or EBV-infected mice (PBS n = 15, EBV n = 15). k-l, Frequencies of HLA-DR+CD8+ (p-value < 0.0001) and HLA-DR+CD4+ T cells of PBS or EBV-infected mouse spleens (PBS n = 20, EBV n = 23). m-n, Frequency of CD4+ (naïve p-value = 0.0001, effector memory p-value < 0.0001) and CD8+ (naïve p-value < 0.0001, effector memory p-value < 0.0001) T cell differentiation subtypes of PBS or EBV-infected mouse spleens. o-p, Correlation between HLA-DR+CD4+ T cells and alanine aminotransferase activity or enzymatic creatinine concentrations in blood (n = 18). q, ELISA of neurofilament light chain (NfL) in plasma before (Baseline) and 6 weeks post-EBV infection (Week 6; p-value < 0.0001). r, Fold increase of plasma NfL levels before and 6 weeks post-EBV infection or PBS injection. For all correlations, solid line represents trend line obtained by linear regression, the dotted lines indicate 95% confidence bands, two-tailed p-value by Spearman correlation. Unless otherwise indicated, p-values by two-tailed MWU test. a, was generated from one experiment using mice reconstituted from one human CD34+ hematopoietic progenitor cells donor. b-m, were generated from 3 independent experiments with 4 donors. d, was created with crusty.humanitas.it. Error bar indicates SEM; each dot represents one single animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 2 Characteristics and origin of EBV expanded T-bet+CXCR3+ B cells.
a, Representative flow cytometric contour plots of T-bet+CXCR3+ and T-bet+CD11c+ B cell gating of splenocytes from PBS or EBV-infected humanized mice. b, Frequencies of CD19+CD11c+ B cells in blood, spleen and brains of PBS or EBV-infected humanized mice (PBS n = 21, EBV n = 28). c, Frequencies of CD19+CD11c+T-bet+ B cells in blood (p-value = 0.0277), spleen and brains of PBS or EBV-infected humanized mice (PBS n = 21, EBV n = 28). d, Total numbers of CD19+T-bet+CD11c+ B cells in blood, spleen and brains of PBS or EBV-infected humanized mice (PBS n = 21, EBV n = 28). e, Frequency of T-bet+CXCR3+ and T-bet-CXCR3- cells among CD11c+ B cells in blood (p-value = 0.0004), spleen (p-value < 0.0001) and brains of EBV infected mice. f, Frequency of T-bet+CXCR3+ and T-bet-CXCR3- cells among CD11c+ B cells in blood (p-value < 0.0001), spleen (p-value < 0.0001) and brains of PBS mice. g, In vitro EBV infection of CXCR3+ and CXCR3- B cells monitoring T-bet/CXCR3 co-expression frequency over time (p-values from left to right: 0.0211, 0.0160, 0.0016, 0.0007, 0.0039, <0.0001). h, In vitro EBV infection of CXCR3+ and CXCR3- B cells monitoring CD11c frequency over time (p-value = 0.0461). i, Growth assay of established LCLs derived from CXCR3+ or CXCR3- B cells over time depicted as total numbers. g-i, p-values were calculated using mixed-effects model with Geisser-Greenhouse correction followed by Sidak’s multiple comparisons test. Unless otherwise indicated, p-values were calculated using the two-tailed MWU test. Data from figures b-f, was generated from 5 independent experiments with mice reconstituted from 6 different human CD34+ hematopoietic progenitor cells donors. Error bar indicates SEM; each dot represents one single animal. Data from Figure g-i was generated from 3 independent experiment using 3 B cells donors. Error bar indicates SEM; dots represent mean of all 3 donors. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 3 Spatial analysis of T-bet+CXCR3+ B cells and activated T cells.
a, ChipCytometry of DNA (Helix-NP), CD39, CD45, CD11c and Ki67 on splenic section of PBS and EBV-infected mice. Scale bar=500μm. b, Frequency of T-bet+CXCR3+ of total B cells in spleen of PBS and EBV-infected mice (PBS n = 1, EBV n = 1). c, Frequency of EBNA2+ among T-bet+CXCR3+ and non-T-bet+CXCR3+ (other) B cells in spleen of EBV infected mice (PBS n = 1, EBV n = 1). d, Cell type enrichment per calculated tissue neighbourhood using HLA-DR as activation marker and e, frequency of respective tissue neighbourhoods (N-1 to N-5) in PBS and EBV-infected spleens (PBS n = 1, EBV n = 1). f-g, Frequency of activated CD4+ and CD8+ T cells (HLA-DR+) in the immediate microenvironment (ME; 100μm) of T-bet+CXCR3+ B cells in PBS and EBV-infected spleens (PBS n = 6, EBV n = 92 microenvironments of 1 experiment). P-values were calculated using two-sided MWU test (p-values = 0.00062 & 0.000045 respectively). h-i, Frequency of proliferative CD4+ and CD8+ T cells (Ki67+) in the immediate microenvironment (100μm) of T-bet+CXCR3+ B cells in PBS and EBV-infected (PBS n = 6, EBV n = 92 microenvironments of 1 experiment). P-values were calculated using two-sided MWU test (p-values = 0.0001 & 0.00014 respectively). Box plots define the medium value (centre), the interquartile range (25th and 75th percentiles) as the boundaries of the box and whiskers which extend to 1.5 times the 25th and 75th percentiles. Outliers outside the boundaries of the whiskers are displayed as individual points. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. j, Cell type enrichment per calculated tissue neighbourhood using Ki67 as a T cell proliferation marker and k, frequency of respective tissue neighbourhoods (N*-1 to N*-6) in PBS and EBV-infected spleens. l, 3D multiplex imaging by light sheet microscopy of cleared brains of two EBV-infected mice (EBV n = 3, PBS n = 1) stained for human CD3 (magenta), human CD45 (cyan) and mouse CD31 (yellow).
Extended Data Fig. 4 Transcriptional characteristics of T-bet+CXCR3+ B cells.
a, UMAP of T-bet+CXCR3+ among splenic B cells of EBV-infected humanized mice by scRNA-seq. b-c, Stacked bar plots showing (left) the CD19+ B cell population frequency and (right) immunoglobulin isotype distribution within T-bet+CXCR3+ and non-T-bet+CXCR3+ (other) cells. d, Expression of indicated Ig isotypes and e, expression of indicated EBV transcripts. f, Heatmap of shared clones between B cell subtypes with Morisita overlap index. g-h, Violin plot of expression of MKI67 (adjusted p-value = 1.41e-260) and pro-proliferative gene module (MKI67, PCNA, TOP2A, CCNB1, CCNB2, CDK1; adjusted p-value = 2.64e-157) between infected (EBV+) and un-infected (EBV-) T-bet+CXCR3+ B cells. i-j, Violin plot of expression of MKI67 (adjusted p-value = 2.105e-86) and pro-proliferative gene module (MKI67, PCNA, TOP2A, CCNB1, CCNB2, CDK1; adjusted p-value = 2.86e-32) between clonally expanded and diverse BCR expressing T-bet+CXCR3+ B cells. k, Violin plot of expression of TBX21 between infected (EBV+) and un-infected (EBV-) CXCR3+ B cells (adjusted p value = 1.706e-29). l, IGHV gene expression among the top 50 expanded B cell clones. IGHV4-34 and IGHV4-39 expressing clones are highlighted. The black line defines the mean expression of each group from g-j. P-values for differential gene expression analysis between groups were calculated using the two-sided MWU test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 5 CNS accumulation and EBV antigen recognition of oligoclonally EBV expanded B cells.
a, tSNE of single cell RNA sequencing (scRNA-seq) of sorted CD19+ B cells from spleen and perfused brains of EBV infected humanized BRGS-A2DR2 mice (n = 10) 6 weeks after EBV infection with manually annotated B cell differentiation clusters. b, BCR receptor clonality analysis. Clones were defined by Single (0 < X < = 1), Small (1 < X < = 5), Medium (5 < X < = 20), Large (20 < X < = 50), Hyperexpanded 1 (50 < X < = 200). c, Z-scored heatmap of 20 most expanded BCRs in matched perfused brain and spleen samples of the 10 analyzed mice (mouse #9, 12, 14, 17, 2, 21, 23, 4, 13, 8). d, Z-scored heatmap of IgM responses to the indicated EBV gene products in blood plasma of the same 10 by scRNA-seq analyzed EBV infected mice and three PBS controls (mouse #1, 6, 10). Plasma of mouse 1 and 10 was pooled due to low individual blood plasma volume.
Extended Data Fig. 6 Oligoclonal B cell expansion and autoantigen recognition after EBV infection.
a, Stacked bar plot showing B cell population frequency in spleen and brain of EBV-infected mice. b, tSNE of combined expression of all EBV transcripts split by organ of origin (spleen and brain). c, Stacked bar plot showing clonality distribution of non-naïve clusters (i.e. memory B cells, cycling B cells, plasmablasts & plasma cells) in spleen and brain of EBV-infected mice only. d-e, tSNE of sorted CD19+ B cell population colored by B cell populations (d) and BCR clonality (e), split by organ of origin (spleen and brain) of PBS mice only. Clones were defined by Single (0 < X < = 1), Small (1 < X < = 5), Medium (5 < X < = 20), Large (20 < X < = 50), Hyperexpanded 1 (50 < X < = 200). f, Shared spleen and brain BCR clones heatmap of individual PBS mice (most abundant 20 BCRs are plotted). BCR frequencies were calculated from total B cells per organ per mouse and were log2 normalised. g, TNFSF9 (encodes 4-1BBL/CD137L) expression in spleen and brain memory B cells. h, Distribution of IGHV gene in spleen versus brain B cells. The linear regression line (which represents the fitted values/mean predicted response from the linear model) and 95% confidence interval (in grey) are plotted. i, Blood plasma IgM antibody specificities against known autoantigens of all mice used for the scRNA-seq analysis. j, Replicate serum antibody specificities against EBV proteins and known autoantigens from pooled mice of previous EBV infection experiments. Pool #1 consisted of 2 mice (EBV infected), pool #2 and #3 consisted of 3 mice each (EBV infected), pool #4 from 3 mice (PBS controls). Pooled mice originated from individual CD34+ hematopoietic progenitor cell donors.
Extended Data Fig. 7 CNS homing and T cell attraction by EBV infected T-bet+CXCR3+ B cells.
a, Heatmap of indicated LCL marker frequency. b, Representative T-bet staining on LCL. c, Total mouse numbers with neuroinvasion. d-e, IHC staining of EBNA2 in indicated (f) meningeal and parenchymal brain areas. Scale bar=50μm. g, EBNA2+ cell frequency (n = 3 per region). h, T-bet and CXCR3 frequency of T-betlo and T-bethi. i, Correlation between blood CXCR3 frequency and neuroinvasion (n = 23). j, Correlation between blood T-bet frequency and neuroinvasion (n = 23). k, T-bet and CXCR3 frequency of LCL used for CXCR3 inhibition (TAK-779). l, EBV viral loads (IU/brain; T-betlo n = 12, T-bethi n = 11). m-n, Brain viral loads by bioluminescence after CXCR3 inhibition (TAK-779; PBS n = 6, CXCR3 antagonist n = 7) or Natalizumab treatment (p-value = 0.0401; PBS n = 8, Natalizumab n = 7). o, Transwell migration assays with indicated LCL supernatants. p, Frequency of migrated HLA-DR+CD8+ T cells (LCL spleen p-value = 0.0015). q, Transwell migration assays with LCL supernatant (p-value = 0.0015) with CCL3/4/5 (p = 0.0366) or CXCR3/CCR5 inhibition (TAK-779; p-value = 0.0125). r, Transwell migration assays with CXCL9+ medium or wild-type vs. EBNA3B deficient LCL supernatants (p-value = 0.0292). s, ELISA on wild-type and EBNA3B deficient LCL supernatants (p-value = 0.0004). t, Transwell migration assays with T-bethi (p-value = 0.0372) or T-betlo LCL supernatants. Each dot represents one well. For all correlations, solid line represents trend line obtained by linear regression, the dotted lines indicate 95% confidence bands two-tailed p-value was calculated using Spearman correlation. Data from figure a-c was generated from 3 independent experiments and h-n from 2 independent experiments with 2 LCL donors. o-r and t were tested with =>3 T cells donors. p-values were calculated using one-way ANOVA with Dunnett’s multiple comparison test. s, was generated using supernatant of n = 3 wild-type (WT) and n = 3 EBNA3B deficient LCLs (EBNA3A KO) in 2 independent experiments, p-value was calculated with two-way ANOVA followed by Sidak’s multiple comparisons test. Error bar indicates SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 8 CD20 targeted B cell depletion.
a-b, Weekly total numbers of CD45+CD19+ or CD20+ B cells in blood of EBV-infected mice with and without Rituximab treatment (PBS n = 5, EBV n = 4, Rituximab n = 7). p-values from left to right: CD20:0.0026, 0.0003,0.029,0.0120CD19: 0.0014,0.0004.0.0235,0.0110, (two-way mixed effects model with Geisser-Greenhouse correction with Tukey’s multiple comparisons test). Dots present mean and error bars SEM. c, Frequency and d, total numbers of CD19+ B cells in spleen (p-value < 0.0001) and brain with and without Rituximab treatment. e, Frequency of CD20+ B cells in spleen (p-value = 0.0007) and brain (p-value = 0.0011) with and without Rituximab treatment. f, Total numbers of CD20+ B cells in spleen (p-value = 0.0007) and brain (p-value = 0.0217) with and without Rituximab treatment. g, In vivo bioluminescence signal of perfused brains (PBS n = 5, EBV n = 4, Rituximab n = 7). h, Total numbers of CD19+CD11c+T-bet+CXCR3+ B cells in the blood (p-value < 0.0001), spleen (p-value = 0.0002), and brain (p-value = 0.0201) with and without Rituximab treatment (PBS n = 5, EBV n = 4, Rituximab n = 7). i, Total numbers of CD4+ T cells in the brain with and without Rituximab treatment (EBV n = 4, Rituximab n = 7). j, Correlation between activated HLA-DR+ CD4+ T cells and T-bet+CXCR3+ B cells in the brains of EBV infected humanized mice. Solid line represents trend line obtained by linear regression, the dotted lines indicate 95% confidence bands (n = 12). k, Total numbers of CD4+ T cell subtypes, including naïve (CD45Ra+CCR7+), effector memory (CD45Ra-CCR7-), central memory (CD45Ra-CCR7+) and Temra (CD45Ra+CCR7-) cells, with and without Rituximab treatment (EBV n = 4, Rituximab n = 7). a-k, were generated from 2 independent experiments with mice reconstituted from 2 different human CD34+ hematopoietic progenitor cell donors. Unless otherwise indicated, p-values were calculated using the two-tailed MWU test. Error bar indicates SEM; each dot represents one single animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 9 Gating strategy for human T and B cell populations ex vivo.
a, Representative flow cytometric contour plots showing FACS gating strategy used for gating on lymphocytes, single cells, live cells and human CD45+ cells followed by CD3 and CD19 gating. b, Representative flow cytometric contour plots following gating of Extended Data Fig. 9a for T cell populations. Depicted gating strategy was used for following FACS data panels: Fig. 1h–p and Fig. 5k–p and Extended Data Figs. 1g–p and 8i-k. c, Representative flow cytometric contour plots for gating of Extended Data Fig. 9a for B cell populations. Depicted gating strategy was used for following FACS data panels: Figs. 2a–d, 4e and 5b,c,h–j and Extended Data Figs. 1f, 2b–h, 7a,h,k and 7a–f,h.
Extended Data Fig. 10 Gating strategy for human T and B cell populations for in vitro experiments.
a, Representative flow cytometric contour plots showing FACS gating strategy used for sorting CD19+ B cells for data in Fig. 3 and Extended Data Figs. 4–6. b, Representative flow cytometric contour plots showing FACS gating strategy used for sorting CD19+CXCR3+ B cells for Extended Data Fig. 2g,h. c, Representative flow cytometric contour plots showing FACS gating strategy used for migration assays in Fig. 4h,i,k,l and Extended Data Fig. 7o–r,t.
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Läderach, F., Piteros, I., Fennell, É. et al. EBV induces CNS homing of B cells attracting inflammatory T cells. Nature 646, 171–179 (2025). https://doi.org/10.1038/s41586-025-09378-0
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DOI: https://doi.org/10.1038/s41586-025-09378-0
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