GDP-l-fucose synthase is a CD4+ T cell–specific autoantigen in DRB3*02:02 patients with 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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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)....
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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)....
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Frequently Asked Questions (12)
Q2. How did the authors integrate the responses from testing dual-defined mixtures into the original scoring matrix?
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.
Q3. What is the role of fucosylated glycans in neuronal development?
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.
Q4. How many peptides were identified that unexpectedly induced a Th1 response?
Fourteenimmunodominant peptides were identified that unexpectedly induced a Th1 response excludinga firm association between GDP-L-fucose synthase reactivity and pattern II demyelination.
Q5. What can be done to improve the understanding of MS?
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).
Q6. What is the peptide that was predicted with harmonic boost frame 2?
The third stimulatory peptide(KLLLHSGVEN) was predicted with harmonic boost frame 2 and belongs to a transmembraneprotein (FLJ37396).
Q7. How many CD4+ cells were grown in 96-well plates?
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.
Q8. What is the main specificity of a brain-infiltrating clonally?
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.
Q9. What is the main specificity of GDP-L-fucose synthas?
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).
Q10. How many peptides were predicted from the uniprot database?
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.
Q11. How many patients responded to GDP-L-fucose synthase?
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).
Q12. What is the likely explanation for the differences in the peptides predicted with harmonic boost?
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.