Dose-Dependent Effects of Murine Anti-Thymocyte Globulin (mATG) on Type 1 Diabetes (T1D) Development in Non-Obese Diabetic (NOD) Mice

Background. Low-dose Anti-Thymocyte Globulin (ATG) shows promise as an immune modulator, hereby
preserving beta cell function in newly diagnosed individuals with Type 1 Diabetes. The Minimum Effective Low
Dose (MELD)-ATG trial investigates the lowest ATG doses in children, adolescents, and young adults, balancing
efficacy in C-peptide preservation with minimal side effects (cytokine release syndrome and serum sickness). To
date, no in vivo studies have compared the kinetics of low mATG doses in murine models. Aim.To test three
murine (m)ATG doses by monitoring diabetes onset in Non-Obese Diabetic (NOD) mice and investigate the
accompanying immune effects using blood cell counting and spectral flow cytometry. Methods. Twelve-week-
old normoglycemic female NOD mice (n=8/group) received two intraperitoneal injections, three days apart, of
125µg, 250µg, or 500µg mATG or were left untreated. Diabetes onset was monitored weekly by checking blood
glucose levels until 30 weeks (w) of age. Animals were considered diabetic when their glycemia rose to
≥200mg/dL on two consecutive days. Peripheral blood samples were collected before treatment and 3 hours, 14
days, and 42 days after the last mATG injection for cell counting and spectral flow cytometry with 40 markers.
Results. A dose of 250µg mATG remarkably reduced diabetes incidence (13%; 1/8), compared to untreated (75%;
6/8), 125µg (38%; 3/8), and 500µg (38%; 3/8) at 30w of age. The earliest diagnosis in the 125µg mATG group
appeared only at 29w of age, a delay not seen with other doses (500µg, 19w; 250µg, 22w; untreated, 18w).
White blood cell counts revealed a greater decrease in the 500µg mATG group (43%), whereas 250µg (7%) and
125µg (18%) had similar depleting effects 3 hours after the last injection. Spectral flow cytometry revealed that
all doses depleted CD3+ cells as effectively 3 hours after the last injection. T-cell numbers in 125µg and 250µg
mATG treated mice returned to pre-treatment levels by day 14, whereas mice that received 500µg only
recuperated their pre-treatment CD3+ cell levels by day 42. CD4+:CD8+ ratios were lowest in the 250µg mATG
group (2.34), followed by the 500µg group (2.83) and the 125µg group (3.22), compared to 1.79 in untreated
mice. Conclusion. This unique study of low doses of mouse-specific ATG demonstrates that lower doses of mATG
delay (125µg) or reduce (250µg) diabetes incidence more than higher doses and regulate immune cell balance
differently in NOD mice.