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CellImmunologyPeer-reviewed journal article

Anoctamin-2-specific T Cells Link Epstein-Barr Virus to Multiple Sclerosis

Olivia G. ThomasiD, Urszula Rykaczewska, Marina GalesiciD, Rianne T. M. van der BurgtiD, Nils Hallen, Filippo Ferro, Mattias BrongeiD, Zoe Marti, Yue LiiD, Alexandra Hill Rique, Jianing LiniD, Aleksa KrsticiD, Alicja Gromadzka, Andras Levente Szonder, Chiara SoriniiD, Maria Reina-CamposiD, Ting SuniD, Leslie A. KirbyiD, Özge DumraliD, Rasmus BerglundiD, Majid Pahlevan KakhkiiD, Milena Z. Adzemovic, Manuel ZeitelhoferiD, Birce Akpinar, Katarina TengvalliD, , Tomas P OlssoniD

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Abstract

Multiple sclerosis (MS) occurs when the central nervous system (CNS) is damaged by misguided adaptive immune responses, likely caused by a combination of environmental factors in genetically susceptible individuals. A known prerequisite for disease is Epstein-Barr virus (EBV) infection, and previous studies have demonstrated elevated Epstein-Barr virus nuclear antigen 1 (EBNA1) antibodies which cross-react with the calcium-activated chloride channel anoctamin-2 (ANO2) in persons with MS (pwMS). ANO2-reactive antibodies have been associated with greater neuroaxonal damage in MS, although their exact effector function is still uncertain. Here, we demonstrate that ANO2 is also the target of IFN{gamma}-producing CD4+ T cells, which are more frequent in untreated and natalizumab-treated pwMS compared to control individuals. Immunisation of SJL/J mice with either ANO2 or EBNA1 elicited cross-reactive CD4+ T cell and antibody responses in vivo. Pre-immunisation of young mice with ANO2 worsened proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which in older mice included atypical clinical phenotype, immune infiltration into the brain and reduced survival. EAE exacerbation was recapitulated with the adoptive co-transfer of ANO2 and myelin antigen-specific CD4+ T cells, and ANO2-specific T cells alone could induce the cell death of ANO2-expressing glial cells in vitro. T cell clones with cross-reactivity to both EBNA1 and ANO2 antigens could be isolated from natalizumab-treated pwMS. Single cell sequencing of EBNA1 and ANO2-specific T cell receptors (TCR) from four pwMS revealed a significant overlap between their antigen-specific expanded TCR repertoires within donors and transcriptomic analysis showed cross-reactive T cells to have predominantly activated and cytotoxic phenotypes. In summary, we report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS-associated autoantigen ANO2, thereby establishing a link between EBV infection and development of autoimmune neuroinflammatory disease.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (5 Dec 2025)

CD4+ T cells cross-reactive between Epstein-Barr virus EBNA1 and the central nervous system channel anoctamin-2 (ANO2) are elevated in multiple sclerosis and exacerbate experimental autoimmune encephalomyelitis in mice.

Epstein-Barr virus (EBV) infection is a prerequisite for multiple sclerosis (MS) and cross-reactive antibodies between EBNA1 and anoctamin-2 (ANO2) associate with neuroaxonal injury, yet antibodies alone cannot initiate demyelinating disease. This study shows that EBNA1-primed CD4+ T cells cross-react with ANO2 in MS patients and drive CNS vascular leakage, glial killing, and disease exacerbation in mice.

Key results

  1. ShownANO2-reactive pro-inflammatory T cells are significantly elevated in untreated and natalizumab-treated persons with multiple sclerosis and strongly discriminate cases from controls.

    In FluoroSpot assays with ANO2(79-168) beads, 56.7% of untreated pwMS (n = 30) had >10 IFN-gamma SFU per 2.5x10^5 PBMCs vs healthy controls (n = 20) and OND (n = 19), yielding ROC AUCs of 0.77 for IFN-gamma (P = 0.0002), 0.75 for IL-17A (P = 0.0005), and 0.85 for IL-22 (P < 0.0001), with spectral flow cytometry confirming higher CD4+ IFN-gamma+ ANO2 responses in natalizumab-treated pwMS (n = 12 vs 9 HC). · T cell responses targeting Anoctamin-2 are…; T cell responses to other regions of ANO2 are…; Figure legends

  2. ShownImmunisation of SJL/J mice with ANO2 or EBNA1 reciprocally cross-primes CD4+ T cells in vivo and exacerbates proteolipid protein-induced experimental autoimmune encephalomyelitis independently of autoantibodies.

    Immunising SJL/J mice with ANO2(1-275) or EBNA1(380-641) proteins, peptide pools, or homologous minimal peptides ANO2(137-151) and EBNA1(428-444) cross-primed CD4+ IFN-gamma+ T cells to the reciprocal antigen; pre-immunisation with either ANO2(1-275) protein or overlapping 15-mer peptides worsened subsequent PLP(139-151)-induced EAE scores and AUC at day 31 despite lacking anti-ANO2 or anti-EBNA1 IgG in peptide mice. · ANO21-275 and EBNA1380-641 immunisation…; ANO21-275 pre-immunisation cross-primes…; Protein versus peptide ANO21-275…; Figure legends

  3. ShownANO2 localizes to CD31+ vasculature in the mouse cerebellum, septum, and inferior olivary nucleus, where ANO2-specific CD4+ T cells exacerbate atypical EAE, blood-brain barrier breakdown, and oligodendrocyte apoptosis.

    In 6-month-old SJL/J mice (n = 23 ANO2/PLP vs 22 adjuvant/PLP), ANO2(1-275) pre-immunisation reduced survival and doubled atypical EAE; RNAscope and iDISCO+ (n = 9 brains) showed ANO2 co-expression with CD31+ endothelium in cerebellum (76%), septum (85%), and inferior olivary nucleus (72%); co-transferring ANO2- and MOG-specific CD4+ T cells increased BBB leakage, and ANO2 T cells killed oligodendrocytes in vitro. · ANO21-275 pre-immunisation of adult mice gives…; ANO2 co-localises with CD31 staining in the…; Adoptive transfer of ANO21-275-specific T cells…; ANO21-275-specific T cells can directly target…

  4. ShownSingle-cell TCR and transcriptome sequencing of CD4+ T cells from pwMS reveals substantial repertoire overlap between EBNA1 and ANO2 with activated and cytotoxic phenotypes.

    In HLA-DRB1*15:01+ pwMS, isolated T cell clones and HLA-DR2a/DR2b-restricted lines recognized both EBNA1(380-641) and ANO2; across 27,524 single-cell TCRs from 4 natalizumab-treated pwMS, ~25-26% of expanded EBNA1 and ANO2 TCRs were shared (55 clonotypes shared exclusively between EBNA1 and ANO2 and 43 tri-cross-reactive with CRYAB), mapping to activated (S1PR1, CCR6, RORC) and cytotoxic (GZMB, PRF1, GNLY) clusters. · Dual-specific T cell clones with reactivity to…; Single cell TCR sequencing of EBNA1- and…; EBNA1-, ANO2-specific and cross-reactive T cells…; Figure legends

Methods, data and reproducibility

Measured ex vivo T cell and IgG reactivity to recombinant ANO2 and EBNA1 proteins and peptides in untreated and natalizumab-treated pwMS and controls via FluoroSpot, Cytek Aurora spectral flow cytometry, and Luminex bead arrays. Assayed in vivo cross-priming, EAE severity, iDISCO+ clearing, Atto647-plasma BBB leakage, and Incucyte glial killing in mice, plus 10x 5' scRNA/TCR-seq of 4 MS-Nat donors.

  • Human PBMCs from untreated MS (MS-Un), natalizumab-treated MS (MS-Nat), healthy controls (HC), and other neurological diseases (OND) for FluoroSpot and spectral flow cytometry · human · n = Cohort 1: MS-Un n=30, HC n=20, OND n=19; Cohort 2: MS-Nat n=61, HC n=28
  • Plasma samples from the Swedish Epidemiological Investigation of Multiple Sclerosis (EIMS) cohort for suspension bead array autoantibody profiling · human · n = 713 pwMS and 722 matched population controls
  • 10x Chromium 5' single-cell RNA and TCR sequencing of CFSE-dim CD45RA-CD4+ T cells stimulated with anti-CD3, ABD, EBNA1(380-641), ANO2(1-275), and CRYAB · human · n = 4 natalizumab-treated pwMS; 27,524 TCRs and 27,076 single-cell transcriptomes
  • Mouse antigen recall, EAE pre-immunisation, adoptive transfer, iDISCO+ whole-mount brain imaging, and primary glial/neuronal co-cultures · mouse (C57BL/6Ntac, NOD, SJL/J, and SJL/J x C57BL/6-GFP F1) · n = 9 brains for iDISCO+; 6-mo SJL/J EAE: n=22 adjuvant/PLP, n=23 ANO2/PLP · https://mouse.brain-map.org/
  • Public MS blood and CSF bulk TCRbeta CDR3 repertoire datasets (Gottlieb et al. and Amoriello et al.) · human · GSE250037
Methods & tools
CellRanger, Seurat, DropletUtils, Scirpy, Scanpy, Muon, Harmonypy, PyDESeq2, fgsea, FlowJo, ImageJ (Fiji), Imaris
Software & environment
R 4.2.3, Python 3.10, Scirpy 0.19.0, Scanpy 1.10.3, Muon 0.1.7, DropletUtils 1.18.1, Seurat 4.3.0, AnndataR 0.99.0, vegan 2.6-4, msigdbr 7.5.1, ImageJ (Fiji) 1.54j, Imaris 10.2.0, Amira 3D 2024.1, GraphPad Prism v10
Reproducibility gaps
Full analysis code is stated to be supplied upon request; no public accession identifier is printed for the newly generated 10x single-cell RNA and V(D)J sequencing dataset.
Limitations (3)
  • Single-cell TCR and transcriptome sequencing was restricted to in vitro antigen-expanded CD4+ T cells from 4 natalizumab-treated pwMS and did not include healthy controls or patients with other EBV-associated autoimmune diseases. · stated by the authors
  • Natalizumab blocks lymphocyte trafficking into the CNS and gut and promotes peripheral lymphocyte activation, which could enrich autoreactive T cells and influence ex vivo and expansion assays. · stated by the authors
  • Overlap analysis with external blood and CSF TCR repertoires relied on bulk TCRVbeta CDR3b chains without paired TCRValpha sequences, preventing definitive confirmation of shared antigen specificity. · stated by the authors

Read this if you investigate Epstein-Barr virus molecular mimicry, autoreactive T cell repertoires, or neuroinflammation in multiple sclerosis and experimental autoimmune encephalomyelitis.

Citations
40
FWCI
182.97
Field percentile
top 1%

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