Antibody-Mediated Targeting of a Hybrid Insulin Peptide Toward Neonatal Thymic Langerin-Positive Cells Enhances T-Cell Central Tolerance and Delays Autoimmune Diabetes

Abstract

Thymic presentation of self-antigens is critical for establishing a functional yet self-tolerant T-cell population. Hybrid peptides formed through transpeptidation within pancreatic β-cell lysosomes have been proposed as a new class of autoantigens in type 1 diabetes (T1D). While the production of hybrid peptides in the thymus has not been explored, due to the nature of their generation, it is thought to be highly unlikely. Therefore, hybrid peptide-reactive thymocytes may preferentially escape thymic selection and contribute significantly to T1D progression. Using an antibody-peptide conjugation system, we targeted the hybrid insulin peptide (HIP) 2.5HIP toward thymic resident Langerin-positive dendritic cells to enhance thymic presentation during the early neonatal period. Our results indicated that anti-Langerin-2.5HIP delivery can enhance T-cell central tolerance toward cognate thymocytes in NOD.BDC2.5 mice. Strikingly, a single dose treatment with anti-Langerin-2.5HIP during the neonatal period delayed diabetes onset in NOD mice, indicating the potential of antibody-mediated delivery of autoimmune neoantigens during early stages of life as a therapeutic option in the prevention of autoimmune diseases.

Figure 1

Anti–Langerin-fluorophore conjugate demonstrates feasibility and duration of approach. A: Representative flow plot and quantification of thymic Langerin-positive DC frequency in 2-day-old NOD neonates (n = 9). B: In vivo targeting of thymic Langerin-positive DCs by anti-Langerin antibody conjugated to the fluorophore APC. NOD neonates (2 days old) of both sexes were given 3 μg of anti-Langerin antibody conjugated to the fluorophore APC. Thymi were harvested at 24, 48, and 72 hours posttreatment. Data were pooled from three independent experiments; gated on MHCII⁺CD11c⁺; Ctrl = untreated; n = 5–7 per group). Data are mean ± SD. Mann-Whitney U test. **P < 0.01.

Figure 2

Anti–Langerin-2.5HIP alters thymocyte development, increasing thymocyte apoptosis and Foxp3⁺ thymocyte population in BDC2.5 neonatal thymus. A: Total thymus cell count (n = 4–8 mice per group). B: Frequency and total number of thymocytes in the CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP), and CD4⁺CD8⁻ (CD4SP) stages of thymocyte development 72 h posttreatment. C: Representative flow plot and quantification of Foxp3⁺ cell percentage and number within CD4SP TCRβ⁺ thymocytes (n = 4–7 mice per group). SSC-A, side scatter area. D: Representative flow plot of apoptotic cell percentage within CD4SP TCRβ⁺ Foxp3⁻ thymocytes (n = 5–6 mice per group). Data were pooled from at least two independent experiments and are reported as mean ± SD. Kruskal-Wallis and Dunn tests. *P < 0.05, **P < 0.01.

Figure 3

Ab-2.5HIP–induced BDC2.5 Tregs are thymically derived and are of the CD25− lineage. A: Representative flow plot and quantification of CD73 expression in Ab-2.5HIP–induced Foxp3⁺ thymocytes 72 h posttreatment (n = 5–6 per group). B: Representative flow plot and quantification of Foxp3⁺CD25⁺ and Foxp3⁺CD25⁻ thymocytes (n = 7–10 per group). C: Representative flow plot and quantification of apoptotic cell frequency (gated on CD4SP Foxp3⁺ cells; n = 8–9 per group). D: Representative flow plot and quantification of Foxp3⁺ T cells within recent thymic emigrants (Rag2GFP⁺ cells) 6 days after Ab-2.5HIP treatment in BDC2.5.Rag2GFP neonates (n = 5–7 per group). SSC-A, side scatter area. Data represent three independent experiments (A–C) and two independent experiments (D) and are reported as mean ± SD. Kruskal-Wallis and Dunn Tests (A–C), and Mann-Whitney U test (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Ab-2.5HIP treatment leads to reduced diabetes incidence in a BDC2.5 thymus transplant model. A: Thymus transplant experimental design. B: Diabetes incidence of the thymus transplant recipients (n = 9 per group). C: Insulitis evaluation of thymus transplant recipients at 2 weeks posttransplant (insulitis score: 0 = no infiltrates, 1 = peri-islet infiltrates, 2 = intraislet infiltrates to ≤50%, 3 = intraislet infiltrates to ≥50%; n = 5–6 per group; data from two independent experiments). D: Quantification of donor CD4 T-cell percentage in the spleen, aLN, and pLN 2 weeks after thymus transplant (n = 6 per group). E: Representative flow plot and quantification of Foxp3⁺ cell percentage in the spleen, aLN, and pLN 2 weeks after thymus transplant (n = 5–6 per group). Data are reported as mean ± SD. Mann-Whitney U test (C–E) and Gehan-Breslow-Wilcoxon test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Ab-2.5HIP treatment in NOD wild-type neonates enhances T-cell central tolerance and delays diabetes onset. A: 2.5HIP tetramer analysis of CD4SP thymocytes 72 h after treatment of 1-day-old NOD wild-type neonates with Ab-alone or Ab-2.5HIP (n = 5 per group). Each data point represents three to five pooled thymi enriched for 2.5HIP-reactive cells by tetramer; data represent 5 independent experiments. B: Frequency of Foxp3⁺ cells within tetramer-positive cells. SSC-A, side scatter area. C: Diabetes incidence of NOD wild-type female mice treated with a single 2.5-μg dose of Ab-alone or Ab-2.5HIP at 1 day old (n = 18–20 per group). D: Quantification of CD4 T cells and 2.5HIP tetramer-positive T cells in the pLN at 10 weeks after treatment. E: Quantification of Foxp3⁺ cells within 2.5HIP tetramer-positive T cells in the pLN at 10 weeks after treatment. F: Quantification of CD4 T cells and 2.5HIP tetramer-positive T cells in the pancreatic islets 10 weeks after treatment. G: Quantification of Foxp3⁺ cells within 2.5HIP tetramer-positive T cells in the pancreatic islets at 10 weeks after treatment. (D–G: n = 5–7 per group; data from two independent experiments). Data are reported as mean ± SD. Paired t test (A and B), Gehan-Breslow-Wilcoxon Test (C), and Kruskal-Wallis and Dunn test (D–G). *P < 0.05, **P < 0.01.