Research ArticleTYPE 1 DIABETES

Association of HLA-dependent islet autoimmunity with systemic antibody responses to intestinal commensal bacteria in children

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Science Immunology  01 Feb 2019:
Vol. 4, Issue 32, eaau8125
DOI: 10.1126/sciimmunol.aau8125
  • Fig. 1 Overview of ACAb assay and pediatric cohorts in this study.

    (A) Schematic representation of the ACAb assay. In a first step, bacteria targets of a specific strain pool are incubated with four serial dilutions of the serum sample, allowing serum antibodies to bind bacteria surface antigens. Next, fluorescently labeled, secondary anti-isotype antibodies (anti-IgA, IgG1, IgG2, and total Ig) are added, allowing the visualization of the bacteria through flow cytometry. (B) An example of the signal measured by flow cytometry for one bacterial pool and four serial dilutions of a serum sample. The first four panels show contour plots of the anti-isotype intensity versus forward scatter (FSC-A) parameter. The last panel shows a histogram overlay of the anti-isotype signal intensities. (C) Schematic representation of the response index calculation. For every serum sample, bacterial strain pool, and isotype, the dilution curve of the MFI versus dilution factor is generated on a log-log scale (left, gray lines). The mean dilution curve is generated using the average MFI values for all serum samples (left, black line). For every sample, the difference between its dilution AUC and the mean dilution AUC is calculated (ΔAUC; right). The resulting response index is calculated as 2ΔAUC. (D) Schematic representation of the NET and TrialNet cohort participants analyzed in this study. Pediatric serum samples (age ≤ 18 years) from the NET study were collected from patients with recent onset CD (n = 32), patients with recent onset T1D (n = 49), and age-matched HCs (n = 90). Serum samples were also obtained from TrialNet individuals ≤18 years of age. Samples defined as cases (n = 68) were collected before T1D diagnosis from individuals with ≥2 positive islet autoantibodies who developed T1D during the study follow-up. Samples defined as controls (n = 62) were collected from age-, sex-, and HLA-matched individuals who did not receive a T1D diagnosis within the follow-up period.

  • Fig. 2 ACAb responses in CD (n = 32) and HC (n = 90) serum samples from NET pediatric participants (CD, red; HCs, blue).

    (A) Distribution of ACAb responses, separated by six commensal strain pools (columns, top) and antibody isotype total Ig or IgA (rows, right) displayed as violin plots. The width of plotted area indicates the density distribution of the responses. q = Wilcoxon log-rank test FDR-adjusted q values. (B) PCA of ACAb responses of all participants to all bacterial targets (70.1% of total variance explained). (C) Bootstrapped-rarefied twofold cross-validation of LDA of ACAb responses by patients with CD and HCs. The y axis displays the number of bootstrapped serum samples in the analysis, of which 78.8% were correctly classified.

  • Fig. 3 ACAb response in T1D (n = 49) and HC (n = 90) serum samples from NET pediatric participants (T1D, red; HCs, blue).

    (A) Distribution of ACAb responses, separated by 11 commensal strain pools (columns, top), antibody isotype total Ig and IgA (rows, right). Width of plotted area indicates the density distribution of the responses. q = Wilcoxon log-rank test FDR-adjusted q values. (B) PCA of ACAb responses of all participants to all bacterial targets (49% of total variance explained). (C) Bootstrapped-rarefied twofold cross-validation of LDA of ACAb responses by patients with T1D and HCs. The y axis displays the number of bootstrapped serum samples in the analysis, of which 82.3% were correctly classified.

  • Fig. 4 ACAb response indices displaying interaction effects between HLA haplotype and clinical status in TrialNet samples (cases, red; controls, blue).

    (A) Box-and-whiskers plot of anti–MET-2 IgG2 response indices (y axis), segregated by HLA haplotype (x axis). (B) Box-and-whiskers plots of anti–R. faecis IgG2 response indices (y axis), segregated by HLA haplotype (x axis). (C) Interaction plot of anti–MET-2 IgG2 response indices. (D) Interaction plot of anti–R. faecis IgG2 response indices. (E) Linear regression of anti–MET-2 IgG2 response indices by age. (F) Linear regression of anti–R. faecis IgG2 response indices by age. (A and B) The horizontal line represents the median, and the rectangle represents the interquartile range (25th to 75th percentile). Whiskers extend to the minimum and maximum values that were not outliers. Outliers were defined as points outside of median ± 1.5× interquartile range. (C and D) Data are shown as means ± SE, and P and q values for the interaction effect significance are shown for the responses without (IgG2 MET-2: P = 0.009, q = 0.16; IgG2 R. faecis: P = 0.01, q = 0.16) and with (IgG2 MET-2: P = 0.005, q = 0.11; IgG2 R. faecis: P = 0.007, q = 0.11) age adjustment. (E and F) Regression lines for cases and controls as well as age effect–associated P and q values are shown (IgG2 MET-2: P = 0.0004, q = 0.005; IgG2 R. faecis: P = 0.01, q = 0.06).

  • Fig. 5 Correlations between isotype-specific ACAb responses and islet autoantibody–positive status in TrialNet case samples (n = 68).

    Correlations between isotype-specific ACAb responses (columns, bottom) and islet autoantibody–positive status (rows, left) are shown in heatmap colors. ACAb responses and islet autoantibodies were each clustered by similarity by hierarchical clustering (top and right dendrograms, respectively). ACAb isotypes are as follows: IgA, blue; IgG1, green; IgG2, purple.

  • Fig. 6 ACAb responses displaying an association with islet autoantibody–positive status in TrialNet case samples (n = 68).

    (A) Box-and-whiskers plots of isotype-specific anticommensal response indices (y axis), separated by autoantibody statuses (x axis). (B) Box-and-whiskers plot of the isotype-specific anticommensal response indices (y axis), autoantibody status, and HLA-DR3 genotype status (x axis). (C) Box-and-whiskers plot of the isotype-specific anticommensal response indices (y axis), autoantibody status, and HLA-DR4 genotype status (x axis). (D) Interaction plots for isotype-specific anticommensal response indices with significant HLA-DR3/DR4 genotype status additive effect. (A to C) The horizontal line represents the median, and the rectangle represents the interquartile range (25th to 75th percentile). The whiskers extend to the minimum and maximum values that were not outliers. Outliers are defined as points outside of median ± 1.5× interquartile range. (A) P values were generated by a linear regression model with only the autoantibody positivity as the explanatory variable and indicate the significance of the autoantibody main effect. (B and C) P values were generated by a linear regression model with autoantibody positivity and DR3/DR4 genotype as the explanatory variables and indicate the significance of the autoantibody effect in the HLA-adjusted model. (D) Data are shown as means ± SE. P values were generated by ANOVA comparing the additive linear model to the model with an interaction term (see Materials and Methods).

  • Fig. 7 Graphical summary of HLA haplotype–dependent associations of ACAb responses with islet autoimmunity and future T1D diagnosis.

    HLA class II haplotypes are the strongest genetic determinants of T1D risk and with islet autoantibody specificities (black arrows). In a pediatric cohort of at-risk individuals, we uncovered HLA-DR3/DR4–dependent associations between systemic ACAbs (lower circle) and future T1D diagnosis (right curved red arrow, upper right circle). In the same participants, we found that islet autoantibody specificities (upper left circle) were associated with ACAb responses both in an HLA-dependent (left curved orange arrow) and HLA-independent (left straight blue arrow) manner.

  • Table 1 Characteristics of NET cohort study participants.

    CD (n = 32)T1D (n = 49)Controls
    (n = 90)
    Sex (% males)n = 17 (53%)n = 31 (62%)n = 30 (33%)
    Age at diagnosis
    (years; mean ± SD)
    12.30 (±2.96)10.68 (±3.95)N/A
    Age at ACAb test
    (years; mean ± SD)
    12.96 (±3.03)11.08 (±3.98)14.91 (±2.10)
    Time between ACAb
    test and diagnosis
    (days; mean ± SD)
    241 (±480)135 (±94)N/A
  • Table 2 Characteristics of TrialNet cohort study participants.

    Cases (n = 68)Controls (n = 62)
    Sex (% males)n = 36 (53%)n = 32 (52%)
    Age at T1D diagnosis
    (years; mean ± SD)
    11.61 (±3.76)N/A
    Age at ACAb test
    (years; mean ± SD)
    10.67 (±3.82)10.92 (±3.65)
    Time between ACAb
    test and diagnosis
    (days; mean ± SD)
    344 (±120)N/A
    Follow-up time (days;
    mean ± SD)
    1045 (±711)2646 (±1051)
    HLA-DR3 positive/
    DR4 negative
    n = 20 (29%)n = 18 (29%)
    HLA-DR3 negative/
    DR4 positive
    n = 22 (32%)n = 21 (34%)
    HLA-DR3 positive/
    DR4 positive
    n = 12 (18%)n = 10 (16%)

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/32/eaau8125/DC1

    Fig. S1. Age of NET participants at time of serum sample collection.

    Fig. S2. Sex effects on ACAb responses in CD (n = 32) and HC (n = 90) serum samples from NET participants.

    Fig. S3. Sex effects on ACAb responses in T1D (n = 49) and HC (n = 90) serum samples from NET participants.

    Fig. S4. Age of TrialNet participants at time of serum sample collection.

    Fig. S5. Follow-up period and time of serum sample collection from TrialNet participants.

    Fig. S6. ACAb responses in serum samples from TrialNet participants.

    Fig. S7. All ACAb responses, separated by HLA haplotype and clinical status in TrialNet serum samples.

    Fig. S8. ACAb responses displaying interaction effects between HLA haplotype and clinical status in TrialNet serum samples.

    Fig. S9. Correlation between anti-commensal Ig and IgA, IgG1, and IgG2 response indices against MET-2 and R. faecis in TrialNet serum samples.

    Fig. S10. HLA dependence of anti-GAD65 and anti-insulin seropositivity in TrialNet serum samples.

    Fig. S11. ACAb responses associated with islet autoantibody-positive status in TrialNet case samples (n = 68).

    Table S1. List of bacterial strain pools used in this study.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Age of NET participants at time of serum sample collection.
    • Fig. S2. Sex effects on ACAb responses in CD (n = 32) and HC (n = 90) serum samples from NET participants.
    • Fig. S3. Sex effects on ACAb responses in T1D (n = 49) and HC (n = 90) serum samples from NET participants.
    • Fig. S4. Age of TrialNet participants at time of serum sample collection.
    • Fig. S5. Follow-up period and time of serum sample collection from TrialNet participants.
    • Fig. S6. ACAb responses in serum samples from TrialNet participants.
    • Fig. S7. All ACAb responses, separated by HLA haplotype and clinical status in TrialNet serum samples.
    • Fig. S8. ACAb responses displaying interaction effects between HLA haplotype and clinical status in TrialNet serum samples.
    • Fig. S9. Correlation between anti-commensal Ig and IgA, IgG1, and IgG2 response indices against MET-2 and R. faecis in TrialNet serum samples.
    • Fig. S10. HLA dependence of anti-GAD65 and anti-insulin seropositivity in TrialNet serum samples.
    • Fig. S11. ACAb responses associated with islet autoantibody-positive status in TrialNet case samples (n = 68).
    • Table S1. List of bacterial strain pools used in this study.

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