Hybrid Insulin Peptides Are Autoantigens in Type 1 Diabetes

Abstract

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester-based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

Figure 1

HIP responders in T1D and nondiabetic control cohorts. Freshly isolated PBMCs were cultured in the presence or absence of antigen for 96 h, washed, and transferred to an IFN-γ–coated plate for overnight culture. After 18 h, plates were developed according to the manufacturer’s protocol, and spots were enumerated. A: To test the biological significance of HIP responses, HIP responders (total number of spots/10⁶ PBMCs >20) were reported (purple squares) as well as responses to left peptides (red squares) and right peptides (blue squares). Cumulative data are shown for 35 patients with diabetes and 19 control subjects. B: Responses to HIPs and corresponding control peptides for subjects #3099 and #3544, showing an increased response to HIPs compared with the native control peptides. Data are from one experiment per subject.

Figure 2

Identification of disease-relevant HIPs. Freshly isolated PBMCs were cultured in the presence or absence of antigen for 96 h, washed, and transferred to an IFN-γ–coated plate for overnight culture. After 18 h, plates were developed according to the manufacturer’s protocol, and spots were enumerated. Cumulative data are shown for 35 patients with diabetes and 19 control subjects. A: Responses to HIP4, HIP11, ins_49–64, or ins_82–97 in patients with T1D. B: Responses to HIP4, HIP11, ins_49–64, or ins_82–97 in control subjects without diabetes. The total number of spots per 10⁶ PBMCs is indicated for each condition. P values, when significant (P < 0.05), are reported and were obtained using a two-tailed t test. Ag, antigen.

Figure 3

Longitudinal analysis of responses to Pediarix or HIPs in one patient with T1D. Freshly isolated PBMCs from patient #3196 were collected 2 weeks, 14 weeks, 26 weeks, 40 weeks, or 79 weeks after diagnosis and cultured in the presence or absence of antigen for 72 h, washed, and transferred to an IFN-γ–coated plate for overnight culture. After 18 h, plates were developed according to the manufacturer’s protocol, and spots were enumerated. The total number of spots per 10⁶ PBMCs is indicated for each condition. Data are cumulative of five independent experiments.

Figure 4

Comparison of proliferation assay and ELISPOT assay. Freshly isolated PBMCs from patient #3196 were isolated 26 weeks after diagnosis. A and B: Cells were labeled with CFSE and cultured in the presence or absence of antigen. After 6 days of culture, cells were harvested and stained for CD4, CD25, and a fixable viability dye. Gates were set on live, single CD4⁺ cells, and the percentage of CFSE^dim CD25⁺ cells is reported. C: Cells were cultured in the presence or absence of antigen for 72 h, washed, and transferred to an IFN-γ–coated plate for overnight culture. After 18 h, plates were developed according to the manufacturer’s protocol, and spots were enumerated. The total number of spots per 10⁶ PBMCs is indicated for each condition. Data are representative of two independent experiments.

Figure 5

Characterization of HIP14-reactive T-cell clones. Freshly isolated PBMCs from patient #3196 were isolated 40 weeks after diagnosis, labeled with CFSE, and cultured with HIP11, HIP12, or HIP14. After 7 days, CFSE^dim CD25⁺ cells were single-cell sorted by FACS and expanded in vitro to generate the HIP-reactive T-cell clones. A and B: The HIP-reactive T-cell clone E2, B6, or G10b (1 × 10⁵ cells) was challenged with HIP11, HIP12, or HIP14, respectively, at indicated concentrations. After 48 h, cells were harvested and stained with CD4 and CD25 before flow cytometry analysis (A), and supernatants from cell cultures were harvested and an IFN-γ ELISA was performed (B). C: The HIP-reactive T-cell clones (1 × 10⁵ cells) were challenged with indicated HIP in the presence or absence of antigen, an anti-DR blocking antibody, or an anti-DQ blocking antibody. After overnight culture, cells were harvested and stained with CD4 and CD25 before flow cytometry analysis. D: The HIP-reactive T-cell clones E2, B6, and G10b (5 × 10⁵ cells) were cultured in the presence of HIPs and indicated left or right peptides. Data are representative of three independent experiments.