Protocol of the adaptive study of IL-2 dose frequency on regulatory T cells in type 1 diabetes (DILfrequency): a mechanistic, non-randomised, repeat dose, open-label, response-adaptive study

Lucy A Truman, Marcin L Pekalski, Paula Kareclas, Marina Evangelou, Neil M Walker, James Howlett, Adrian P Mander, Jane Kennet, Linda S Wicker, Simon Bond, John A Todd, Frank Waldron-Lynch, Lucy A Truman, Marcin L Pekalski, Paula Kareclas, Marina Evangelou, Neil M Walker, James Howlett, Adrian P Mander, Jane Kennet, Linda S Wicker, Simon Bond, John A Todd, Frank Waldron-Lynch

Abstract

Introduction: Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, leading to insulinopenia and hyperglycaemia. Genetic analyses indicate that alterations of the interleukin-2 (IL-2) pathway mediating immune activation and tolerance predispose to T1D, specifically the polymorphic expression of the IL-2 receptor-α chain (CD25) on T lymphocytes. Replacement of physiological doses of IL-2 could restore self-tolerance and prevent further autoimmunity by enhancing the function of CD4(+) T regulatory cells (Tregs) to limit the activation of auto reactive T effector cells (Teffs). In this experimental medicine study, we use an adaptive trial design to determine the optimal dosing regimen for IL-2 to improve Treg function while limiting activation of Teffs in participants with T1D.

Methods and analysis: The Adaptive study of IL-2 dose frequency on Tregs in type 1 diabetes(DILfrequency) is a mechanistic, non-randomised, repeat dose open-label, response-adaptive study of 36 participants with T1D. The objective is to establish the optimal dose and frequency of ultra-low dose IL-2: to increase Treg frequency within the physiological range, to increase CD25 expression on Tregs, without increasing CD4(+) Teffs. DILfrequency has an initial learning phase where 12 participants are allocated to six different doses and frequencies followed by an interim statistical analysis. After analysis of the learning phase, the Dose and Frequency Committee will select the optimal targets for Treg frequency, Treg CD25 expression and Teff frequency. Three groups of eight participants will be treated consecutively in the confirming phase. Each dose and frequency selected will be based on statistical analysis of all data collected from the previous groups.

Ethics: Ethical approval for DILfrequency was granted on 12 August 2014.

Results: The results of this study will be reported, through peer-reviewed journals, conference presentations and an internal organisational report.

Trial registration numbers: NCT02265809, ISRCTN40319192, CRN17571.

Keywords: DIABETES & ENDOCRINOLOGY; GENETICS; IMMUNOLOGY.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Figures

Figure 1
Figure 1
Study design of the learning phase of DILfrequency. The study has 12 visits and starts with a screening visit followed by a treatment period of 10 visits and a follow-up visit that will be carried out approximately 4 weeks after the final dose of Proleukin. Twelve participants will be allocated in the learning phase to two different doses and four frequencies of administration of Proleukin to measure the change from baseline of CD4+ T regulatory cells (Tregs), CD4+ T effectors and CD25 expression on Tregs during treatment with ultra-low dose interleukin-2. At the first two dosing visits, 90 min time points are measured to access early immune activation and the effects of repeat dosing on these events.
Figure 2
Figure 2
Clinical fluorescence-activated cell sorting (FACS) assays on whole blood to measure absolute lymphocyte counts, proportions of lymphocytes and CD25 on T regulatory cells. Fluorescently labelled beads are added to whole blood and analysed to accurately count the absolute number of lymphocytes (A), beads (B), CD3+ T cells (C) CD4+ and CD8+ T cells (D) CD19+ B cells and CD19−, CD16+, CD56+ NK cells as a percentage of all lymphocytes (E). In a parallel whole blood FACS assay, a lymphocyte gate is drawn to include all events (F) and doublets are excluded (not shown). The CD3+, CD4+ T-cell gate excludes CD8+ T cells and B cells (G). CD127−, CD25+ T regulatory cells (Tregs) are separated from non-Tregs since the non-Tregs are heterogeneous for CD127 and CD25 and this percentage is used to calculate the absolute Treg count out of CD4+ (H), naïve, effector memory, central memory and total effector memory CD45 RA+(TEMRA) Tregs (I) and non-Tregs (J) are measured according to CD62L and CD45RA expression, as shown. A cocktail of six standardised beads labelled with different amounts of fluorescent allophycocyanin (APC) are measured by FACS daily to accurately measure CD25-APC on the surface of Tregs (K) and a standard curve plotted (L). The mean fluorescence intensity of CD25+ on Tregs can be accurately read from the curve, minimising interassay variation.

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