Research ArticleIMMUNOLOGY

Cytokine release and gastrointestinal symptoms after gluten challenge in celiac disease

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Science Advances  07 Aug 2019:
Vol. 5, no. 8, eaaw7756
DOI: 10.1126/sciadv.aaw7756
  • Fig. 1 Activation of immune response by gluten-derived immunodominant peptides.

    (A) Heatmap of average baseline-adjusted fold change response in groups of patients either treated with first dose of Nexvax2 (60, 90, 150, or 300 μg) or matched placebo control. Only IL-2 and IL-8 showed significant elevations as early as 2 hours. Temporal response typically peaked at 4 hours except for some cytokines, such as IP-10 and G-CSF, which peaked at 6 hours after dose. (B) Heatmap of average baseline-adjusted fold change response in groups of patients either treated with first dose of Nexvax2 (150 μg) or third weekly dose. (C) Heatmap of average baseline-adjusted fold-change response in groups of patients either treated with first dose of Nexvax2 (150 μg) or 16th twice-weekly dose. VEGF, vascular endothelial growth factor.

  • Fig. 2 Assessment of select immune response by gluten peptides using a sensitive multiplex assay.

    (A) Baseline-adjusted fold-change response assessed in 150 μg of Nexvax2-treated cohort. Median and IQRs are shown. (B) Responses in placebo-treated patients. (C) Differences in activation response of IL-2, IL-8, and IFN-γ as judged by cytokine concentrations. (D to F) Pearson’s correlation analysis of IL-2 concentration at 4 hours after dose with IL-8 at 4 hours (D), MCP-1 at 4 hours (E), and IP-10 at 6 hours after dose (F) are shown. Green dots indicate cytokine response in placebo-treated patients (n = 7). (G) Baseline-adjusted fold-change response at onset of vomiting in Nexvax2- and placebo-treated patients. Median values and IQRs are shown. Response in participants who vomited was compared to placebo response using a Mann-Whitney U test. Significant cytokines are indicated with asterisks (***P < 0.001; *P < 0.05). (H) Kinetics of cytokine elevation (on left y axis) overlaid on incidence of vomiting (on right y axis). Concentration profiles were normalized by peak concentration value and expressed as a percentage. Median values and IQRs are shown. (I) IL-2 concentration stratified by either patient-reported nausea score or occurrence of vomiting is shown. For nausea scores, a P value was estimated by Kruskal-Wallis test. For vomiters and nonvomiters, a P value was computed by Mann-Whitney U test, and significance was further confirmed by regression modeling. (J) A sigmoidal dose-response relationship is observed between levels of plasma IL-2 and magnitude of self-reported nausea score after first dose of Nexvax2 (60, 90, 150, or 300 μg). Blue line represents a 4-parameter logistic dose-response curve. Red dot indicates that patient vomited after receiving Nexvax2. Significant occurrence of high-grade nausea and vomiting are observed at IL-2 > 10 pg/ml (model-based threshold estimate). Intensity of IL-2 induction and self-reported attenuated scores are attenuated after third weekly dose (K) and absent after 16th twice-weekly dose (L).

  • Fig. 3 Validation of IL-2 activation in gluten-induced recall responses.

    (A to C) Fold-change responses assessed in CeD patients in a randomized double-blind 3-g gluten food challenge study with matched controls. Median and IQRs are shown. (D) IL-2 response to gluten challenge from stored plasma samples collected in a previously reported open-label 5.7-g study. Median and IQRs of response in HLA-DQ2.5+ patients are shown. (E and F) Pearson’s correlation analysis of IL-2 activation with frequency of circulating gluten-specific CD4+ T cells in HLA-DQ2.5+ CeD patients (E) and villous height–to–crypt depth (Vh:CrD) ratio (F) at baseline before oral gluten challenge. (G) Self-reported nausea scores collected every 2 hours after gluten challenge. Median with IQRs is shown. P value was estimated by paired Wilcoxon signed-rank test. (H and I) Dose-response analysis of IL-2 concentration with patient-reported nausea scores and occurrence of vomiting in double-blind 3-g gluten food challenge study (H) and previously reported gluten challenge study (I). Red dot indicates that patient vomited after gluten challenge. (J) Cytokine response of CD4+ T cell clones, specific for HLA-DQ2.5–restricted epitopes in Nexvax2 and derived from peripheral blood or intestinal biopsies of CeD patients, after incubation with anti-CD3 and anti-CD28 antibody in the absence of other cell types for 24 hours. (K) Cytokine response of short-term CD4+ T cell lines, derived from peripheral blood or intestinal biopsies of CeD patients, after incubation with anti-CD3 and/or anti-CD28 antibody in the absence of other cell types for 24 hours. (L) Cytokine response in fresh whole blood, drawn from HLA-DQ2.5+ CeD patients, incubated with Nexvax2 peptides for 24 hours. Stimulation index estimated as fold change relative to incubation with media only. Median value and IQR are shown. Significance of cytokine induction estimated by paired Wilcoxon signed-rank test between Nexvax2 and Nil incubations. Asterisks denote cytokines with significant differences (***P < 0.0001).

  • Table 1 Plasma cytokines after intradermal Nexvax2 or gluten challenge assessed with ECL assay.

    Median, IQR values are shown. ND, not determined; NA, not applicable.

    #Patients (N)Phase 1 trials of Nexvax2 i.d. in HLA-DQ2.5 CeD on GFDSham-controlled food
    challenge in HLA-DQ2.5
    CeD on GFD
    Open-food challenge muesli bar (5.7 g) in
    CeD on GFD
    1st dose
    Nexvax2
    All
    cohorts
    Any dose
    (60–300
    μg)
    Nexvax2 150 μg 3-dose
    study
    Nexvax2 150 μg 16-dose
    study
    3-g gluten
    protein
    (vital
    wheat
    gluten
    flour
    slurry)
    Matched
    gluten-
    free
    challenge
    (rice flour
    slurry)
    All
    participants
    HLA-
    DQ2.5+
    HLA-DQ8+
    (−ve for
    HLA-DQ2.5)
    1st dose3rd
    (final)
    dose
    1st dose16th
    (final)
    dose
    5488151511819163
    IL-2Baseline,
    pg/ml
    0.1 (0.1–0.2)0.2 (0.1–0.3)0.1 (0.1–0.3)0.1 (0.1–0.2)0.1 (0.1–0.2)0.1 (0.1–0.1)0.1 (0.1–0.2)0.1 (0.1–0.3)0.2 (0.1–0.3)0.1 (0.1–0.2)
    Peak, pg/ml33 (1.3–92)40 (6.9–106)0.9 (0.5–2.0)53 (26–101)0.2 (0.1–0.2)1.8 (0.7–2.2)0.2 (0.1–0.2)1.0 (0.2–7.2)0.9 (0.2–4.2)8.0 (2.1–23)
    Peak, fold
    change
    272 (12–597)248 (34–707)8.4 (2.3–22)323 (131–915)1.0 (1.0–1.3)15 (5.3–27)1.0 (1.0–1.0)10 (1.8–27)7.3 (2.0–19)29 (8.4–246)
    Fisher’s
    exact, P
    value
    8.25E-081.55E-040.00705.86E-061.00.0001NA0.00060.00130.0545
    Responder
    (%)
    96100751000910747567
    Correlation
    with IL-8,
    r value
    0.840.98ND0.92ND0.81NA0.710.740.17
    Correlation
    with IL-8,
    P value
    1.66E-153.25E-05ND1.23E-06ND0.0027NA0.00060.00110.8902
    Correlation
    with
    IL-10, r
    value
    0.63ND0.98ND0.430.84NA0.960.990.85
    Correlation
    with
    IL-10, P
    value
    5.80E-07ND1.30E-05ND0.11200.0013NA6.26E-112.64E-140.3532
    IL-8Baseline,
    pg/ml
    4.0 (3.1–4.9)3.4 (2.9–5.0)ND3.2 (2.5–3.8)ND8.5 (7.2–12)5.8 (5.2–8.6)4.8 (3.5–5.5)4.9 (3.8–5.8)3.4 (2.1–4.6)
    Peak, pg/ml43 (12–112)34 (27–76)ND68 (35–168)ND34 (12–46)6.5 (5.4–11)7.0 (5.0–18)7.0 (5.2–17)15 (5.7–28)
    Peak, fold
    change
    11 (2.7–31)12 (4.7–24)ND13 (11–38)ND2.4 (1.3–4.7)1.1 (1.0–1.2)1.5 (1.1–4.2)1.3 (1.1–3.3)4.5 (2.2–6.1)
    Fisher’s
    exact, P
    value
    1.15E-040.0014ND2.11E-04ND0.0128NA0.02580.05380.0545
    Responder
    (%)
    7888ND87ND640474467
    IL-10Baseline,
    pg/ml
    6.1 (3.2–13)ND0.3 (0.2–1.1)ND0.2 (0.1–0.2)0.2 (0.2–0.6)0.3 (0.2–0.7)0.3 (0.2–0.3)0.3 (0.2–0.4)0.3 (0.2–0.3)
    Peak, pg/ml15 (6.4–41)ND2.3 (0.8–8.3)ND0.2 (0.2–0.3)0.6 (0.4–0.7)0.3 (0.2–0.7)0.3 (0.3–0.7)0.3 (0.3–0.9)0.4 (0.2–0.4)
    Peak, fold
    change
    1.2 (1.0–3.4)ND7.7 (1.9–28)ND1.1 (1.0–1.3)1.5 (1.1–1.9)1.0 (1.0–1.0)1.2 (1.0–1.6)1.2 (1.0–2.3)1.2 (1.2–1.3)
    Fisher’s
    exact, P
    value
    0.0844ND0.0014ND0.52270.0445NA0.28550.26221.0
    Responder
    (%)
    39ND88ND2045021250

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw7756/DC1

    Fig. S1. Activation of immune response by gluten peptides.

    Fig. S2. Effects of HLA genotype and previous gluten exposure on cytokine response and cytokine response stratified by Nausea scores or occurrence of vomiting.

    Fig. S3. Cytokine release assessed in plasma and serum.

    Fig. S4. Gating strategy for the generation of primary human CD4+ T cell lines.

    Fig. S5. Cytokine release in gluten-specific CD4+ T cell clones and short-term CD4+ T cell lines.

    Fig. S6. Cytokine release in gluten-specific CD4+ T cell clones, short-term CD4+ T cell lines, and antigen-presenting cells.

    Fig. S7. Cytokine release in fresh whole blood incubated with Nexvax2 peptides for 24 hours.

    Table S1. Plasma cytokines after intradermal Nexvax2 assessed with 38-plex magnetic bead assay.

    Table S2. Plasma cytokines after intradermal Nexvax2 assessed with 18-plex ECL assay.

    Table S3. Characteristics of patients enrolled in masked, 3-gram gluten food challenge study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Activation of immune response by gluten peptides.
    • Fig. S2. Effects of HLA genotype and previous gluten exposure on cytokine response and cytokine response stratified by Nausea scores or occurrence of vomiting.
    • Fig. S3. Cytokine release assessed in plasma and serum.
    • Fig. S4. Gating strategy for the generation of primary human CD4+ T cell lines.
    • Fig. S5. Cytokine release in gluten-specific CD4+ T cell clones and short-term CD4+ T cell lines.
    • Fig. S6. Cytokine release in gluten-specific CD4+ T cell clones, short-term CD4+ T cell lines, and antigen-presenting cells.
    • Fig. S7. Cytokine release in fresh whole blood incubated with Nexvax2 peptides for 24 hours.
    • Table S1. Plasma cytokines after intradermal Nexvax2 assessed with 38-plex magnetic bead assay.
    • Table S2. Plasma cytokines after intradermal Nexvax2 assessed with 18-plex ECL assay.
    • Table S3. Characteristics of patients enrolled in masked, 3-gram gluten food challenge study.

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