Endocrine Abstracts

Background and aims: The initiation of verapamil, a calcium channel blocker with beta cell protective and T cell targeting properties, exhibits significant promise for the preservation of beta cell function at symptom onset in type 1 diabetes (T1D). Nonetheless, an immunogenic process leads to the progressive destruction of beta cells. Potent systemic immunomodulatory agents, like low-dose anti-thymocyte globulin (ATG), have shown promising effects as single agents at T1D onset. Our aim was to combine the beta cell protective properties of verapamil with the potent immunomodulatory effects of low-dose ATG to establish enduring immune tolerance and sustained T1D remission.

Materials and methods: We combined continuous administration of verapamil (1 mg/ml in drinking water) with low-dose murine (m)ATG (250 µg per day on day 0 and 3; i.v.) to study their efficacy in new-onset diabetic non-obese diabetic (NOD) mice.

Results: Verapamil stably reversed disease in 20% of mice (n = 3/15). Low-dose mATG reversed T1D in 39% of mice (n = 7/18) 7 days after therapy start, but the effect waned to 22% of mice (n = 4/18) by 8-weeks follow-up. Verapamil combined with low-dose mATG induced durable disease remission in 45% of mice (n = 9/20). Only combination therapy was able to preserve C-peptide levels and decrease insulitis severity compared to untreated mice. Mechanistically, either low-dose mATG-treated group induced lymphocyte and monocyte depletion 3 days after therapy start, recovering by day 14. Flowcytometry analysis revealed a decreased percentage of CD8+ T cells in the blood of either low-dose mATG-treated group at day 3, with a recuperation towards a CD8+ effector memory (CD44highCD62L-) phenotype by day 14. Moreover, all treated (verapamil, low-dose mATG or the combination) groups were associated with an increased frequency of FoxP3+(CD25+) regulatory T cells (Tregs) by day 3, that persisted until day 14 in the peripheral blood and pancreatic draining lymph nodes (PLN). Interestingly, while all low-dose mATG-treated groups were associated with an increased ratio of Tregs over activated (CD44high) CD8+ T cells in the peripheral blood by day 3, this effect did not persist until day 14. However, in the PLN, where an unbalanced immune state serves as one of the main drivers of T1D, only combination therapy resulted in an increased ratio of Tregs over activated (CD44high) CD8+ T cells that persisted until day 14.

Conclusion: Here we present the first evidence that continuous administration of verapamil in combination with a short course of low-dose mATG protected beta cells and induced a transient imbalance in immune cell frequency favouring Tregs, which may be sufficient to establish long-term tolerance and confer permanent T1D remission. This innovative approach shows significant therapeutic promise and represents a paradigm shift towards combination therapies in the future management of T1D.