Verapamil and low-dose anti-mouse thymocyte globulin combination therapy stably reverses recent-onset type 1 diabetes in NOD mice by acting on the beta cell and immune axes

Abstract

Aims/hypothesis

Verapamil, a calcium channel blocker, and low doses of anti-thymocyte globulin (ATG) have individually shown efficacy in preserving beta cell function in people with recent-onset symptomatic type 1 diabetes (stage 3). We hypothesised that combining interventions with complementary modes of action and different targets would increase their efficacy in arresting beta cell demise and promoting disease recovery.

Methods

Continuous administration of verapamil via drinking water, combined with a short course of low-dose rabbit-anti-mouse ATG (mATG), was studied in female recent-onset diabetic NOD mice for its potential to induce disease remission and mechanism of action.

Results

Verapamil stably reversed diabetes in 3 out of 15 mice (20%) by day 56 after therapy start. Low-dose mATG reversed diabetes in 7 out of 18 mice (39%) by day 7 after therapy start, yet the effect waned to 3 out of 18 mice (17%) by day 56. The combination of verapamil with mATG induced durable diabetes reversal in 9 out of 20 mice (45%) by day 56, which was associated with preserved beta cell function, higher pancreatic insulin content and increased total beta cell volume with decreased severe insulitis. mATG, both alone and in combination, induced a temporary depletion of lymphocytes in peripheral blood on day 3 after therapy start, which largely recovered by day 14, when naive cells had shifted to a memory phenotype in both CD4⁺ and CD8⁺ T cells. Only in combination-treated mice was a higher CD4⁺ regulatory T cell to CD8⁺ effector memory T cell ratio observed in the pancreatic draining lymph nodes. The expression of the glucose-induced gene encoding thioredoxin-interacting protein (Txnip), a key regulator of beta cell apoptosis and dysfunction, was reduced in pancreatic beta cells in reversed mice, irrespective of whether they received verapamil or not.

Conclusions/interpretation

The combination of verapamil and low-dose mATG outperformed monotherapy in reversing recent-onset type 1 diabetes in NOD mice. This approach targets both the beta cell and immune axes, suggesting a promising strategy for disease reversal in human type 1 diabetes.

Graphical Abstract

Supplementary Information

The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-025-06490-8.

Introduction

We are entering a new era in type 1 diabetes treatment, with disease-modifying therapies aimed at preventing or arresting the decline in beta cell mass in pre-symptomatic (stage 1–2) and recent-onset (stage 3) individuals, respectively. To optimise outcomes, emerging strategies include associating agents working on the beta cell axis, to support beta cell function or increase beta cell mass through regeneration, with low doses of immunomodulatory drugs to re-educate the immune axis [1–4].

Verapamil, a non-dihydropyridine calcium channel blocker and approved anti-hypertensive drug, has been shown to temporarily preserve stimulated C-peptide secretion in small studies in children, adolescents and adults with stage 3 type 1 diabetes [5, 6], and is being further studied in a clinical trial for type 1 diabetes reversal (ClinicalTrials.gov registration no. NCT04545151). These trials were instigated by preclinical observations that orally administered verapamil reduced the expression of the thioredoxin-interacting protein (TXNIP), a ubiquitously expressed cellular redox regulator that is induced in pancreatic beta cells under hyperglycaemic conditions [7–9], leading to reduced beta cell apoptosis and enhanced endogenous insulin production [8]. While TXNIP overexpression promoted mitochondria-dependent beta cell apoptosis [10–12], TXNIP deficiency stimulated beta cell survival and protected against streptozocin-induced diabetes [13]. Additionally, dose-dependent immunosuppressive effects of verapamil had previously been observed [14–16], and recent studies demonstrated that verapamil blocked T cell activation by interfering with the activation of calcium-dependent transcription factors needed to stimulate gene transcription [14, 17]. A multi-omics approach established that verapamil, besides displaying direct beta cell protective effects, decreased circulating proinflammatory IL-21 values and surface expression of follicular helper T cell (Tfh) markers [18], both implicated in the pathogenesis of type 1 diabetes [19–21].

Polyclonal anti-thymocyte globulin (ATG) has a unique disease-modifying profile. It not only depletes conventional (Tconv) T cells, including naive, effector memory (TEM) and central memory (TCM) T cells, through complement-dependent lysis and T cell activation and apoptosis, but also modulates dendritic cell function and induces regulatory (Treg) T cells in non-diabetic individuals [22]. In stage 3 type 1 diabetes, low-dose ATG (2.5 mg/kg) attenuated the decline in beta cell function for at least 1 year compared with placebo [23, 24], accompanied by a significant reduction in the CD4⁺:CD8⁺ ratio and increased Treg:Tconv ratio [24]. A recent study further identified response signatures to low-dose ATG, including a temporary rise in inflammatory cytokines and durable CD4⁺ T cell exhaustion phenotypes [25]. Results of a study testing even lower ATG doses for efficacy and safety in children, adolescents and young adults with recent-onset stage 3 type 1 diabetes will read out soon (ClinicalTrials.gov registration no. NCT04509791) [26], and a study assessing its potential to delay disease progression from stage 2 to stage 3 type 1 diabetes has been started (ClinicalTrials.gov registration no. NCT04291703). Based on these complementary qualities, the combination of verapamil and low-dose ATG has been included in multi-arm intervention platforms in stage 3 type 1 diabetes that are recruiting (isrctn.com registration no. ISRCTN45965456) or will recruit soon (ClinicalTrials.gov registration no. NCT06455319).

Methods

Animals

NOD mice have been inbred at the KU Leuven animal facility since 1989, maintained under semi-barrier conditions and housed according to protocols approved by the KU Leuven Animal Care and Use Committee. This study was approved by the local ethics committee (Leuven, Belgium; project number 151-2020). Mice were screened three times a week for glucosuria using Diastix (Ascensia Diabetes Care, Machelen, Belgium). Diabetes was confirmed when glucosuria was detected, followed by non-fasting blood glucose concentrations exceeding 11.1 mmol/l on two consecutive days (AccuCheck, Roche Diagnostics International, Basel, Switzerland).

Treatments and follow-up

Female NOD mice with recent-onset diabetes were randomly assigned to one of four treatment groups: untreated, verapamil+IgG, rabbit-anti-mouse ATG (mATG) or verapamil+mATG, with 15–26 mice per group. mATG (PharmAbs, Leuven, Belgium) or the corresponding IgG isotype control (BioXcell, West Lebanon, NH) was administered intravenously at 250 μg per day on days 0 and 3 after disease onset (total dose of 500 µg). Verapamil (Thermo Fisher Scientific, Waltham, MA) was dissolved in sterile drinking water and administered ad libitum at a concentration of 1 mg/ml throughout the 56 day follow-up period. Daily verapamil dosing ranged from 4 to 44 mg per day, based on an average daily water consumption of 6.6 ± 1.4 ml (n=10) for normoglycaemic and 39.9 ± 13.5 ml (n=11) for diabetic NOD mice (data not shown). Mice were monitored three times per week for body weight and blood glucose concentrations, and follow-up continued for 56 days after therapy start. Disease reversal was defined by the absence of glucosuria and normalisation of blood glucose concentrations (<11.1 mmol/l). Mice were withdrawn from the study before the 56 day endpoint if their blood glucose concentrations exceeded 33.3 mmol/l in two consecutive measurements. The experimental design is illustrated in electronic supplementary material (ESM) Fig. 1.

Complete blood counts and plasma cytokine and C-peptide analyses

EDTA-anticoagulated peripheral blood was collected via submandibular bleeding on days 0, 3 and 14 after therapy start. Whole blood samples were analysed using a Vet abc Plus+ (Scil Animal Care Company, Viernheim, Germany) to determine the percentages (%) and absolute numbers (10³/mm³) of white blood cells (WBCs), lymphocytes, monocytes and granulocytes. Whole blood samples were centrifuged at 4°C for 10 min at 2000 g, and plasma was collected to detect the presence of the following cytokines: IFN-γ, IL-2, IL-10, IL-15 and IL-21 (cat. no. K15069L-1, Meso Scale Diagnostics, Rockville, MD), and random C-peptide concentrations by ELISA (Merck Millipore, Burlington, MA).

Pancreatic insulin content

Pancreases were harvested on days 0 and 14 after therapy start for insulin content determination. Pancreases were homogenised in acidic ethanol (91% ethanol, 9% 1 mol/l H₃PO₄) at 4°C overnight and then sonicated (30 s). Insulin content in the supernatant was determined by ELISA (cat. no. 10-1247-10, Mercodia, Uppsala, Sweden) and normalised to the weight of the pancreas.

Multi-parameter flow cytometry

EDTA-anticoagulated peripheral blood was collected via submandibular bleeding on days 0, 3 and 14 after therapy start. Pancreatic draining lymph nodes (PLNs) and pancreases were harvested on days 0 and 14. The pancreas was mechanically disrupted using surgical blades, followed by enzymatic digestion with collagenase-D and DNAse I at 37°C, and purified using 40% Percoll. PLNs were crushed through a 70 µm cell strainer. Single-cell preparations were stained with the following antibodies: CD3 (RRID: AB_1107000, clone 145-2C11, cat. no. 45-0031-82, eBioscience, Thermo Fisher Scientific, Waltham, MA, 1:100 or RRID: AB394595, clone 145-2C11, cat. no. 553062, BD Biosciences, Erembodegem, Belgium, 1:100), CD4 (RRID: 1645235, clone GK1.5, cat. no. 560181, BD Biosciences, 1:160), CD8 (RRID: 1272198, clone 53-6.7, cat. no. 48-0081-82, eBioscience, Thermo Fisher Scientific, 1:300), CD25 (AB_469608, clone PC61.5, cat. no. 25-0251-82, eBioscience, Thermo Fisher Scientific, 1:625), CD44 (AB_465044, clone IM7, cat. no. 11-0441-81, eBioscience, Thermo Fisher Scientific, 1:400), CD62L (AB_469409, clone MEL-14, cat. no. 17-0621-81, eBioscience, Thermo Fisher Scientific, 1:400) and FOXP3 (AB_465935, clone FJK-16s, cat. no. 12-5773-80, eBioscience, Thermo Fisher Scientific, 1:100). Zombie Aqua (BioLegend, San Diego, CA) was used as viability marker. FOXP3 detection was performed using a staining buffer set (cat. no. 00-5523-00, eBioscience, Thermo Fisher Scientific), following the manufacturer’s instructions. Cell acquisition was performed on a BD Canto II AIG flow cytometer (BD Biosciences) and analysed using FlowJo software v.10.10 (FlowJo, Ashland, OR). All analyses were conducted on a fixable viability dye-negative singlet population. The gating strategy is shown in ESM Fig. 2.

Insulitis grading

Formalin-fixed paraffin-embedded (FFPE) pancreases collected on day 14 after therapy start were sectioned, deparaffinised and rehydrated. Tissue sections were stained with Mayer’s Haematoxylin Solution (1 g/l) followed by 0.2% eosin. H&E-stained pancreas sections were examined using a BA310 LED trinocular microscope equipped with a Moticam S3 (MoticEurope, Barcelona, Spain). Islets were graded by an independent investigator (P-JM). At least 25 islets per pancreatic sample were assessed for immune infiltration, and the degree of insulitis was scored as follows: 0, no infiltration; 1, peri-insulitis; 2, lymphocyte infiltration in <50% of the islet area; and 3, lymphocyte infiltration in >50% of the islet area or complete islet destruction.

Pancreas immunohistochemistry

First, 5 µm sections from FFPE pancreas tissues collected on days 0 and 14 after therapy start were mounted on Superfrost Plus slides (VWR International, Radnor, PA) and dried overnight at 40°C. The slides were baked for 60 min at 60°C, deparaffinised in xylene (2 × 5 min), rehydrated in 95% ethanol (2 × 2 min) and rinsed for 2 min under running distilled water. For immunostaining, non-specific binding sites were blocked for 30 min with Blocker Casein in PBS (cat. no. 37528, Thermo Fisher Scientific) diluted 1:4 in PBS + 0.1% Tween-20 (PBST). Sections were incubated overnight at 4°C with primary antibody for insulin (guinea pig anti-insulin, Diabetes Research Center [DRC], Vrije Universiteit Brussel [VUB], Brussels, Belgium) diluted in BOND Primary Antibody Diluent (cat. no. AR9352, Leica Biosystems, Nussloch, Germany). After incubation, slides were rinsed 3 × 20 min in PBST and subsequently incubated for 30 min with biotinylated secondary antibody (RRID: AB_2340451, biotin-SP-labelled donkey anti-guinea pig, Jackson ImmunoResearch), diluted 1:1,000 in BOND Primary Antibody Diluent. Slides were rinsed 3 × 10 min in PBST and subsequently incubated for 30 min with Vectastain Elite ABC-AP Standard (Vector AK-5000, Vector Laboratories, Burlingame, CA). Slides were rinsed 3 × 10 min in PBST and subsequently incubated for 10 min with ImmPACT Vector Red (Vector SK-5105, Vector Laboratories). Slides were rinsed for 2 min under running distilled water. Finally, slides were dried for 60 min at 60°C and mounted with Pertex mounting medium (VWRKAM-0801, VWR International). Brightfield image acquisition for beta cell volume analysis was done with a Leica Aperio GT450 (Leica Biosystems). Image analysis was conducted with Fiji image processing software v.1 (NIH, Bethesda, MD).

Pancreas RNA in situ hybridisation

RNA in situ hybridisation (RNA-scope), immunostaining and imaging were performed by the Visual and Spatial Tissue Analysis (VSTA) core facility at VUB (Brussels, Belgium) (https://vsta.research.vub.be) using the RNA-scope Multiplex Fluorescent v2 Assay kit (cat no. 323100, Advanced Cell Diagnostics [ACD], Newark, CA) according to the manufacturer’s instructions. The probe used was Mus musculus Txnip (cat no. 457221, ACD). For immunostaining after in situ hybridisation, sections were blocked for 30 min with Blocker Casein in PBS (cat no. 37528, Thermo Fisher Scientific) diluted 1:4 in PBST. Primary antibodies diluted in BOND Primary Antibody Diluent (Leica Biosystems) were guinea pig anti-insulin (DRC, VUB, 1:5,000) and rabbit anti-glucagon (RRID: AB_10561971, clone EP3070, cat no. ab92517, Abcam, 1:5,000), incubated overnight at 4°C, and detected using anti-guinea pig AlexaFluor 488-conjugated IgG (RRID: AB_2337438, Jackson ImmunoResearch, 1:500) and anti-rabbit DyLight 755-conjugated IgG (RRID: AB_2556615, Thermo Fisher Scientific, 1:200), respectively. Nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St Louis, MO, B2261) added at 2 µg/ml to the secondary antibodies. Fluorescence image acquisition was done with a Zeiss Axio Scan.Z1 (Zeiss, Jena, Germany) at ×20 magnification.

Blinding/masking

Experimenters were not blinded throughout the study.

Statistical analysis

Statistical comparisons were performed using GraphPad Prism v.9.3.0 (GraphPad Software, La Jolla, CA). The respective number of replicates (n) is indicated in the figures or figure captions. Power analyses were used to predetermine appropriate sample sizes for animal experiments (power 0.8, α 0.05). Kaplan–Meier life table analysis with logrank Mantel–Cox regression was applied to compare the survival curves between experimental groups. p values were determined by an appropriate statistical test such as unpaired two-tailed Student’s t test or Mann–Whitney U test if the data did not assume Gaussian distribution, as indicated in the figure legends. Differences between paired data were determined by a mixed-effects model test with post hoc Tukey’s honestly significant difference test. Outliers were determined by the Grubbs test (alpha 0.05). p values ≤0.05 were considered significant (*p≤0.05, **p≤0.01, ***p≤0.001).

Results

Combination therapy with verapamil and low-dose mATG provides superior and sustained hyperglycaemia reversal in recent-onset diabetic NOD mice

We compared the effectiveness of combination therapy with verapamil and low-dose mATG against either verapamil or mATG monotherapy in reversing hyperglycaemia in recent-onset diabetic NOD mice (Fig. 1a, ESM Fig. 3a–d). In untreated control mice, hyperglycaemia persisted in nearly all cases (25 out of 26 mice). Verapamil monotherapy led to hyperglycaemia reversal in 20% of mice (3 out of 15 mice), which was sustained throughout the 56 day follow-up period. Similarly, low-dose mATG monotherapy resulted in 17% reversal (3 out of 18 mice), with peak efficacy between days 4 and 21 after therapy start (normoglycaemia in 39% of mice).

Fig. 1.

Effect of verapamil combined with a short course of low-dose mATG on diabetes reversal. (a) Kaplan–Meier survival curves depicting the percentage of diabetic animals over time in all NOD mice included in the study. (b, c) Analysis of plasma C-peptide (pmol/l) (b) and chromogranin A (pg/ml) (c) concentrations at day 14 after therapy start. Data are represented as median with interquartile range (box) with minimum and maximum non-outlier values (whiskers). Symbols represent individual mice. Red dotted line represents median concentration at diabetes onset. (d–g) Kaplan–Meier survival curves depicting the percentage of diabetic animals over time subdivided into those with a starting blood glucose level of <19.4 mmol/l and age <16 weeks (d), a starting blood glucose level of <19.4 mmol/l and age ≥16 weeks (e), a starting blood glucose level of ≥19.4 mmol/l and age <16 weeks (f) and a starting blood glucose level of ≥19.4 mmol/l and age ≥16 weeks (g). *p≤0.05; ***p≤0.001. IgG was used as the isotype control. d, day; Untr, untreated; V, verapamil

In contrast, the combination of verapamil and low-dose mATG induced immediate hyperglycaemia reversal in 20% of the mice, with efficacy progressively increasing to 45% (9 out of 20 mice) by the end of the follow-up period (Fig. 1a, ESM Fig. 3d). Notably, by day 14 after therapy start, only combination therapy-treated mice demonstrated significantly better preservation of C-peptide secretion compared with untreated controls (Fig. 1b, ESM Fig. 4). Although previous studies suggested that normalised chromogranin A concentrations indicated restored beta cell integrity [18], we observed no significant differences in plasma chromogranin A concentrations across treatment groups at day 14 (Fig. 1c). All treatment regimens were well tolerated, with no signs of weight loss in treated animals (data not shown).

Next, we stratified recent-onset diabetic NOD mice based on the timing of diabetes onset (acute, <16 weeks of age; or late, ≥16 weeks of age) and initial blood glucose levels (<19.4 mmol/l or ≥19.4 mmol/l at therapy start). Monotherapies were most effective in mice with late-onset diabetes, whereas the combination therapy showed efficacy irrespective of diabetes onset timing and was most effective in mice with mild hyperglycaemia at therapy start (Fig. 1d–g).

Transient lymphocyte depletion with increased CD4⁺ to CD8⁺ ratio and cytokine release in peripheral blood is mainly due to the short course of low-dose mATG therapy

Consistent with previous findings in mouse and human [24, 27], we observed that a short course of low-dose mATG, whether administered alone or alongside continuous verapamil treatment, led to a marked decrease in WBC, lymphocyte and monocyte counts by day 3 after therapy start. These cell counts returned to baseline values by day 14 (Fig. 2a–c). Granulocyte counts remained unaffected by either treatment (Fig. 2d).

Fig. 2.

Effect of verapamil combined with a short course of low-dose mATG on cell counts and T lymphocytes in whole blood. (a–d) Absolute cell numbers per mm³ of total WBCs (a), lymphocytes (b), monocytes (c) and granulocytes (d) over time. (e–g) Frequency of CD4⁺ (e) or CD8⁺ (f) within CD3⁺ T cells, and the calculated CD4⁺ to CD8⁺ ratio (g) in whole blood on days 3 and 14 after therapy start. (h–j) Frequency of CD44^LowCD62L⁺ (naive) (h), CD44^HiCD62L⁺ (TCM) (i) and CD44^HiCD62L⁻ (TEM) (j) within CD4⁺ T cells in whole blood on days 3 and 14 after therapy start. (k–m) Frequency of CD44^LowCD62L⁺ (naive) (k), CD44^HiCD62L⁺ (TCM) (l) and CD44^HiCD62L⁻ (TEM) (m) within CD8⁺ T cells in whole blood on days 3 and 14 after therapy start. (n–p) Frequency of FoxP3⁺ (n), FoxP3⁺CD25⁺ (o) and FoxP3⁺CD25⁻ (p) within CD4⁺ T cells in whole blood on days 3 and 14 after therapy start. Data are represented as median with interquartile range (box) with minimum and maximum non-outlier values (whiskers). (e–p). Symbols represent group median (a–d) or individual mice (e–p). Red dotted line represents median frequency at diabetes onset (e–p). *p≤0.05; **p≤0.01; ***p≤0.001: asterisks above each bar show the comparison vs baseline (red line); bracketed asterisks are for the comparison shown. IgG was used as the isotype control. d, day; Untr, untreated; V, verapamil

By day 3, the percentage of CD4⁺ T cells in peripheral blood increased relative to baseline, whereas the percentage of CD8⁺ T cells declined in both mATG-treated groups, resulting in an elevated CD4⁺:CD8⁺ T cell ratio (Fig. 2e–g). However, by day 14, the percentage of CD4⁺ T cells decreased, while CD8⁺ T cells remained largely unchanged, leading to a reduced CD4⁺:CD8⁺ T cell ratio compared with baseline and untreated controls (Fig. 2e–g). Furthermore, both CD4⁺ and CD8⁺ T cells displayed shifts in activation and differentiation markers, with mATG therapy, either alone or combined with verapamil, reducing the frequency of naive T cells (CD44^LowCD62L⁺) and increasing the proportion of TEMs (CD44^HiCD62L⁻) in peripheral blood (Fig. 2h–m). The proportions of TCM (CD44^HiCD62L⁺) CD4⁺ and CD8⁺ T cells in peripheral blood remained unaffected (Fig. 2i, l). Although the frequency of FoxP3⁺, mainly CD25⁺ CD4⁺ Tregs, was initially decreased by both mATG therapies in peripheral blood at day 3 after therapy start, higher frequencies were observed at day 14 (Fig. 2n–p).

Additionally, low-dose mATG therapy has been shown to trigger a temporary cytokine release syndrome. In line with observations from both animal and human studies [25, 28], we observed significant increases in plasma values of IFN-γ, IL-2 and IL-10 on day 3 after therapy start in both mATG-treated groups, compared with baseline, verapamil monotherapy and untreated controls. IL-15 values, however, remained unchanged. By day 14, these cytokine concentrations showed partial or complete recovery (ESM Fig. 5a–d).

Combination therapy with verapamil and low-dose mATG reduces insulitis severity and increases the ratio of CD4⁺ Tregs:CD8⁺ TEM cells in PLNs

To evaluate the effect of the different treatment regimens on pancreatic inflammation, we quantified immune cell infiltration in the pancreatic islets on day 14 after therapy start (Fig. 3a–c). Mice receiving combination therapy exhibited a modest increase in the proportion of insulitis-free islets (Fig. 3b) and a reduced proportion of heavily infiltrated islets (Fig. 3c), compared with untreated controls.

Fig. 3.

Effect of verapamil combined with a short course of low-dose mATG on insulitis. (a) Bar plot of insulitis score with percentages of defined insulitis severity. (b, c) Percentage of insulitis-free (b), and severe insulitis (c) scored islets on day 14 after therapy start. Islets with lymphocyte infiltration in <50% of the area were scored as mild insulitis, and islets with lymphocyte infiltration in ≥50% of the area or destroyed islets were scored as severe insulitis. *p≤0.05. IgG was used as the isotype control. d, day; Untr, untreated; V, verapamil

Immune phenotyping of the pancreas by flow cytometry revealed an increased frequency of TEM (CD44^HiCD62L⁻) and TCM (CD44^HiCD62L⁺) CD4⁺ T cells in combination-treated mice at day 14, relative to untreated controls (Fig. 4a–c). In contrast, CD8⁺ T cell populations were largely unaffected (ESM Fig. 6a–c), and the percentage of Tregs in the pancreas did not differ between treatment and control groups (Fig. 4d–f).

Fig. 4.

Effect of verapamil combined with a short course of low-dose mATG on T cell subpopulations in pancreas and PLNs. (a–c) Frequency of CD44^LowCD62L⁺ (naive) (a), CD44^HiCD62L⁺ (TCM) (b) and CD44^HiCD62L⁻ (TEM) (c) within CD4⁺ T cells in the pancreas on day 14 after therapy start. (d–f) Frequency of FoxP3⁺ (d), FoxP3⁺CD25⁺ (e) and FoxP3⁺CD25⁻ (f) within CD4⁺ T cells in the pancreas on day 14 after therapy start. (g–i) Frequency of CD44^LowCD62L⁺ (naive) (g), CD44^HiCD62L⁺ (TCM) (h) and CD44^HiCD62L⁻ (TEM) (i) within CD4⁺ T cells in PLNs on day 14 after therapy start. (j–l) Frequency of FoxP3⁺ (j), FoxP3⁺CD25⁺ (k) and FoxP3⁺CD25⁻ (l) within CD4⁺ T cells in PLNs on day 14 after therapy start. Data are represented as median with interquartile range (box) with minimum and maximum non-outlier values (whiskers). Symbols represent individual mice. Red dotted line represents median frequency at diabetes onset. *p≤0.05; **p≤0.01; ***p≤0.001: asterisks above each bar show the comparison vs baseline (red line); bracketed asterisks are for the comparison shown. IgG was used as the isotype control. d, day; Untr, untreated; V, verapamil

While T cell activation occurs in the pancreatic islets, T cell proliferation is known to take place primarily in the PLNs [29]. By day 14, combination therapy induced a shift from naive to memory (both TEM and TCM) CD4⁺ T cells in the PLNs, compared with baseline and untreated controls (Fig. 4g–i). Additionally, there was a notable expansion of FoxP3⁺CD4⁺ Tregs, both CD25⁺ and CD25⁻, in the PLNs of combination-treated mice (Fig. 4j–l). CD8⁺ T cell frequencies in the PLNs remained unchanged compared with baseline and untreated controls (ESM Fig. 6d–f). Interestingly, the combination therapy resulted in an increased ratio of CD4⁺ Tregs to CD8⁺ TEM cells in the PLNs, although this increase was not observed in peripheral blood or pancreas (Fig. 5a–c).

Fig. 5.

Effect of verapamil combined with a short course of low-dose mATG on the ratio of FoxP3⁺CD25⁺CD4⁺ Tregs to CD8⁺ TEM cells. The ratio of FoxP3⁺CD25⁺CD4⁺ Tregs to CD8⁺ TEM cells in blood (a), pancreas (b) and PLNs (c) on day 14 after therapy start. Data are represented as median with interquartile range (box) with minimum and maximum non-outlier values (whiskers). Symbols represent individual mice. Red dotted line represents median frequency at diabetes onset. *p≤0.05; **p≤0.01; ***p≤0.001: asterisks above each bar show the comparison vs baseline (red line); bracketed asterisks are for the comparison shown. IgG was used as the isotype control. d, day; Untr, untreated; V, verapamil

Verapamil treatment reduces Txnip expression in pancreatic beta cells, supporting improved beta cell function

The enhanced preservation of beta cell function in combination therapy-treated mice at day 14 after therapy start (Fig. 1b) was associated with an increased pancreatic insulin content (Fig. 6a) and greater insulin-positive volume relative to pancreatic volume (Fig. 6b) compared with untreated controls. Given that previous studies have identified TXNIP as a critical regulator of beta cell death induced by glucotoxicity and inflammation [10, 30], and that verapamil has been shown to reduce Txnip expression in rat beta cell lines, as well as in human and mouse islets [31], we examined Txnip expression in pancreatic beta cells across the different treatment groups at day 14.

Fig. 6.

Effect of verapamil combined with a short course of low-dose mATG on beta cells. (a, b) Analysis of pancreatic insulin content (ng/mg pancreas) (a) and total insulin volume/pancreas volume (%) (b) on day 14 after therapy start. (c) Representative images of RNA-scope (Txnip) and immunostaining (INS, GCG and Hoechst) on pancreatic sections on day 14 after therapy start. Scale bar = 100 μm. Data are represented as median with interquartile range (box) with minimum and maximum non-outlier values (whiskers). Symbols represent individual mice. Red dotted line represents median frequency at diabetes onset. *p≤0.05. IgG was used as the isotype control. d, day; GCG, glucagon; HO, Hoechst; INS, insulin; Untr, untreated; V, verapamil

In hyperglycaemic mice, Txnip expression co-localised with insulin-positive cells, indicative of beta cell stress. This upregulation was absent in mice becoming normoglycaemic by day 14 after therapy start, regardless of treatment regimen (Fig. 6c). These findings suggest that the reduced expression of Txnip, particularly in the combination therapy-treated mice, may contribute to improved beta cell survival and function.

Discussion

Type 1 diabetes is a complex metabolic disorder driven by both inherent beta cell vulnerability and immune-mediated beta cell destruction. This dual pathology suggests that single-target treatments may be inadequate for achieving lasting remission. Clinical trials have shown that both verapamil and ATG can preserve C-peptide production in individuals with recent-onset type 1 diabetes [5, 6, 18, 23, 24]. However, their effects were short-lived, with C-peptide preservation diminishing over time. Combining these agents, which have complementary mechanisms of action and distinct targets, may enhance their ability to halt beta cell destruction and lead to more durable therapeutic outcomes.

This study provides the first evidence that continuous administration of verapamil combined with a short course of low-dose mATG significantly improves glucose control in recent-onset diabetic NOD mice, outperforming either treatment alone. Notably, the addition of verapamil to the mATG regimen prevented the loss of therapeutic efficacy over time, with more mice achieving long-term normoglycaemia. While mATG monotherapy initially led to higher rates of diabetes reversal, the effect was temporary. In contrast, combination therapy, although slower in onset, demonstrated superior long-term outcomes, suggesting a synergistic effect in which mATG rapidly modulates the immune response, allowing verapamil to preserve beta cell function more effectively. Since the combination therapy was most efficacious in mice with mild hyperglycaemia, we highlight the importance of early intervention either at clinical diagnosis (stage 3 type 1 diabetes) or prior to the acute decline in beta cell function (stage 2 type 1 diabetes), when a larger population of beta cells may still be viable and responsive to therapeutic strategies. Given the variability in disease progression, some individuals, especially those diagnosed at older age, may maintain significant C-peptide levels up to 1 year post diagnosis, further suggesting that timely intervention with beta cell supportive therapies could still benefit certain individuals [32].

The well-documented anti-apoptotic properties of verapamil are central to its therapeutic potential [33]. By reducing the expression of TXNIP, a key regulator of glucose-induced beta cell stress, verapamil prevents the pro-apoptotic pathways activated by hyperglycaemia [30, 34]. TXNIP binds to and inhibits thioredoxin, a critical component of the intracellular antioxidant redox system [35], thereby promoting oxidative stress and caspase 3-dependent apoptosis [36]. Conversely, overexpression of thioredoxin has been shown to delay type 1 diabetes onset in NOD mice and alleviate streptozocin-induced diabetes severity [8]. Beta cells residing in the islets of Langerhans are particularly susceptible to oxidative stress due to their weak antioxidant defences, making them vulnerable to the upregulated Txnip expression induced by hyperglycaemia. Verapamil mitigates these effects by reducing the binding of carbohydrate response element-binding protein (ChREBP), a key transcription factor that controls Txnip transcription. By reducing Txnip transcription in beta cells, verapamil protects beta cells against reactive oxygen species (ROS) production and apoptosis, thus preserving insulin-producing cells. Additionally, verapamil upregulates the expression of thioredoxin reductase (Txnrd1) and sulfiredoxin (Srxn1), genes that help conserve the cellular redox potential [18]. These beta cell protective effects of verapamil form the foundation for promising preclinical [8, 37] and clinical [5, 6] trials, indicating that verapamil may preserve beta cell function when initiated at the onset of type 1 diabetes symptoms.

Our results demonstrate that the combination therapy of verapamil and mATG, but not the monotherapies, was associated with significant preservation of pancreatic insulin content, beta cell volume and beta cell function, as reflected by higher circulating C-peptide concentrations compared with untreated controls. RNA-scope revealed lower Txnip expression in the beta cells of mice that became normoglycaemic, regardless of the treatment arm, suggesting that Txnip may contribute to the preservation of beta cell function. In human studies, a downregulation of HLA class I and II genes and reduced inflammatory signalling observed in islet samples of verapamil-treated individuals likely also contributed to the protective effects of verapamil in recent-onset type 1 diabetes by preventing immune cell infiltration into the islets and chronic beta cell destruction [18]. Additionally, combination-treated mice exhibited a shift in the insulitis profile, with more insulitis-free islets and fewer severely inflamed islets compared with untreated controls. Although human studies have suggested that plasma chromogranin A could serve as a therapeutic marker for verapamil efficacy [18], we did not observe significant differences in chromogranin A values between our experimental groups.

We successfully generated high-quality mATG through the immunisation of rabbits with murine thymocytes, allowing us to study its effects in a type 1 diabetes mouse model. As seen in human studies, mATG therapy induced shifts in T cell subpopulations, characterised by an initial increase in the CD4⁺:CD8⁺ T cell ratio, which normalised by day 14 [24]. mATG reduced the number of naive T cells in peripheral blood, limiting the activation of new self-reactive T cells and thereby reducing autoimmune activity. Interestingly, not all T cell subtypes were equally affected by mATG. For instance, Tregs and antigen-primed T cells were less susceptible to depletion [27, 38]. In the human situation, high-dose (i.e. 6.5 mg/kg) ATG was unable to preserve beta cell function in individuals with recent-onset type 1 diabetes in the START (Study of Thymoglobulin to Arrest Type 1 Diabetes) phase 2 randomised placebo-controlled trial, due to Treg depletion and retention of refractory TEM cells, resulting in a decreased Treg to TEM ratio [39]. In contrast, low-dose (i.e. 2.5 mg/kg) ATG preserved Tregs in a subsequent clinical trial ATG-GCSF (ATG and pegylated granulocyte colony stimulating factor), increasing the Treg to Tconv ratio [24]. Although a subtherapeutic dose of mATG was used in our study, a significant decrease in FoxP3⁺CD4⁺ T cells in peripheral blood was observed in the mATG monotherapy group, primarily due to the depletion of FoxP3⁺CD25⁻CD4⁺ Tregs. This likely contributed to the temporary impact of mATG monotherapy on disease reversal in recent-onset diabetic NOD mice, lasting only 7 to 21 days after therapy start before most of the mice relapsed to hyperglycaemia.

Consistent with previous findings on immune cell repopulation following ATG therapy [40, 41], we found that both CD4⁺ and CD8⁺ TEM cells in peripheral blood repopulated after mATG treatment, either alone or in combination, with Tregs eventually increasing in proportion. The rise in Tregs suggests a temporary restoration of peripheral regulation due to the initial immune cell depletion and preferential sparing of Tregs by mATG. However, this effect is likely transient and may fail without further intervention, as creating space and removing an inhospitable environment has been shown to offer only short-term benefits. Importantly, the combination therapy resulted in a significant expansion of FoxP3⁺CD25⁺CD4⁺ Tregs in PLNs at days 3 and 14 after therapy start, with an increased ratio of Tregs to activated CD8 TEM cells. This shift suggests enhanced immune regulation in the PLNs, a key site for T cell proliferation and activation. CD25 (IL-2Rα chain) is an activation marker highly expressed in Tregs, playing a critical role in their proliferation, survival and suppressive function [42].

In addition to its beta cell protective effects, verapamil also appears to have immunomodulatory properties. Recent studies have shown that verapamil can counteract the increase in Tfh cells and proinflammatory cytokine IL-21 values in individuals with recent-onset type 1 diabetes [18]. Since IL-21 plasma concentrations were below the detection limit in our assay, we could not confirm the formerly observed downregulation of this proinflammatory cytokine after verapamil therapy, yet we observed an increase in plasma concentrations of IFN-γ and IL-10 compared with baseline, even in the absence of mATG. While plasma cytokine analysis provides insights into systemic immune responses, it may not fully reflect the local immune environment in the pancreas. Still, the elevation in T helper 1 (Th1) cytokines, coupled with a shift from naive to TEM cells in peripheral blood, suggests a transient phase of T cell activation. The concomitant rise in IL-10 might positively influence the Treg population; however, to our knowledge, no previous studies have reported on direct effects of verapamil on Tregs.

In conclusion, our study is the first to demonstrate the combined efficacy of verapamil and low-dose mATG in reversing type 1 diabetes in recent-onset diabetic NOD mice. The combination therapy stably reversed type 1 diabetes in nearly half of the mice and was associated with both immune-modulatory and beta cell protective effects, suggesting a synergistic interaction between the two interventions. These observations are timely as clinical trials of verapamil monotherapy (VER-A-T1D [Verapamil SR in Adults with Type 1 Diabetes]; ClinicalTrials.gov registration no. NCT04545151) and low-dose ATG (MELD-ATG [Minimum Effective Low-Dose Anti-Thymocyte Globulin]; ClinicalTrials.gov registration no. NCT04509791) are reading out soon, and trials on combination therapies are being initiated in people with recent-onset stage 3 type 1 diabetes globally (isrctn.com registration no. ISRCTN45965456; ClinicalTrials.gov registration no. NCT06455319).

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ATG

Anti-thymocyte globulin

FFPE

Formalin-fixed paraffin-embedded

mATG

Rabbit-anti-mouse ATG

PBST

PBS + 0.1% Tween-20

PLN

Pancreatic draining lymph node

RNA-scope

RNA in situ hybridisation

TCM

Central memory T cell

Tconv

Conventional T cell

TEM

Effector memory T cell

Tfh

Follicular helper T cell

Treg

Regulatory T cell

TXNIP

Thioredoxin-interacting protein

WBC

White blood cell

Acknowledgements

We thank the KU Leuven Flow and Mass Cytometry core facility (Leuven, Belgium) and the VUB VSTA core facility (Brussels, Belgium). Some of the data were presented as an oral presentation by P-JM at the 60th Meeting of the EASD in September 2024, and as a poster presentation by P-JM at the 20th Meeting of the Immunology of Diabetes Society (IDS) in November 2024.

Data availability

Data supporting the results from this study are available from the corresponding author on reasonable request.

Funding

This work was funded by the Innovative Medicine Initiative 2 Joint Undertaking (IMI2 JU) under grant agreement no. 115797 (INNODIA [Innovative approach towards understanding and arresting type 1 diabetes]). This Joint Undertaking received support from the European Union’s Horizon 2020 research and innovation programme and EFPIA, Breakthrough T1D (formerly known as JDRF) and The Leona M. and Harry B. Helmsley Charitable Trust. This work was supported by grants from the KU Leuven C1 funding (C1/18/006 and C16/24/012) and a small research infrastructure grant (KA/20/077). WS holds a Research Foundation Flanders (FWO) senior clinical investigator grant (1.8064.21N) and a Breakthrough T1D Career Development Award (CDA-2024-1491-S-B).

Authors’ relationships and activities

CM serves or has served on the advisory panels for AstraZeneca, Avotres, Boehringer Ingelheim, Eli Lilly and Company, Imcyse, Insulet, Mannkind, Medtronic, Merck Sharp and Dohme Ltd, Novartis, Novo Nordisk, Pfizer, Precigen Actobio, Roche, Sandoz, Sanofi, Vertex Pharmaceuticals and Zealand Pharma. Financial compensation for these activities has been received by KU Leuven. The authors declare that there are no other relationships or activities that might bias, or be perceived to bias, their work.

Contribution statement

CG designed the study. P-JM, LD and CG wrote the initial draft of the manuscript. P-JM, LD and MV performed mouse treatments and follow-up. YH performed RNA-scope staining. GL performed histological analyses. NG developed mATG to deplete T cells in vivo, enabling precise modulation of the immune system, and provided feedback on the manuscript. NDL and WS provided the necessary expertise to interpret the histological analyses and provided critical feedback on the manuscript. All authors critically improved the manuscript. CM and CG conceptualised the research goals, acquired major funding and discussed the data. CM and CG are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors approved the final version of this manuscript.