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Journal ArticleDOI

GDP-l-fucose synthase is a CD4+ T cell–specific autoantigen in DRB3*02:02 patients with multiple sclerosis

TL;DR: GDP-l-fucose synthase is an autoantigen recognized by cerebrospinal fluid–infiltrating CD4+ T cells from HLA-DRB3*–positive patients with multiple sclerosis, and the possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis is suggested.
Abstract: Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4 + T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)–l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid–infiltrating CD4 + T cells from HLA-DRB3*–positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.

Summary (3 min read)

INTRODUCTION

  • Multiple sclerosis (MS) is considered a demyelinating autoimmune disease of the central nervous system (1-4).
  • Several studies have reported distinct fecal microbial community profiles in MS patients compared to healthy controls (13-16), which might affect autoimmune responses by regulating permeability of the blood brain barrier (BBB), by modulating the maturation, activation and function of immune cells (12, 15-17), or by controlling the maturation and function of microglia (18) and astroglia (19).
  • T cells in MS may improve their understanding of disease mechanisms, help in diagnostic classification of MS patients and enable the development of antigen-specific immunotherapies (20).
  • Using the above methodological approach, the authors have identified GDP-L-fucose synthase as the main specificity of TCC21.1.

RESULTS

  • TCC isolated from CSF-infiltrating cells and clonally expanded in two active white matter demyelinating MS lesions (LI and LIII) from a SPMS patient (1154SA) with pattern II demyelination (26).
  • A similar but not identical response pattern was obtained with both readouts.
  • For frame 2 matrix, the top 40 peptides were previously predicted (Table S1).

T cell responses to immunodominant GDP-L-fucose synthase peptides are associated

  • With reactivity against MBP 83-99 T cells in CIS/MS patients against GDP-L-fucose synthase peptides, the authors compared it with reactivity against the seven immunodominant/encephalitogenic myelin peptides, which had previously been shown to be the targets of high avidity T cells in MS (34) (Fig.5A).
  • Four out of six (67%) of the high responder patients to GDP-L-fucose synthase also responded to myelin peptides, while only one of the six (17%) moderate responders and two of the 19 (11%) nonresponders did (Fig.5A).

T cell response to GDP-L-fucose synthase is associated with DRB3* alleles

  • Table 2 summarizes demographical and clinical characteristics of the CIS/MS patients and their designation as nonresponders, moderate responders, or high responders to GDP-L-fucose synthase peptides.
  • Interestingly, all six of the high responder patients are DRB3*02:02, while only one of the six (17%) moderate responders and four of the 19 (21%) nonresponders express this class II molecule.
  • Cross-recognition of human and bacterial GDP-L-fucose synthase peptides by CD4+ CSF- infiltrating T cells GDP-L-fucose synthase is a cytosolic enzyme that converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose that then is used to fucosylate oligosaccharides (35) including sugars and glycoproteins on mucosal intestinal cells.
  • Blast (NCBI) analysis of these bacterial peptides (Fig. S3C) revealed that peptide 3 was shared by bacteria from the genera Akkermansia and Prevotella, which both have been reported to be altered in MS patients (14).

DISCUSSION

  • T cell-mediated autoimmune disease it is crucial to elucidate the target antigens responsible for inducing CD4+.
  • Reactivity against GDP-L-fucose synthase peptides was also identified in bulk CSF-infiltrating CD4+.
  • The identification of TCC21.1 specificity using positional scanning libraries was less efficient than in previous studies (23, 25) due to recognition by this TCC of peptides with identical binding/contact motif but variable flanking C- and N-terminal AA.
  • Brain fucosylated glycans have been implicated in the molecular mechanisms that underlie neuronal development, survival and function (55, 58- 60) as well as in modulating immune responses.
  • T cells from four CIS/MS patients that were reactive against the human GDP-L-fucose synthase peptide 161-175 also recognized two gut microbial GDP-L-fucose synthase peptides from bacterial genera that have been associated with MS (13-16, 62-65) and that in addition showed the highest homology with the human peptide.

Patient Material

  • CSF-derived mononuclear cells and PBMCs were obtained from a SPMS patient with pattern II demyelinating lesions as previously described (26), also known as Patient 1154SA.
  • Demographical and clinical characteristics of the patients are summarized in Table 2.
  • All CIS patients had CSF-specific oligoclonal bands detected by isoelectric focusing (IEF).
  • Informed consent was obtained from all patients or relatives.
  • Demographical and clinical characteristics of MS patients and non-MS controls are summarized in Table S7.

Transcriptomic and Proteomic Analysis

  • Transcriptomic analysis of brain lesions was performed as previously described (26).
  • Reduction and alkylation was applied on the homogenate before samples were digested with Lys-C and trypsin.
  • Peptides were desalted on solid phase extraction columns (C18 Finisterre, Wicom Germany), vacuum dried, re- GDP-L-fucose synthase as putative autoantigen in MS 17   dissolved and measured (Nanodrop 1000, spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
  • Data analysis was performed with MASCOT software using a human UniProtKB/Swiss-Prot protein database (March 22, 2016 with 40912 entries).
  • Carbamidomethylation at cysteine was set as a fixed modification, and oxidation of methionine, n-terminal acetylation as variable modifications.

Positional Scanning Peptide Libraries and Individual Peptides

  • A synthetic N-acetylated, C-amide L-amino acid (AA) decapeptide combinatorial library in a positional scanning format (200 mixtures) and twenty-two dual defined mixtures were prepared as previously described (69).
  • Individual peptides (Table S1 and S2) were synthesized by Peptides and Elephants GmbH (Potsdam, Germany).

Cells and culture conditions

  • Bulk CSF-derived mononuclear cells from patient 1154SA were expanded as previously reported (26).
  • Medium consisted of IMDM (PAA) containing 100 U/ml penicillin/streptomycin (PAA), 50 µg/ml gentamicin (BioWhittaker, Cambrex), 2 mM L-glutamine (Gibco, Invitrogen, Carlsbad, CA) and 5% heat-decomplemented human serum (PAA).
  • After two weeks, cells were pooled (short expansion) or expanded by eight rounds of restimulation.
  • TCL-39 was generated from CSF-derived CD4+ CD4+ fractions were then seeded at 1500 cells per well in 96- well U-bottom microtiter plates together with 1.5 x 105 allogeneic irradiated PBMC, 1 µg/ml of PHA-L and IL-2 supernatant.

T cell stimulation

  • TCC responses to single/dual defined peptide mixtures or individual decapeptides were tested by seeding in duplicate 2 x 104 T cells and 5 x 104 irradiated BLS cell lines or autologous BCL or 1 x 105 irradiated PBMC (as indicated) with or without combinatorial peptide mixtures or individual decapeptides.
  • T Cell Activation Kit was used as positive control.

Cytokine measurement

  • Cytokines in the supernatant of stimulated and unstimulated TCC21.1 or expanded CSF- infiltrating T cells were measured after 48 h of culture using the Human T Helper Cytokine Panel LEGENDplex bead-based immunoassay , GM-CSF ELISA (BD Biosciences, Franklin Lakes, NJ) and IL-3 ELISA according to the manufacturer´s instructions.
  • For intracellular cytokine staining, TCC21.1 was analyzed 48 h after stimulation.
  • After 5 h in presence of GolgiStop protein transport inhibitor (BD Biosciences), T cells were labeled with Live/Dead® Aqua .
  • Following fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences), cells were stained with antibodies against CD4 (APC-Cy7, Biolegend), IFN-γ (FITC, Biolegend), IL-4 (PE, BD Bioscience), GM-CSF (APC, Biolegend) and IL-3 (PE, Biolegend) in PBS containing saponin and BSA, and analyzed by flow cytometry.

Proliferative Responses

  • Proliferation was measured 72 h after stimulation by 3H-thymidine (Hartmann Analytic, Braunschweig, Germany) incorporation in a scintillation counter (Wallac 1450, PerkinElmer, Rodgau-Jürgesheim, Germany).
  • The stimulatory index (SI) was calculated as follows: SI = Median (replicates cpm peptide) / Median (replicates cpm without peptide).
  • Proliferation was also measured using a Click-iTTM EdU Flow Cytometry Assay Kit (APC, Molecular Probes, Invitrogen) following manufacturer’s instructions.
  • Cells were stained with the following antibodies anti-CD3 (PE-Cy-7, e-Bioscience, San Diego, CA) and anti-TRBV-21 (FITC, Beckman Coulter, Brea, CA) and analyzed by flow cytometry.

RT-PCR and Sequencing of TCR Rearrangements

  • RNA extraction, reverse transcription and TCRα/β-chain (TRA/BV) sequencing of TCC21.1 and TCL-39 was assessed as previously reported (26).
  • TCR gene designations are in accord with IMGT nomenclature (ImMunoGeneTics, www.IMGT.org).
  • 20   Individuals were typed for HLA-class I and II molecules at Histogenetics LLC, NY, USA.
  • Isolation of DNA from whole blood with a final concentration of 15 ng/µl was performed with a standard DNA isolation protocol using a Triton lysis buffer and Proteinase K treatment.
  • The MHC class II binding predictions were made using the IEDB analysis resource Consensus tool (74, 75).

Statistical analysis

  • Three-cluster k-means analysis was performed on patient scores to group patients into three categories.
  • Associations between response levels of peptides, patients, and HLA status were all performed using Fisher’s Exact Test with Bonferroni-Holm correction applied as appropriate, with 5% significance.

FIGURE LEGENDS

  • Combination with biometric analysis to identify the specifity of TCC21.1.
  • In the CEF peptide pool and control beads graphs, each dot represents the median SIs of four wells for one individual patient.

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ZurichOpenRepositoryand
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www.zora.uzh.ch
Year:2018
GDP-l-fucosesynthaseisaCD4+Tcell–specicautoantigeninDRB3*02:02
patientswithmultiplesclerosis
Planas,Raquel;Santos,Radleigh;Tomas-Ojer,Paula;Cruciani,Carolina;Lutterotti,Andreas;
Faigle,Wolfgang;Schaeren-Wiemers,Nicole;Espejo,Carmen;Eixarch,Herena;Pinilla,Clemencia;
Martin,Roland;Sospedra,Mireia
Abstract:Multiple sclerosisis animmune-mediated autoimmune diseaseof the central nervoussys-
temthatdevelopsingeneticallysusceptibleindividualsandlikelyrequiresenvironmentaltriggers.The
autoantigensandmolecularmimicstriggeringtheautoimmuneresponseinmultiplesclerosisremainin-
completelyunderstood.Byusingabrain-inltratingCD4<jats:sup>+</jats:sup>Tcellclonethatis
clonallyexpandedinmultiplesclerosisbrainlesionsandasystematicapproachfortheidenticationofits
targetantigens,positionalscanningpeptidelibrariesincombinationwithbiometricalanalysis,wehave
identiedguanosinediphosphate(GDP)–<jats:sc>l</jats:sc>-fucosesynthaseasanautoantigenthatis
recognizedbycerebrospinaluid–inltratingCD4<jats:sup>+</jats:sup>TcellsfromHLA-DRB3*–
positivepatients.SignicantassociationswerefoundbetweenreactivitytoGDP-<jats:sc>l</jats:sc>-
fucosesynthasepeptidesandDRB3*02:02expression,alongwithreactivityagainstanimmunodominant
myelinbasicproteinpeptide.Theseresults,coupledwiththecross-recognitionofhomologouspeptides
fromgutmicrobiota,suggestapossibleroleofthisantigenasaninducerordriverofpathogenicautoim-
muneresponsesinmultiplesclerosis.
DOI:https://doi.org/10.1126/scitranslmed.aat4301
PostedattheZurichOpenRepositoryandArchive,UniversityofZurich
ZORAURL:https://doi.org/10.5167/uzh-158835
JournalArticle
AcceptedVersion
Originallypublishedat:
Planas,Raquel;Santos,Radleigh;Tomas-Ojer,Paula;Cruciani,Carolina;Lutterotti,Andreas;Faigle,
Wolfgang;Schaeren-Wiemers, Nicole;Espejo, Carmen;Eixarch, Herena;Pinilla, Clemencia;Martin,
Roland;Sospedra,Mireia (2018).GDP-l-fucose synthase is aCD4+ T cell–specic autoantigen in
DRB3*02:02patientswithmultiplesclerosis.ScienceTranslationalMedicine,10(462):eaat4301.
DOI:https://doi.org/10.1126/scitranslmed.aat4301

GDP-L-fucose synthase as putative autoantigen in MS
!
!
1!
GDP-L-Fucose Synthase As A Novel CD4
+
T Cell-Specific Autoantigen in DRB3*02:02
Multiple Sclerosis Patients
Authors: Raquel Planas
1
, Radleigh Santos
2
, Paula Tomas-Ojer
1
, Carolina Cruciani
1
, Andreas
Lutterotti
1
, Wolfgang Faigle
1
, Nicole Schaeren-Wiemers
3
, Carmen Espejo
4
, Herena Eixarch
4
,
Clemencia Pinilla
2
, Roland Martin
1
, Mireia Sospedra
1*
Affiliations:
1 Neuroimmunology and MS Research (nims), Department of Neurology, University Zurich,
Frauenklinikstrasse 26, 8091 Zürich, Switzerland
2 Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway Port St. Lucie, FL
34987, USA.
3 Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4031 Basel,
Switzerland
4 Servei de Neurologia-Neuroimmunologia, Centre d'Esclerosi ltiple de Catalunya, Vall
d'Hebron Institut de Recerca, Hospital Universitari Vall d'Hebron, 08035 Barcelona, Spain;
Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain.
*Corresponding author:
Mireia Sospedra, PhD
Neuroimmunology and MS Research (nims)
Department of Neurology
University Hospital Zürich
Frauenklinikstrasse 26
8091 Zürich
Switzerland
Tel. +41442553905
Fax. +41442558864
e-mail:
Mireia.SospedraRamos@usz.ch
One Sentence Summary: Using a brain-infiltrating T cell clone that is clonally expanded in
multiple sclerosis brain lesions and positional scanning peptide libraries in combination with
biometrical analysis, we have identified GDP-L-fucose synthase as a putative autoantigen that
is recognized by cerebrospinal fluid-infiltrating CD4
+
T cells from HLA-DRB3*-positive patients.

GDP-L-fucose synthase as putative autoantigen in MS
!
!
2!
ABSTRACT
Multiple sclerosis is a CD4
+
T cell-mediated autoimmune disease of the central nervous system
that develops in genetically susceptible individuals and likely requires environmental triggers.
The autoantigen/s and molecular mimic/s triggering the autoimmune response in multiple
sclerosis remain incompletely understood. By using a brain-infiltrating T cell clone that is
clonally expanded in multiple sclerosis brain lesions and an unbiased approach for the
identification of its target antigens, positional scanning peptide libraries in combination with
biometrical analysis, we have identified GDP-L-fucose synthase as a novel autoantigen that is
recognized by cerebrospinal fluid-infiltrating CD4
+
T cells from HLA-DRB3*-positive patients.
Significant associations were found between reactivity to GDP-L-fucose synthase peptides and
DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein
peptide. These results, coupled with the cross-recognition of homologous peptides from gut
microbiota, suggest a possible role of this antigen as inducer or driver of pathogenic
autoimmune responses in MS.

GDP-L-fucose synthase as putative autoantigen in MS
!
!
3!
INTRODUCTION
Multiple sclerosis (MS) is considered a demyelinating autoimmune disease of the central
nervous system (1-4). The inflammatory infiltrate in demyelinating brain lesions, the intrathecal
production of oligoclonal immunoglobulin G, the genetic trait consisting of multiple immune-
related genes (5, 6), the positive effect of immunotherapies targeting B and T cells, and the
similarities with the animal model experimental autoimmune encephalomyelitis, all support that
MS is an immune-mediated disease. The dominance of CD8
+
T cells in MS brain lesions and
the predisposition conferred by the HLA-A*03:01 allele and protection by HLA-A*0201 (6, 7) as
well as evidence in experimental animal models (8) hint at a relevant role of these cells in MS
pathogenesis. However, the fact that the HLA-DR15 haplotype is by far the strongest genetic
risk factor associated with MS and that immunization with myelin components induces relapsing
or chronic demyelinating disease models, which can be transferred by myelin-specific CD4
+
T
cells to naive animals, underscore that autoreactive CD4
+
T cells play a central role in MS
pathogenesis (1). Although myelin protein/peptides are considered relevant autoantigens in MS
(9), the full spectrum of target antigen(s) driving the immune response in this disease has yet to
be defined.
The etiology of MS involves both a complex genetic trait with more than 100 quantitative trait
loci (5, 6) and several environmental risk factors including vitamin D, smoking (10), viral
infections (11) and gut microbiota (12). Several studies have reported distinct fecal microbial
community profiles in MS patients compared to healthy controls (13-16), which might affect
autoimmune responses by regulating permeability of the blood brain barrier (BBB), by
modulating the maturation, activation and function of immune cells (12, 15-17), or by controlling
the maturation and function of microglia (18) and astroglia (19). In order to be able to cross the
BBB and infiltrate the brain, autoreactive T cells first have to be activated outside the CNS.
Cross-reactivity between autoantigens and peptides from pathogens or gut microbiota, i.e.
molecular mimicry, has been considered a putative trigger of the autoimmune reaction in MS.
The identification of autoantigen/s and molecular mimic/s targeted by pathogenic CD4
+
T cells
in MS may improve our understanding of disease mechanisms, help in diagnostic classification
of MS patients and enable the development of antigen-specific immunotherapies (20). In the
past, the search for candidate autoantigen/s in MS has concentrated on myelin components
based on the fact that demyelination is a hallmark of MS brain lesions. Furthermore,
immunological research in MS patients focused on studying myelin-specific T cells from the
peripheral blood, since the target tissue, i.e. brain and spinal cord, is rarely available for
immunological studies. Autoimmune diseases like primary biliary cirrhosis can be exquisitely

GDP-L-fucose synthase as putative autoantigen in MS
!
!
4!
organ- or tissue-specific, and nevertheless T cell autoreactivity be directed against ubiquitous
autoantigens such as pyruvate dehydrogenase (21). Thus, the focus on myelin-specific CD4
+
T
cells and on cells circulating in the peripheral blood has had limitations in the search for
candidate autoantigen/s and molecular mimic/s in MS. Non-myelin target antigens should
therefore also be considered in MS. To overcome these limitations, we (a) applied the unbiased
identification of stimulatory peptides using positional scanning peptide libraries (22, 23) and
biometrical analysis (24, 25), and (b) concentrated on putatively disease-relevant T cells that
infiltrated brain lesions and were clonally expanded locally. We focused on T cell clone (TCC)
21.1, a CD4
+
TCC that was identified in active pattern II demyelinating brain lesions from a
secondary progressive (SP) MS patient using deep T cell receptor (TCR) β-chain sequencing
and that was isolated from autologous cerebrospinal fluid (CSF)-infiltrating cells as previously
described (26). The release of Th2 cytokines and the ability to help B cells (26) supports a
pathogenic role of this TCC in pattern II demyelination that is mediated by antibodies and
complement (27). Using the above methodological approach, we have identified GDP-L-fucose
synthase as the main specificity of TCC21.1. Reactivity against this protein as well as against
myelin and putative molecular mimic/s has then been examined more broadly in CSF-infiltrating
CD4
+
T cells from MS patients and led to the identification of a novel target autoantigen in MS.

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Cites background from "GDP-l-fucose synthase is a CD4+ T c..."

  • ...In addition to myelin (150) and EBV (6), other antigenic targets of locally produced IgG and infiltrating T cells have been suggested, such as sperm-associated antigen 16 [SPAG16 (163)], neurofilament light, RAS guanyl-releasing protein 2 [RASGRP2 (4)], αB-crystallin and GDP-l-fucose synthase (135)....

    [...]

Journal ArticleDOI
TL;DR: The cellular immunology of relapsing multiple sclerosis was considered to be principally T-cell driven; however, this process is now understood to involve multiple cell types and their functionally distinct subsets as mentioned in this paper.
Abstract: Novel insights from basic and translational studies are reshaping concepts of the immunopathogenesis of multiple sclerosis and understanding of the different inflammatory responses throughout the disease course. Previously, the cellular immunology of relapsing multiple sclerosis was considered to be principally T-cell driven; however, this process is now understood to involve multiple cell types and their functionally distinct subsets. Particularly, relapsing multiple sclerosis appears to involve imbalanced interactions between T cells, myeloid cells, B cells, and their effector and regulatory subpopulations. The major contributors to such imbalances differ across patients. Several emerging techniques enable comprehensive immune cell profiling at the single-cell level, revealing substantial functional heterogeneity and plasticity that could influence disease state and response to treatment. Findings from clinical trials with agents that successfully limit new multiple sclerosis disease activity and trials of agents that inadvertently exacerbate CNS inflammation have helped to elucidate disease mechanisms, better define the relevant modes of action of current immune therapies, and pave the way for new therapeutic strategies.

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"GDP-l-fucose synthase is a CD4+ T c..." refers background in this paper

  • ...The release of T helper 2 (TH2) cytokines and its ability to help B cells (22) support a pathogenic role of this TCC in pattern II demyelination that is mediated by antibodies and complement (23)....

    [...]

  • ...In pattern II lesions, demyelination is mediated by deposits of antibody and complement in addition to macrophages and T cells (23)....

    [...]

Journal ArticleDOI
10 Aug 2011-Nature
TL;DR: In this article, a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, they have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci.
Abstract: Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

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Frequently Asked Questions (12)
Q1. How many peptides were identified in braintissue from other MS patients?

Seventeen GDP-L-fucose synthase peptides were identified in white and grey matter braintissue from other MS patients and non-MS controls by proteomic analysis. 

In order to integrate the responses from testing dual-defined mixtures into the original scoring matrix using the harmonic mean model, the authors first retested single defined mixtures along with the dual defined mixtures and used them to normalize all activity values to a single scale using linear regression. 

Brain fucosylated glycans have been implicated inthe molecular mechanisms that underlie neuronal development, survival and function (55, 58-60) as well as in modulating immune responses. 

Fourteenimmunodominant peptides were identified that unexpectedly induced a Th1 response excludinga firm association between GDP-L-fucose synthase reactivity and pattern II demyelination. 

T cellsin MS may improve their understanding of disease mechanisms, help in diagnostic classificationof MS patients and enable the development of antigen-specific immunotherapies (20). 

The third stimulatory peptide(KLLLHSGVEN) was predicted with harmonic boost frame 2 and belongs to a transmembraneprotein (FLJ37396). 

CD4+ fractions were then seeded at 1500 cells per well in 96-well U-bottom microtiter plates together with 1.5 x 105 allogeneic irradiated PBMC, 1 µg/ml ofPHA-L and IL-2 supernatant. 

In conclusion, using positional scanning peptide libraries and biometrical analysis the authors identifiedGDP-L-fucose synthase as the main specificity of a brain-infiltrating clonally expanded TCCmost likely involved in MS pathogenesis. 

Usingpositional scanning peptide libraries in combination with biometric analysis, the authors have identifiedGDP-L-fucose synthase as the main specificity of TCC21.1, a brain-infiltrating and clonallyexpanded TCC from a SPMS patient with pattern II demyelination (26). 

Of the 40 predicted natural brain peptides with highest scores for the frame 1matrix, 38 peptides were already predicted from the unbiased UniProt human database. 

Four out of six (67%) of the high responderpatients to GDP-L-fucose synthase also responded to myelin peptides, while only one of the six(17%) moderate responders and two of the 19 (11%) nonresponders did (Fig.5A). 

The twomost stimulatory peptides, NVLHSAFEVG predicted with harmonic boost frame 1 andDNVLHSAFEV predicted with harmonic boost frame 2, belong to GDP-L-fucose synthaseencoded by the TSTA3 gene, and overlap by 9 AAs.