Background: To arrest the immune-mediated destruction of the insulin-producing pancreatic beta cells in type 1 diabetes (T1D), interventional studies have predominantly focussed on two distinct approaches: arresting the ongoing immune response or protecting the pancreatic beta cells. Recently, both approaches using respectively low-dose anti-thymocyte globulin (ATG), an immune-depleting agent, and verapamil, a calcium channel blocker with beta cell protective and immune targeting properties, have demonstrated promising effects as single agents at T1D onset. However, the primary challenge remains the achievement of a durable response, primarily attributable to the highly immunogenic response and the restricted availability of functional beta cells at symptom onset. Consequently, integrating general immunomodulatory with beta cell supportive therapy represents a promising approach to achieve superior and long-lasting outcomes, particularly when initiated at symptom onset.
Methods: We combined low-dose murine (m)ATG (250 µg per day on day 0 and 3; i.v.) with continuous administration of verapamil (1 mg/ml in drinking water) to study their efficacy in newly diagnosed diabetic non-obese diabetic (NOD) mice.
Results: 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 by 8-weeks follow-up. Verapamil stably reversed disease in 20% of mice (n=3/15). However, combining low-dose mATG with verapamil induced durable disease remission in 45% of mice (n=9/20; P<0.0001 vs. control). Especially in mice with mild hyperglycaemia (<350 mg/dl) at disease onset combination reversed 75% (n=6/8) of mice compared to 33% in either low-dose mATG (n=3/9 mice; P<0.05) or verapamil (n=2/6 mice; P<0.05). While combination therapy resulted in a more pronounced disease reversal, this was not mirrored in the levels of pancreatic insulin content. However, preservation of beta cell function, as indicated by C-peptide levels, was observed only in combination-treated mice. Regarding pancreas inflammation, only combination therapy reduced insulitis severity. Mechanistically, either low-dose mATG-treated group induced lymphocyte depletion (69% reduction vs. control) 3 days after therapy start, recovering by day 14. Flow cytometry analysis revealed a decreased percentage of CD8+ T cells in the blood of either low-dose mATG-treated group at day 3, followed by a recovery of effector memory (EM) CD44highCD62L-CD8+ T cells by day 14. Moreover, low-dose mATG was associated with an increased frequency of FoxP3+(CD25+) regulatory T cells (Tregs) in the blood and pancreatic-draining lymph nodes (PLN) by day 14. Interestingly, only combination therapy had an increased ratio of Tregs-to-activated CD44highCD8+ T cells in PLN.
Conclusion: This is the first study to demonstrate that a short course of lowdose mATG in combination with verapamil protects the beta cells and mechanistically induces a transient imbalance in the frequency of immune cells favouring Tregs. This has to potential to establish enduring immune tolerance and confer sustained T1D remission in future clinical trials.