Endocrine Abstracts

Background and aims: An interesting approach in the pursuit for a cure of type 1 diabetes is restoration of immune tolerance by a combination treatment of low-dose aCD3 with the islet antigen proinsulin (PINS) and the pro-tolerogenic cytokine IL-10 administered orally via genetically modified Lactococcus lactis (L. lactis) bacteria. The purpose of adding PINS is to expand antigen-specific Tregs as they are believed to migrate preferentially to disease-related target organs and be more powerful in dampening overactive immune responses. The aim of this study is to prove antigen-specificity of the L. lactis-based antigen immunotherapy.

Materials and methods: Newly diagnosed diabetic NOD mice were injected with alloxan (90 mg/kg i.v., Sigma) in order to completely deplete residual endogenous beta cell mass. After 48 h, all mice received 500 insulitis-free syngeneic islets and were either 1) left untreated (CTRL), 2) treated with aCD3 alone (aCD3), 3) aCD3 combined with L. lactis bacteria secreting PINS with IL-10 (CT), or 4) aCD3 combined with L. lactis secreting a non-islet antigen, ovalbumin with IL-10 (aCD3+LL-OVA). Flow cytometry analysis was done with insulin-reactive (e.g., InsB12–20 (TEGVEALYLVC-GGGS) and InsB13–21 (TEGEALYLVCGEGGS) PE- and APC-labeled MHC/peptide tetramers, used at a final concentration of 10 mg/ml in FACS buffer for 1 hour at room temperature.

Results: The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (OVA) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Flow cytometry data indicate that Foxp3+ Tregs, both CD25- and CD25+, in the islet grafts of mice, treated with aCD3 combined with L. lactis secreting PINS and IL-10, contained significantly more InsB12–20+ cells compared to those in the islet grafts of mice under anti-CD3 (with or without OVA) therapy alone or the untreated controls (Figure 1). Interestingly, increased numbers of Foxp3+ Tregs detected in the islet grafts of the combination therapy-treated mice were reactive to InsB12–20 and less to InsB13–21 (data not shown). Only in the mice treated with the add-on of the islet antigen proinsulin, a higher degree of insulin-reactive Tregs in the islet grafts was observed. Moreover, these insulin-reactive Tregs were preferentially observed in the islet grafts but not in the kidney draining lymph nodes (KLN) (Figure 1), indicating that these cells trafficked to the inflammatory sites where they may suppress persistent effector T cell function.

Figure 1. Combination therapy increases numbers of InsB12–20⁺Foxp3⁺CD4⁺ T cells in islet grafts. Newly diagnosed diabetic NOD mice with disease duration of < 2 days were injected i.v. with alloxan (90 mg/kg) and transplanted with 500 syngeneic islets after 48 h. Mice were left untreated (CTRL; = 4–5) or given a short-term low-dose aCD3 therapy (aCD3; n = 7–8) either alone or combined with L. lactis bacteria secreting the irrelevant antigen ovalbumin (LL-OVA; n = 3–5) or secreting beta cell antigen (PINS; n = 7–8) combined with IL-10 (CT). Absolute numbers of tetramer positive (tet+) InsB12–20 cells per 100 Foxp3+ Tregs, either CD25+ or CD25−,are shown 3 weeks after islet substitution and therapy initiation in the kidney draining lymph nodes (KLN) (A) and islet grafts (B) of diabetic NOD mice. Symbols represent individual mice, and line and error bars reflect group mean±S.E.M. Statistical significance between groups was calculated by Mann-Whitney U test; *P<0.05, **P<0.01, ***P<0.001.

Conclusion: This study provides for the first time strong evidence for the antigen specificity of our L. lactis-based antigen immunotherapy as PINS was needed for Foxp3+ Tregs to become insulin-reactive and home to insulin-containing islet grafts.