Research ArticleAutoimmunity

Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes

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Science Translational Medicine  09 Aug 2017:
Vol. 9, Issue 402, eaaf7779
DOI: 10.1126/scitranslmed.aaf7779
  • Fig. 1. Study design and treatment groups.

    Graphical representation of study design shows timing of treatments and evaluation of stimulated C-peptide. Enrolled subjects were allocated randomly with double blinding to receive high-frequency (10-μg proinsulin C19-A3 peptide every 2 weeks) or low-frequency (10-μg proinsulin C19-A3 peptide every 4 weeks alternating with saline every 4 weeks) active treatment or placebo (saline injections every 2 weeks) by intradermal injection for a total of 12 administrations over 6 months. Residual C-peptide production was evaluated at baseline and 3, 6, 9, and 12 months thereafter.

  • Fig. 2. Changes in C-peptide.

    (A) Natural log of mean change in normalized mixed-meal tolerance test (MMTT) stimulated AUC C-peptide from baseline is shown in groups receiving placebo (open squares), low-frequency (triangles) and high-frequency (inverted triangles) C19-A3 peptide over 12 months. Error bars represent SEM. (B) Change in normalized MMTT stimulated AUC C-peptide values from baseline versus levels at 3, 6, 9, and 12 months in groups receiving placebo (open squares), low-frequency (triangles), and high-frequency (inverted triangles) C19-A3 peptide over 12 months. Comparisons were made using paired t tests. The mean % change was calculated at each time point for each study group.

  • Fig. 3. Changes in insulin use and HbA1c.

    (A) Mean change in average insulin dose, (B) mean change in HbA1c, and (C) mean change in insulin dose–adjusted HbA1c (IDAA1c) values from baseline over 12 months are shown in groups receiving placebo (open squares), low-frequency (triangles), and high-frequency (inverted triangles) C19-A3 peptide over 12 months. Error bars indicate SDs.

  • Fig. 4. Analysis of T cell responses according to treatment and in peptide-treated C-peptide responders and nonresponders.

    (A) Cumulative mean CD4 T cell IL-10 and IFN-γ responses to proinsulin stimulation measured over the duration of the 6-month treatment period shown according to treatment group. (B) Mean CD4 T cell IL-10 and (C) IFN-γ responses to proinsulin stimulation measured at each month during therapy in peptide-treated subjects divided according to C-peptide responder status. Bars and symbols represent mean stimulation index (SI) at each time point, and error bars are the 95% confidence intervals (CI). For analysis over the treatment period, longitudinal measurements of the SI were transformed using the natural logarithm (“Ln”) and were analyzed with linear models having visit and treatment as main factors and a repeated-measures error structure. Estimates of the mean SI across visits were computed using model-based estimates (least-squares means). (D) Change in FOXP3 expression levels [mean fluorescence intensity (MFI)] on all Treg subsets (CD4+CD25hiFOXP3+), (E) on memory (CD45RA) adaptive Tregs, and (F) on memory CD39+ Tregs in peptide-treated subjects divided according to C-peptide responder status. (G) Change in Helios expression by Tregs in the same period and same groups. (H) Mean percentage levels of antigen-experienced (CD57+) CD8 T cells stained with peptide-HLA tetramers loaded with β cell peptides at baseline and at 6 months in peptide-treated C-peptide responders, compared with placebo and nonresponder subjects. Error bars show means and SEM. (B to H) C-peptide responders/nonresponders defined as having a post-baseline value that is 100% or more of the baseline value of C-peptide AUC during the treatment period. There were 9 peptide-treated C-peptide responders (6 of 9 subjects in the low-frequency and 3 of 7 in the high-frequency groups) and 10 nonresponders.

  • Fig. 5. Analysis of proinsulin/C-peptide ratio in peptide-treated C-peptide responders and nonresponders.

    Proinsulin/C-peptide ratio measured at fasting and at 90 min during the MMTT in peptide-treated subjects who are C-peptide nonresponders (A and B) and responders (C and D). P values are for comparisons against the corresponding baseline. Error bars show means and SEM.

  • Table 1. Baseline characteristics of the study subjects.
    C19-A3 PIT
    CharacteristicPlaceboLow frequencyHigh frequency
    Number of subjects8109
    Mean age (years; ±SD)28.9 ± 8.226.6 ± 5.530 ± 5.7
    Gender (female:male)2:64:63:6
    Body mass index (kg/m2; ±SD)23.1 ± 2.624.2 ± 5.525.6 ± 5.4
    Number of autoantibodies (GAD65Ab, IA-2Ab, and ZnT8Ab):
      Single antibody–positive
    12.5%50.0%11.1%
      Double antibody–positive25.0%30.0%11.1%
      Triple antibody–positive62.5%20.0%77.8%
    Mean time from diagnosis to first dose (days; ±SD)95 ± 22.882.5 ± 16.091 ± 15.5
    Mean glycated hemoglobin (mmol/mol; ±SD)62.5 + 13.758.4 + 14.951.7 + 6.83*
    Average total daily insulin dose (IU Kg−1 day−1; ±SD)0.42 + 0.200.38 + 0.180.30 + 0.07
    Stimulated C-peptide AUC (nM/min; ±SD)0.58 ± 0.250.81 ± 0.760.99 ± 0.73

    *P = 0.02 versus placebo.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/402/eaaf7779/DC1

      Fig. S1. Enrollment, randomization, and follow-up of study subjects.

      Fig. S2. Representative flow cytometry gating strategy for Treg subsets.

      Fig. S3. Representative flow cytometry gating strategy for antigen-specific CD8 T cells.

      Table S1. Trial primary and secondary end points.

      Table S2. Normalized C-peptide AUC at baseline and 3, 6, 9, and 12 months after initiation of treatment.

      Table S3. Percentage change in daily insulin use (unit/kilogram) at baseline and 3, 6, 9, and 12 months after initiation of treatment.

      Table S4. Change in daily insulin use (unit/kilogram) at baseline and 3, 6, 9, and 12 months after initiation of treatment.

      Table S5. Percentage change in HbA1c (mmol/mol) at baseline and 3, 6, 9, and 12 months after initiation of treatment.

      Table S6. Change in HbA1c (mmol/mol) at baseline and 3, 6, 9, and 12 months after initiation of treatment.

      Table S7. In vitro response to proinsulin (or diluent control) according to C-peptide response status in peptide-treated subjects.

      Table S8. Percentage change in Treg FOXP3 and Helios MFI from baseline at months 3 and 12.

      Table S9. Percentage of CD57+ β cell–specific CD8 T cells at baseline and 6 months after initiation of treatment.

      Table S10. In vitro response to proinsulin peptide C19-A3.

      Table S11. Autoantibody levels at baseline and 6 and 12 months.

      Table S12. In vitro response to recall antigen according to treatment group and C-peptide response.

      Table S13. Analysis of proinsulin/C-peptide ratio in peptide-treated C-peptide responders and nonresponders.

    • Supplementary Material for:

      Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes

      Mohammad Alhadj Ali, Yuk-Fun Liu, Sefina Arif, Danijela Tatovic, Hina Shariff, Vivienne B. Gibson, Norkhairin Yusuf, Roman Baptista, Martin Eichmann, Nedyalko Petrov, Susanne Heck, Jennie H. M. Yang, Timothy I. M. Tree, Irma Pujol-Autonell, Lorraine Yeo, Lucas R. Baumard, Rachel Stenson, Alex Howell, Alison Clark, Zoe Boult, Jake Powrie, Laura Adams, Florence S. Wong, Stephen Luzio, Gareth Dunseath, Kate Green, Alison O'Keefe, Graham Bayly, Natasha Thorogood, Robert Andrews, Nicola Leech, Frank Joseph, Sunil Nair, Susan Seal, HoYee Cheung, Craig Beam, Robert Hills, Mark Peakman,* Colin M. Dayan

      *Corresponding author. Email: mark.peakman{at}kcl.ac.uk

      Published 9 August 2017, Sci. Transl. Med. 9, eaaf7779 (2017)
      DOI: 10.1126/scitranslmed.aaf7779

      This PDF file includes:

      • Fig. S1. Enrollment, randomization, and follow-up of study subjects.
      • Fig. S2. Representative flow cytometry gating strategy for Treg subsets.
      • Fig. S3. Representative flow cytometry gating strategy for antigen-specific CD8 T cells.
      • Table S1. Trial primary and secondary end points.
      • Table S2. Normalized C-peptide AUC at baseline and 3, 6, 9, and 12 months after initiation of treatment.
      • Table S3. Percentage change in daily insulin use (unit/kilogram) at baseline and 3, 6, 9, and 12 months after initiation of treatment.
      • Table S4. Change in daily insulin use (unit/kilogram) at baseline and 3, 6, 9, and 12 months after initiation of treatment.
      • Table S5. Percentage change in HbA1c (mmol/mol) at baseline and 3, 6, 9, and 12 months after initiation of treatment.
      • Table S6. Change in HbA1c (mmol/mol) at baseline and 3, 6, 9, and 12 months after initiation of treatment.
      • Table S7. In vitro response to proinsulin (or diluent control) according to C-peptide response status in peptide-treated subjects.
      • Table S8. Percentage change in Treg FOXP3 and Helios MFI from baseline at months 3 and 12.
      • Table S9. Percentage of CD57+ β cell–specific CD8 T cells at baseline and 6 months after initiation of treatment.
      • Table S10. In vitro response to proinsulin peptide C19-A3.
      • Table S11. Autoantibody levels at baseline and 6 and 12 months.
      • Table S12. In vitro response to recall antigen according to treatment group and C-peptide response.
      • Table S13. Analysis of proinsulin/C-peptide ratio in peptide-treated C-peptide responders and nonresponders.

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