Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

Abstract

Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA(g7) in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.

Keywords: MHCII tetramers; antigen presentation; self-antigen.

Fig. 1.

Detection of insulin-specific T cells following in vitro expansion. CD4⁺ T cells from T1D patients, type 2 diabetes (T2D) patients, and healthy control subjects with DQ8 haplotypes were individually stimulated with B:11-23 or modified B:11-23^R22E for 14 d. Antigen-specific T cells were then detected by staining with HLA-DQ8/B:11-23^R22E tetramer. Each panel depicts a representative subject (patient ID listed above each panel) which had a positive tetramer staining result. Cells from subject T1D#10 were stimulated with B:11-23 peptide and the rest were stimulated with B:11-23^R22E. Subjects T1D#5, Healthy#5, and T2D#1 provide examples of negative tetramer staining.

Fig. 2.

HLA-DQ8–restricted T-cell clones isolated using B:11-23^R22E tetramers respond to wild-type insulin peptide and denatured insulin. (A) DQ8/B:11-23^R22E tetramer staining of a representative T-cell clone isolated from subject T1D#3. (B) ³H-Thymidine incorporation of T-cell clone T1D#3 C8 following stimulation with B:1-23^R22E peptide or irrelevant control peptide stimulation in the absence or presence of 5 μg/mL of L243 (HLA-DR blocking antibody), B7/21 (HLA-DP blocking antibody), or SPVL3 (HLA-DQ blocking antibody). Data are mean ± SD of duplicates. (C) Stimulation of a representative T-cell clone T1D#3 C8 by B:11-23^R22E, B:11-23, denatured or native insulin. Data are mean ± SD of duplicates. (D) In vitro binding of B:11-23, B:11-23^R22E, and influenza matrix protein (MP) peptides to DQ8. Competitive binding assays were carried out using a biotinylated reference GAD65 peptide and increasing concentrations of nonbiotinylated B:11-23, B:11-23^R22E, or MP24 (MP_185–204 TAKAMEQMAGSSEQAAEAME). Based on the binding curves, the IC₅₀ values were 10.4 μM for B:11-23, 1.6 μM for B:11-23^R22E, and 1.4 μM for MP24, respectively.

Fig. 3.

Determining the immunogenic register of B:11-23 that is recognized by DQ8-restricted T cells. (A) Stimulation index for T-cell clones activated in vitro using B:11-23^R22E or its substituted derivatives (all clones were stimulated with 3-μM peptides). The B:11-23^R22E peptide sequence is listed on the top of the y axis, followed by substituted peptides, with identical residues marked using the asterisk symbol. Alanine was used to replace each residue of the peptide with the exception of A14 where the wild-type alanine was replaced with F. Clone T1D#10 C8 was generated from wild-type B:11-23 stimulated cultures, and clone T1D#3 C8 and T1D#4 C5 were generated from B:11-23^R22E-stimulated cultures. Data are mean ± SD of duplicates. (B) Stimulation index for T-cell clones activated in vitro using B:11-23 or its substituted derivatives (all clones were stimulated with 30-μM peptides). The B:11-23 peptide sequence is listed on the top of the y axis, followed by substituted peptides, with identical residues marked using the asterisk. Alanine was used to replace each residue of the peptide with the exception of A14 where the wild-type alanine was replaced with F. Data are mean ± SD of duplicates.

Fig. 4.

Stimulation index for a representative T-cell clone activated in vitro using 3 μM of B:11-23^R22E peptide, B:11-23 peptide, or homologous microbial peptides, respectively. Data are mean ± SD of duplicates.