Autoantigen-specific regulatory T cells induced in patients with type 1 diabetes mellitus by insulin B-chain immunotherapy

Abstract

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund's adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499).

Figures

Figure 1

Mean total C-peptide area under the curve (AUC) for the mixed meal tolerance test (MMTT) by group over time. Self-insulin production, as measured by C-peptide during MMTT showed no statistical difference between the drug-treated (IBC-VS01) and placebo (VS02) groups. Four out of six subjects with the lowest stimulated C-peptide (mean total C-peptide AUC

Figure 2

Insulin autoantibodies (IAA) by group over time. IAA titers were significantly increased in the insulin B-chain vaccinated subjects compared to the placebo group (p

Figure 3

T-cell assay stimulation indices (SI) in treatment and placebo groups in response to insulin B-chain (A), B-chain 5–20 peptide (B) and B chain 9–23 peptide (C) stimulation over time. All patients vaccinated with full human insulin B-chain developed a robust T cell response to insulin B-chain. This response peaked at 24 weeks, then slowly declined but remained positive for up to 2 years during follow up (x±SEM; n=6, except for B-chain 9–23 peptide treatment group samples, for which only three showed response over time).

Figure 4

Ratio of the TGF-β1 concentrations of the B-chain stimulated/unstimulated cells in the 7-day assay (x±SEM; p

Figure 5

Suppression assay of two CFSE-B-chain single cell clones with different cell-to-cell ratios of the CD4+CD25- (responder T, 10000) cells and the cells from the clones (2500–5000–10000) of the same subject. The cells were stimulated with bead bound anti-CD-2, anti-CD3 and anti-CD28 (5 µg/ml, Miltenyi Biotec). Responder T cells and cells from the clones were stimulated alone, also. Tritium labeled thymidine (H3 Thymidine) incorporation was assessed after 5 days of culturing. Due to limited cell numbers the cocultures were put together without replicates.