Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM
Authors: Peggy P Ho
Isobel A Hawes
K Christopher Garcia
Beatrix M Ueberheide
Victoria C Cotham
Michael Platten
Jeffrey E Dunn
Jae-Seung Moon
Christopher M Bartley
Lucas B Kipp
Mark S Anderson
R Camille Brewer
Manasi Iyer
J Bradley Zuchero
Gabriel-Stefan Nadj
Blake T Aftab
Joseph L DeRisi
Daniel Fernandez
Rachael J M Bashford-Rogers
Tobias V Lanz
Christopher B Lock
Kevin M Jude
Ryan D Schubert
Bianca Teegen
William H Robinson
Ricardo A Fernandes
Sara E Vazquez
Michael R Wilson
Lawrence Steinman
Alejandro M Gomez
Journal: Nature (2022)
Discipline: Multidisciplinary
Keywords: Multiple sclerosis; autoimmune disease; B lymphocytes; Epstein-Barr virus; molecular mimicry
Affiliations: Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA., Department of Neurology, Mannheim Center for Translational Neurosciences (MCTN), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany., Department of Neurology and National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany., The Geriatric Research, Education, and Clinical Center (GRECC), VA Palo Alto Health Care System, Palo Alto, CA, USA., Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Beckman Center for Molecular Medicine, Stanford, CA, USA., Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Beckman Center for Molecular Medicine, Stanford, CA, USA., Macromolecular Structure Knowledge Center, Stanford ChEM-H Institute, Stanford, CA, USA., Weill Institute for Neurosciences, Department of Psychiatry and Behavioral Sciences, University of California San Francisco, San Francisco, CA, USA., Weill Institute for Neurosciences, Department of Neurology, University of California San Francisco, San Francisco, CA, USA., Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA., Institute of Experimental Immunology, Euroimmun AG, Lubeck, Germany., Division of Neuroimmunology, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA., Department of Biochemistry and Molecular Pharmacology, NYU Perlmutter Cancer Center, NYU School of Medicine, New York, NY, USA., NYU Langone Health Proteomics Laboratory, Division of Advanced Research Technologies, NYU School of Medicine, New York, NY, USA., Preclinical Science and Translational Medicine, Atara Biotherapeutics, South San Francisco, CA, USA., Department of Medicine, Diabetes Center, University of California San Francisco, San Francisco, CA, USA., Chan Zuckerberg Biohub, University of California San Francisco, San Francisco, CA, USA., Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., DKTK Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany., Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. ., The Geriatric Research, Education, and Clinical Center (GRECC), VA Palo Alto Health Care System, Palo Alto, CA, USA. .

Abstract:  Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies. 

Retracted: No

PubPeer comments:
 *[left]*  Fig. 8 Lupeol downregulates p-AKT, p-ERK, and PCNA in PCNA-1 xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification, u00d7400). bu2013d The expression of p-AKT, p-ERK, and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification, u00d7400). *[right]* Figure 7 from [Liu et al (2016)] **[retracted]**.  Oxymatrine upregulation of Bax and caspase 3, and downregulation of Bcl-2 and Ki67 in SMMC-7721 xenografts. A, Hematoxylin and eosin (H E) staining analyses of the pathological features of the tumors from the 4 groups (original magnification, 400u0004). B-E, The expression of caspase 3, Bax, Bcl-2, and Ki67 in xenograft tumors was analyzed by immunohistochemistry . Overlaps marked. Lupeol or Oxymatrin? ![] , 
 *[above]*  Fig. 3 Hoechst staining of PCNA-1 cells treated with 15, 30, and 60 u03bcM lupeol for 36 h. a The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope (original magnification u00d7200). *[below]* Fig 3A from [Liu et al (2016)] **[retracted]**.  Oxymatrine treatment induced apoptosis in human hepatoma cells. A, The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope (original magnification 200u0004) . Two panels appear to be the same. ![file] , 
 *[right]* Fig. 8.  Lupeol downregulates p-AKT, p-ERK, and PCNA in PCNA-1 xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification, u00d7400). bu2013d The expression of p-AKT, p-ERK, and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification, u00d7400). *[left]* Fig. 8 from [Liu et al (2016)] .  Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification x400). bu2013d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification x400). Unexpected overlaps between different conditions and IHC stains. ![] , 
 *[right]* Fig. 7.  Lupeol inhibits the growth of PCNA-1 cells in vivo. The PCNA-1 cells were subcutaneously injected into mouse right axillary fossa to establish xenograft models, and mice were treated with either lupeol (30 and 60 mg kg) or control (positive: 20 mg kg 5-Fu; negative: sterile physiological saline). Lupeol significantly reduced the tumor growth in vivo. Four representatives of eight tumors in each group are shown . *[left]* Fig. 7 from [Liu et al (2016)] .  Lupeol inhibits the growth of GBC-SD cells in vivo. a GBC-SD cells were subcutaneously injected into mouse right axillary fossa to establish xenograft models and all mice were divided into four groups (n = 6 per group): control (sterile physiological saline), 5-Fu (20 mg kg), and lupeol (30 and 60 mg kg) treatment groups. Lupeol significantly reduced the tumor weight in vivo. ![file] Control or 5-FU? , 
 We feel so sorry for the errors which occurred inadvertently when processing and grouping the figures. Because the experiments and immunohistochemistry related to these articles were basically carried out at the same time, there was a possibility of partial overlap in the preservation of images and the selection of representative images, and it was easy to make a mistake. Fig 3 and Fig 8 were confused and duplicated with other pictures, the representative picture selection in Figure 7 was also flawed. Surely, I will ask the journal for the correction soon. Thank you very much for your efforts in making the scientific publication solid and clean. Sincerely. , 
 Top to bottom: Fig 3a from [Liu et al (2016a)] .  Combination treatment with OMT and 5-Fu-induced cell apoptosis in HCC cells. a The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . At right, panels rotated through 90 and 270 deg. Fig. 3a from [Liu et al (2015)] .  Hoechst staining of PCNA-1 cells treated with 15, 30, and 60 u03bcM lupeol for 36 h. a The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . Fig 3a from [Liu et al (2016b)] .  Oxymatrine treatment induced apoptosis in human hepatoma cells. A, The nuclei were stained by Hoechst 33342 and visualized under fluorescence microscope . Fig 3 from [Liu et al (2016c)] .  Apoptotic nuclear morphology changes induced by lupeol (15, 30, and 60 lM) treatment for 36 h, were observed by Hoechst 33342 staining in GBC-SD cell lines . Surprisingly many overlaps, given the differences in cell-lines and conditions. ![file] , 
 #3 *[right]* Fig. 8.  Lupeol downregulates p-AKT, p-ERK, and PCNA in PCNA-1 xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification, u00d7400). bu2013d The expression of p-AKT, p-ERK, and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification, u00d7400). *[left]* Fig. 8 from [Liu et al (2016)] .  Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification x400). bu2013d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification x400). ![] In addition, Fig 6d from [Liu et al (2016b)] .  Representative images of hematoxylin and eosin (H E) staining of tumor sections . PCNA row. ![file] Unexpected overlaps between different conditions and IHC stains. I have added some rectangles for the PCNA rows. , 
 Retracted [1 July 2021] . -------------------------------- The Editor-in-Chief has retracted this article at the request of the first author. Concerns were raised regarding a number of figures in this article, specifically: - Figure 7a: the four right tumors of the Control group appear to be identical to the 5-Fu (20mg kg) group of Figure 7a in [1] - Figure 8b: the lupeol (60mg kg) p-EGFR panel appears to partially overlap with the 5-FU (20mg kg) p-AKT panel of Figure 8b in [1] - Figure 8c: the lupeol (60mg kg) MMP-9 panel appears to partially overlap with the 5-FU (20mg kg) p-ERK panel of Figure 8c in [1] - Figure 8d: the Control PCNA panel appears to partially overlap with the Control PCNA panel of Figure 8d in [1] The first author has stated that some of the data could not be recovered. The Editor-in-Chief therefore no longer has confidence in the reliability of the data reported in the article. All authors agree to this retraction. ,