Immune therapy and β-cell death in type 1 diabetes

Abstract

Type 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing β-cells. The killing of β-cells is not currently measurable; β-cell functional studies routinely used are affected by environmental factors such as glucose and cannot distinguish death from dysfunction. Moreover, it is not known whether immune therapies affect killing. We developed an assay to identify β-cell death by measuring relative levels of unmethylated INS DNA in serum and used it to measure β-cell death in a clinical trial of teplizumab. We studied 43 patients with recent-onset T1D, 13 nondiabetic subjects, and 37 patients with T1D treated with FcR nonbinding anti-CD3 monoclonal antibody (teplizumab) or placebo. Patients with recent-onset T1D had higher rates of β-cell death versus nondiabetic control subjects, but patients with long-standing T1D had lower levels. When patients with recent-onset T1D were treated with teplizumab, β-cell function was preserved (P < 0.05) and the rates of β-cell were reduced significantly (P < 0.05). We conclude that there are higher rates of β-cell death in patients with recent-onset T1D compared with nondiabetic subjects. Improvement in C-peptide responses with immune intervention is associated with decreased β-cell death.

Trial registration: ClinicalTrials.gov NCT00378508.

Figures

FIG. 1.

Measurement of unmethylated INS DNA by real-time PCR. DNA extraction of sera, tissue, and cells and bisulfite treatment. A: DNA was isolated from serum and treated with bisulfite. The treated DNA was amplified using a first-step, methylation-insensitive reaction between bp 329 and 399 from the transcription start site. The products of this reaction were used in a second-step RT-PCR reaction with primers specific for methylated or unmethylated DNA sequences. The difference in the Ct values (methylated − unmethylated) was determined and is represented by the Δ. B: The methylated and unmethylated sequences from 10 clones from human β-cells, 14 clones from human islet cells, and 12 clones from human kidney cells are indicated at eight CpG sites (○, unmethylated cytosines; ●, methylated cytosines). The methylation pattern of the CpG sites in β-cells and kidney has previously been published (10). The bp are indicated downstream from the transcription start site. The majority of the CpG sites in purified β-cells are unmethylated, whereas there is a mixture of methylated and unmethylated sites in the products of the first-step PCR reaction from islet DNA reflecting the mixture of endocrine cells. The products of the PCR reaction with kidney DNA are predominantly methylated. C, cytosine; G, guanine; T, thymine. CG (cytosine-guanine) refers to methylated CpG sites, and TG (thymine-guanine) refers to unmethylated CpG sites.

FIG. 2.

Unmethylated INS DNA in patients with T1D. A: The levels of unmethylated INS DNA were measured in 43 subjects with recent-onset T1D and 13 nondiabetic control (Ctl) subjects of similar age (unpaired t test, ***P = 0.001). In the patients and control subjects, unmethylated Ct values ranged from 29.8 to 37.2 and from 22.3 to 37.3, respectively, and the methylated Ct values ranged from 15.4 to 30.7 and from 15.1 to 24.2. B: The C-peptide responses to a mixed meal (area under the curve [AUC]) and the corresponding Δ are shown for the subjects with recent-onset T1D (r = 0.34, P = 0.03).

FIG. 3.

Effects of teplizumab therapy on clinical responses and levels of unmethylated INS DNA. A and B: Percent decline in C-peptide (*P = 0.03) and increase in insulin use (**P < 0.009) from baseline are shown for drug- and placebo-treated subjects. The baseline C-peptide and insulin in the drug group was 0.53 ± 0.07 pmol/mL/min and 0.38 ± 0.03 units/kg/day, respectively, and in the placebo group 0.53 ± 0.08 pmol/mL/min and 0.38 ± 0.04 units/kg/day (P = 0.18 and 0.94). C: Relative β-cell–derived DNA levels before and after treatment with teplizumab or placebo. There was a significant reduction in the Δ in subjects treated with teplizumab (***P < 0.0001) but not placebo (P = 0.08). At baseline, the Δs in the drug- and placebo-treated groups were similar (P = 0.8). D: Subjects treated with teplizumab had a greater decline in the Δ (*P = 0.04).