Research ArticleMultiple Sclerosis

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

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Science Translational Medicine  10 Oct 2018:
Vol. 10, Issue 462, eaat4301
DOI: 10.1126/scitranslmed.aat4301
  • Fig. 1 Single- and dual-defined decapeptide positional scanning mixtures in combination with biometric analysis to identify the specifity of TCC21.1.

    (A) GM-CSF production by TCC21.1 in response to 20 combinatorial peptide mixtures with amino acids defined at position 5 and presented by BLS-BCL expressing only DRB1*15:01, DRB5*01:01, or DQB1*06:02 class II molecules. x axis, single-letter amino acid code; y axis, cytokine production. (B) GM-CSF (black histograms) and interleukin-10 (IL-10; white histograms) production by TCC21.1 in response to a complete decapeptide positional scanning library (200 mixtures) presented by BLS-BCL expressing only DRB1*15:01. (C) Score matrix designed with the log10 median of GM-CSF production of three independent experiments. Dark gray cells correspond to values ≥2, and light gray cells correspond to values between 1.5 and 2. Bold borders show mixtures selected for dual-defined mixtures, and continuous and dotted borders were based on GM-CSF and IL-10 results, respectively. (D) GM-CSF production by TCC21.1 in response to the 50 peptides with highest scores predicted using the GM-CSF–based score matrix. (E) GM-CSF production by TCC21.1 in response to 22 dual-defined mixtures. In green, mixtures with defined amino acids of the TCR motif in frame 1 and in blue in frame 2. Stimulatory responses of mixtures shown with darker colors (frame 1/2-HM) were integrated into the original matrix using the HM model. (F) TCR motif and dual-defined mixture activity values selected on the basis of the HM model and incorporated into the original matrix, frame 1-HM (green), and frame 2-HM (blue). (G) GM-CSF production by TCC21.1 in response to the 50 peptides with higher scores predicted using harmonic boost frame 1 and frame 2 score matrices. Complete decapeptide library, dual-defined mixtures, and individual decapeptides were presented by BLS-BCL expressing DRB1*15:01. Mixtures and individual decapeptides were tested at 200 and 5 μg/ml, respectively, for 72 hours. Histograms show means ± SEM and dot plots mean of three independent experiments. Cytokine released is expressed as pg/ml released by TCC21.1 in response to stimuli minus pg/ml released in the absence of stimulus (negative control).

  • Fig. 2 Characterization of TCC21.1 response to GDP-l-fucose synthase peptides.

    (A) GM-CSF production by TCC21.1 in response to 11 decapeptides overlapping by nine amino acids presented by BLS-BCL expressing DRA*01:01/DRB1*15:01 and tested at 0.5 and 5 μg/ml. (B and C) GM-CSF production by TCC21.1 in response to GDP-l-fucose synthase peptide (96–105) presented by BLS-BCL expressing DRA*01:01/DRB1*15:01, DRA*01:01/DRB5*01:01, or DQA1*01:02/DQB1*06:02 class II molecules. (D) GM-CSF production by TCC21.1 in response to 13 A scan peptides presented by BLS-BCL expressing DRA*01:01/DRB1*15:01. Alanine substitution is shown in gray. (E and F) Proliferative responses (white bars) and production of cytokines (black bars) by TCC21.1 in response to GDP-l-fucose synthase peptides presented by autologous peripheral blood mononuclear cells (PBMCs) or autologous BCL and nonspecific stimulus (anti-CD3 and PMA; anti-CD3 and anti-CD28; and anti-CD3, anti-CD28, and anti-CD2 beads). (F) Proliferative responses (white bars) and GM-CSF and IL-3 production (black bars) by TCC21.1 and other TCCs from other patients with MS with different functional phenotypes in response to the corresponding specific peptides presented by autologous PBMCs (for TCC21.1, also autologous BCL) and to nonspecific stimuli (anti-CD3 and PMA; anti-CD3 and anti-CD28; and anti-CD3, anti-CD28, and anti-CD2 beads for TCC21.1; and only anti-CD3, anti-CD28, and anti-CD2 beads for the other TCCs). Cytokine release is expressed as pg/ml released by the TCC in response to stimuli minus pg/ml released in the absence of stimulus (negative control). All results show the means ± SEM. Dotted lines show a putative threshold for positivity: stimulatory index (SI) = 2 in proliferation histograms and the amount of interferon-γ (IFN-γ), IL-4, IL-17, IL-9, and IL-22 released by T cells with TH1, TH2, TH17, TH9, or TH22 functional phenotype, respectively. (G) Representative flow cytometry analysis of intracellular IL-4, IFN-γ, GM-CSF, and IL-3 production by TCC21.1 in response to GDP-l-fucose synthase peptide (96–105) and PMA/ionomycin (Io). Numbers represent the percentage of positive cells. (H) Representative flow cytometry analysis of the mean fluorescence intensity of CD28, CCR4, CCR6, and CRTH2 on the surface of resting TCC21.1 (black lines) in comparison to the isotype control staining (gray shaded). Flow cytometry analysis of TCC21.1 was performed three times after independent expansions.

  • Fig. 3 Recognition of GDP-l-fucose synthase peptides by TCC21.1 and CSF-infiltrating CD4+ T cells from patient 1154SA.

    (A) Proliferative responses expressed as SI of TCC21.1 and CSF-infiltrating CD4+ T cells after short and long PHA expansion to GDP-l-fucose synthase, myelin, or CEF peptides presented by autologous PBMCs, as well as to control anti-CD3, anti-CD28, and anti-CD2 beads. TCC21.1 graph shows the mean SI ± SEM, whereas, in the CSF graphs, each dot represents one well. Positive peptides are shown in black. (B) Proliferation of long-expanded CSF-infiltrating CD4+ T cells in response to GDP-l-fucose synthase peptide (91–105), CEF pool, or PHA using BLS-BCL DRA*01:01/DRB1*15:01 as APCs and assessed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Numbers represent the percentage of EdU+ cells. (C) Proliferative responses expressed as stimulation SIs of positive wells to GDP-l-fucose synthase peptide (191–205) for generation of TCL-39. (D) TCRBV sequencing of TCCs present in TCL-39. (E) Production of cytokines of long expanded CSF-infiltrating CD4+ T cells to GDP-l-fucose synthase peptides (91–105) and (191–205) presented by autologous PBMCs and to control beads. Cytokine release is expressed as pg/ml released by stimulated cells minus pg/ml released in the absence of stimulus (negative control). Results show the means ± SEM.

  • Fig. 4 Recognition of GDP-l-fucose synthase peptides by CSF-infiltrating CD4+ T cells from moderate- and high-responder patients with CIS/MS.

    (A) Proliferative responses expressed as SIs of CSF-infiltrating CD4+ T cells (single round of PHA expansion) to GDP-l-fucose synthase or CEF peptides presented by autologous PBMCs, as well as to control anti-CD3, anti-CD28, and anti-CD2 beads. Each dot represents one well, and median SIs are shown as solid lines. Positive peptides (black dots and red line) are peptides with median of all SIs > 1.455 (dotted red line). All peptides have been tested in four wells, except for peptides (156–170) and/or (161-175) in 748UR, 1129RE, 1005ME, 1173DI, 718MA, and 776JE, which were tested also for bacterial peptides by seeding eight wells. (B) Number of patients with CIS/MS with CSF-infiltrating CD4+ T cells that responded to GDP-l-fucose synthase peptides. Immunodominant peptides that were positive in at least three patients are shown in red. Solid red indicates high responder, and light red indicates moderate responder patients. (C) Production of cytokines of CSF-infiltrating CD4+ T cells from three high-responder patients to GDP-l-fucose synthase immunodominant peptides presented by autologous PBMCs.

  • Fig. 5 Recognition of myelin peptides by CSF-infiltrating CD4+ T cells from patients with CIS/MS.

    (A) Proliferative responses expressed as SIs of CSF-infiltrating CD4+ T cells (single round of PHA expansion) to myelin peptides presented by autologous PBMCs. Each dot represents one well, and median SIs are shown as solid lines. Positive peptides (black wells and red line) are peptides with median of all SIs > 1.455 (dotted red line). (B) Checkerboard graph illustrating GDP-l-fucose synthase immunodominant (red) and myelin (blue) peptides recognized by each patient with CIS/MS. Solid colors are GDP-l-fucose synthase and myelin peptides. The number of patients that responded (Y, yes) and that did not respond (N, not) to the peptides and the P values for Fisher’s exact tests with Bonferroni-Holm correction are shown.

  • Fig. 6 Recognition of bacterial GDP-l-fucose synthase peptides by CSF-infiltrating CD4+ T cells from patients with CIS/MS.

    Proliferative responses expressed as SIs of CSF-infiltrating CD4+ T cells (single round of PHA expansion) to eight bacterial GDP-l-fucose synthase peptides, sharing with the human homologous peptide at least 6 of the 15 amino acids (blue), presented by autologous PBMCs. Each dot represents one well, and median SIs are shown as solid lines. Positive peptides (black wells and red line) are peptides with median of all SIs > 2 (dotted red line).

  • Table 1 GDP-l-fucose synthase transcripts and peptides identified in brain tissue.

    RPKM, reads per kilobase of exon model per million mapped reads; PSMs, peptide spectrum matches. Proteome data correspond to white matter (WM) and gray matter (GM) from patients with MS (MS; n = 15) and controls without MS (non-MS; n = 9). In bold are GDP-l-fucose synthase values.

    ProteinGene
    ID
    TranscriptomeUniProt
    ID
    Peptide sequenceResiduesProteome (PSMs)DistinctProtein
    RPKMMSNon-MSTotalPeptidesCoverage
    LI*LIILIIIWMGMWMGM#%
    GDP-l-fucose synthaseTSTA32.172.022.67Q13630ILVTGGSGLVGK10–2112121212481756.0
    VVADGAGLPGEDWVFVSSK26–441212121248
    DADLTDTAQTR45–551212121248
    VQPTHVIHLAAMVGGLFR61–7899927
    YNLDFWR82–881212121248
    YNLDFWRK82–8911
    NVHMNDNVLHSAFEVGAR90–10713131339
    NVHMNDNVLHSAFEVGARK90–10833
    VVSCLSTCIFPDK109–12133
    MIDVQNR149–155224
    RMIDVQNR148–155224
    SSGSALTVWGTGNPR200–21433
    SSGSALTVWGTGNPRR200–21511
    TTYPIDETMIHNGPPHNSNFGYSYAK222–24733
    EYNEVEPIILSVGEEDEVSIK233–25333
    TYLPDFR291–2971212121248
    LRTYLPDFR289–29722
    63878895333
    Myelin basic proteinMBP37.2028.9759.21P0268699387198586537148079.6
    Myelin proteolipid proteinPLP1458.31341.43431.91P602011481531291575871330.6
    Myelin-oligodendrocyte
    glycoprotein
    MOGQ166532161992222168532849.8
    Myelin-associated
    glycoprotein
    MAG16.2910.8326.84P209162432322212439393436.9
    Myelin-associated
    oligodendrocyte
    basic protein
    MOBP14.4811.1619.82Q138752012271473626.2
    Oligodendrocyte myelin
    glycoprotein
    OMG5.215.073.26P235151441551361555901328.8
    2′,3′-cyclic-nucleotide
    3′-phosphodiesterase
    CNPP09543116898511571060437011296.4

    *LI and LIII, active lesions in which TCC21.1 was clonally expanded. LII, inactive lesion in which TCC21.1 was not identified.

    • Table 2

      Demographic and clinical characteristics of patients with CIS/MS and HLA-DR/DQ typing. RRMS, relapsing remitting MS.

      PatientDiseaseSexAgeIgGCSFHLA
      IDIndex# of
      cells/μl
      DRB1*DRB1*DRB3*DRB3*DRB4*DRB4*DRB5*DRB5*DQA1*DQA1*DQB1*DQB1*
      Nonresponders
      66JORRMSF382.89815:0115:011:011:011:021:026:026:02
      103HRCISM370.68915:0115:011:011:011:021:026:026:02
      0204AMRRMSF381.04815:0115:011:011:011:021:026:026:02
      0247PESPMSF391.5315:017:011:031:011:022:016:023:03
      0381MASPMSF450.5315:014:011:011:011:0203:XX6:023:02
      0702ANRRMSM450.73515:011:031:011:025:013:016:02
      0740RACISF350.534:017:011:011:012:013:012:013:01
      0936MARRMSF310.66215:0115:011:011:011:021:026:026:02
      0973JORRMSM220.511815:014:021:011:011:023:013:026:02
      1125PARRMSM410.6364:0214:542:021:011:013:013:025:03
      1292DIRRMSF271.281015:0116:011:012:021:021:025:026:02
      1300EVRRMSF370.5723:018:012:024:015:012:014:02
      0897URRRMSF341.97715:018:011:011:024:014:026:02
      0127ROCISM301.382715:018:011:011:024:014:026:02
      1290JASPMSM270.5815:014:041:011:013:026:021:0203:XX
      0800THRRMSM220.52715:0111:042:021:011:025:013:016:02
      0830OLCISF360.56415:0111:012:021:011:025:013:016:02
      0818MARRMSM370.6733:014:071:011:013:015:012:013:01
      1206CORRMSF371.37515:014:011:011:011:023:013:026:02
      Moderate responders
      0780URRRMSM271.19815:019:011:011:011:023:013:036:02
      0866JERRMSF301.141011:0113:011:012:021:035:013:016:03
      0748URRRMSM531.49715:0115:011:011:011:021:026:026:02
      1346JURRMSF211.09127:0113:011:011:011:032:012:016:03
      1081SACISF490.71231:018:031:016:013:015:01
      0817JDRRMSM280.53615:013:011:011:011:025:012:016:02
      High responders
      1129RERRMSM320.9237:0111:032:021:032:015:013:013:03
      1005MECISF330.76415:033:012:021:011:025:012:016:02
      1173DIRRMSM320.51144:0411:042:021:013:015:013:023:01
      0718MARRMSF360.54115:013:012:021:015:015:012:013:01
      0816DACISF521.141215:0113:012:021:011:031:026:026:03
      0776JECISF280.763:018:032:025:016:012:013:01

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/462/eaat4301/DC1

      Materials and Methods

      Fig. S1. Integration of stimulatory response from testing dual-defined mixtures into the original scoring matrix using the HM model.

      Fig. S2. Response of CSF-infiltrating CD4+ T cells.

      Fig. S3. New patients and phylogeny of bacterial species sharing GDP-l-fucose synthase peptides.

      Table S1. Summary of human decapeptides predicted with the biometrical approach, synthesized, and tested for stimulatory capacity.

      Table S2. GDP-l-fucose synthase, myelin, and CEF peptides.

      Table S3. Peptides from brain proteins identified by proteomic analysis in brain tissue.

      Table S4. Negative bootstrap Monte Carlo summary.

      Table S5. Patient classification and HLA binding.

      Table S6. Bacteria selected for GDP-l-fucose synthase comparison.

      Table S7. Sequence identity between human and bacterial GDP-l-fucose synthase peptides.

      Table S8. Demographic and clinical characteristics of patients with MS and controls without MS.

      Table S9. Primary data.

      References (6775)

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Integration of stimulatory response from testing dual-defined mixtures into the original scoring matrix using the HM model.
      • Fig. S2. Response of CSF-infiltrating CD4+ T cells.
      • Fig. S3. New patients and phylogeny of bacterial species sharing GDP-L-fucose synthase peptides.
      • Table S1. Summary of human decapeptides predicted with the biometrical approach, synthesized, and tested for stimulatory capacity.
      • Table S2. GDP-L-fucose synthase, myelin, and CEF peptides.
      • Table S3. Peptides from brain proteins identified by proteomic analysis in brain tissue.
      • Table S4. Negative bootstrap Monte Carlo summary.
      • Table S5. Patient classification and HLA binding.
      • Table S6. Bacteria selected for GDP-L-fucose synthase comparison.
      • Table S7. Sequence identity between human and bacterial GDP-L-fucose synthase peptides.
      • Table S8. Demographic and clinical characteristics of patients with MS and controls without MS.
      • References (6775)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S9 (Microsoft Excel format). Primary data.

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