Abstract Introduction Engineered regulatory T cell (EngTreg) therapy offers a novel approach to restore immune tolerance in type 1 diabetes (T1D). To support POLARIS, GentiBio’s first-in-human trial of GNTI-122, we developed a biomarker program to monitor drug kinetics, pharmacodynamics, and immunogenicity. Methods Kinetics of GNTI-122 will be quantified by droplet digital PCR (ddPCR) detecting engineered transgenes. An 18-color flow cytometry panel is designed to detect GNTI-122, its Treg profile, and impact on immune subsets. Mechanism of action of GNTI-122 will be assessed by reduction in β cell-specific effector T cell (Teffs) responses using two complementary assays: an Activation-Induced Marker (AIM) assay and a FluoroSpot assay. Immunogenicity will be evaluated by a cell-based anti-drug antibody (ADA) assay targeting the TCR of GNTI-122. Results Biomarker assays demonstrated robust sensitivity, specificity and precision. Validated assays show the ability to detect GNTI-122 at a level of sensitivity < 0.1% of total WBCs. AIM and FluoroSpot assays reproducibly detected β cell-specific Teffs. Our cell-based anti-TCR ADA assay detected anti-TCR antibodies at a sensitivity < 850 ng/ml. Conclusion This comprehensive biomarker strategy provides a rigorous translational framework to monitor cellular kinetics, pharmacodynamics, and immunogenicity, to elucidate biological mechanisms of GNTI-122 for GentiBio’s POLARIS Phase 1 safety trial of GNTI-122 in recently diagnosed T1D. Funding Source T1D Fund Topic Categories Immune Mechanisms of Human Disease (HUM)

Regulatory T cells (Tregs) are essential for preventing autoimmunity. They depend upon interleukin-2 (IL-2) for optimal function and due to high expression of the CD25 subunit of the IL-2 receptor, are 10-fold more sensitive to IL-2 than effector T cells (Teffs). Consequently low-dose IL-2 can be used to preferentially expand Tregs, a therapeutic strategy which has shown promise in a number of autoimmune and inflammatory conditions - including graft-versus-host disease and lupus, where IL-2 driven expansion correlates with improvements in some clinical markers of disease activity. Autoimmunity is a frequent delayed complication of treatment with the lymphocyte-depleting drug alemtuzumab in relapsing-remitting multiple sclerosis (RRMS). Here, using in vitro assays, a pre-clinical mouse model, and an experimental medicine study, we investigated whether low-dose IL-2 could selectively expand Tregs in alemtuzumab-treated RRMS patients. Six months after alemtuzumab treatment, the frequency of patient-derived naïve CD4+ Teffs expressing high-affinity IL-2 receptors increased from 30.11+/-5.09% to 72.88 +/-5.57%, and the density of receptors per cell increased, rendering them six times more sensitive to IL-2 in vitro, at concentrations that typically favour Tregs. Using a human CD52-expressing transgenic mouse model of alemtuzumab treatment, we found that IL-2 was still able to preferentially expand Tregs, but only when administered at a later time point, corresponding to more than 6 months post-treatment in patients. Guided by these findings, we evaluated low-dose IL-2 for Treg expansion in a prospective open-label mechanistic study of RRMS patients who had received alemtuzumab more than 6 months previously. IL-2, at a dose and frequency similar to that previously shown to expand Tregs in autoimmune diabetes (0.3 x 106 IU/m2 twice a week), was well tolerated and safe, but unexpectedly failed to expand Tregs. We discuss the potential reasons underlying this lack of response, ruling out sIL2RA-related neutralisation, and instead considering ceiling effects on Treg proliferation, Treg exhaustion and intrinsic Treg dysfunction in MS as possible contributors. Together, our findings demonstrate that low-dose IL-2 alone is not an effective strategy for promoting Treg expansion post-alemtuzumab, and is therefore not a viable approach by itself for preventing post-treatment autoimmune complications.

Background Type 1 diabetes is an autoimmune disease with significant long-term complications. Variability in the decline of insulin secretion after diagnosis complicates both the development of treatments and disease management. We previously reported that gene expression changes within the first year post-diagnosis were associated with C-peptide decline at two years in the first INNODIA cohort of patients with newly diagnosed type 1 diabetes. Here, we aimed to validate these findings in an independent follow-up cohort and to increase statistical power by combining the data from both cohorts. Methods We analysed transcriptomic data from a follow-up INNODIA cohort of 168 individuals with newly diagnosed type 1 diabetes to assess whether previously identified associations with disease progression could be replicated. We then combined data from the original and follow-up cohorts for integrated analysis. Longitudinal gene expression changes during the first year after diagnosis were examined in relation to disease progression, alongside age and estimated immune cell abundances. Findings Analysis of the follow-up cohort validated the previously observed longitudinal changes in gene expression during the first year after diagnosis. In the combined dataset, transcriptomic analysis identified a large number of genes that were differentially expressed during the first year after disease onset. More rapid disease progression was associated with younger age and a relative decrease in neutrophil abundance. In addition, changes in the expression of several genes were associated with the rate of disease progression. Interpretation These findings support the existence of biological heterogeneity in disease progression after diagnosis of type 1 diabetes and contribute to an improved understanding of the molecular dynamics associated with disease progression. These findings may help future studies aiming to enable patient stratification and design of more targeted and personalised therapeutic approaches in type 1 diabetes. Funding This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115797 (INNODIA) and No 945268 (INNODIA HARVEST).

Low-dose interleukin-2 (IL-2) shows potential for treating autoimmune disorders by expanding regulatory T cells (Treg), but therapeutic utility is limited by short half-life and off-target effects. Here, we describe the molecular design, nonclinical development, and first-in-human study of MHS552, an IL-2 mutein-engrafted antibody designed to selectively expand Treg via high-affinity IL-2 receptors. In vitro, MHS552 selectively induces IL-2 signaling in Treg from healthy donors and autoimmune patients. In cynomolgus monkeys, MHS552 produces dose-dependent Treg expansion, with modest increases in conventional T cells (Tconv) at higher doses. In a randomized, double-blind, placebo-controlled, single-ascending-dose trial in 60 healthy participants (EudraCT 2018-004233-33), evaluating safety and tolerability as the primary objective and pharmacokinetics as the secondary objective, intravenous and subcutaneous administration of MHS552 is well tolerated at lower doses and shows a predictable pharmacokinetic profile. However, a serious adverse event (SAE) is reported at the highest subcutaneous dose. Both administration routes result in dose-dependent Treg expansion, with up to 6-fold increase in total Treg and 60-fold increase in CD25hi subset, without significant conventional T cell activation. Exploratory analyses confirm selective Treg activation, with expanded Treg retaining their suppressive function ex vivo. These findings support IL-2-based therapeutics for autoimmune disorders, while the SAE highlights the need for careful safety evaluation. Although low-dose IL-2 therapy has shown promise in treating autoimmune and inflammatory conditions by supporting the expansion and immunosuppressive activity of regulatory T cells (Treg), robust clinical responses have been lacking. Here, the authors describe the design of the Treg-selective IL-2 agonist MHS552, its preclinical characterization in non-human primates, and a first-in-human dose escalation study in healthy participants reporting efficacy, selectivity and safety of the IL-2 mutein.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results in loss of insulin-secreting pancreatic β-cells in the islets of Langerhans. A diagnosis of T1D is typically associated with children and adolescents, yet half of all diagnoses of T1D are made in adults. In children and adolescents, T1D is often first recognized following hospitalization for diabetic ketoacidosis (DKA), which occurs in approximately 20%-50% of new-onset T1D for people younger than 18 years of age in Europe. For adults with new-onset T1D, DKA rates of up to 24% are estimated. Early-stage T1D, during the asymptomatic period, can be detected through screening for multiple islet autoantibodies in blood samples, including capillary and venous samples, and such programs are made more popular by the availability of disease-modifying therapies for early-stage T1D. For individuals who screen positive for early-stage T1D, participation in monitoring programs can greatly reduce the incidence of DKA once symptomatic hyperglycemia develops, as well as reducing severity of symptoms of T1D at onset. Education and awareness of the clinically relevant features of symptomatic T1D can also support the psychological wellbeing of people with early-stage T1D and minimize distress at the point when insulin treatment is necessary. All of these consequences come with a predicted reduced burden of healthcare costs for managing T1D at a population level, and general population screening for islet autoantibodies is underway. In this European perspective, we discuss the imperatives and the components of implementation of general population screening for early-stage T1D.

Abatacept is a CTLA4-Ig fusion protein that blocks CD80/CD86-dependent T-cell co-stimulation. When administered, Abatacept limits, to a variable degree, loss of stimulated C-peptide secretion in patients with newly-diagnosed type 1 diabetes (T1D), while reducing both circulating memory CD4+ T-cells and T follicular helper (Tfh) cells; however, its precise mechanism of action is not known. To investigate this effect, we studied 12 patients, using multi-parameter flow cytometry, who each self-administered Abatacept in subcutaneous formulation for 6 months within 100 days of diagnosis. Abatacept treatment impacted the CD4+ T cell memory compartment, inducing a reduction in T-effector cells across both conventional (Tconv) and regulatory (Treg) sub-populations. A reduction in activated Tfh cells (CXCR5+PD1+ICOS+), previously described with intravenous therapy, was replicated and extended. An integrated baseline immunological phenotype predicted Abatacept-induced preservation of C-peptide.

Neoantigen vaccines are under investigation for various cancers, including epidermal growth factor receptor (EGFR)-driven lung cancers1,2. We tracked the phylogenetic history of an EGFR mutant lung cancer treated with erlotinib, osimertinib, radiotherapy and a personalized neopeptide vaccine (NPV) targeting ten somatic mutations, including EGFR exon 19 deletion (ex19del). The ex19del mutation was clonal, but is likely to have appeared after a whole-genome doubling (WGD) event. Following osimertinib and NPV treatment, loss of the ex19del mutation was identified in a progressing small-cell-transformed liver metastasis. Circulating tumour DNA analyses tracking 467 somatic variants revealed the presence of this EGFR wild-type clone before vaccination and its expansion during osimertinib/NPV therapy. Despite systemic T cell reactivity to the vaccine-targeted ex19del neoantigen, the NPV failed to halt disease progression. The liver metastasis lost vaccine-targeted neoantigens through chromosomal instability and exhibited a hostile microenvironment, characterized by limited immune infiltration, low CXCL9 and elevated M2 macrophage levels. Neoantigens arising post-WGD were more likely to be absent in the progressing liver metastasis than those occurring pre-WGD, suggesting that prioritizing pre-WGD neoantigens may improve vaccine design. Data from the TRACERx 421 cohort3 provide evidence that pre-WGD mutations better represent clonal variants, and owing to their presence at multiple copy numbers, are less likely to be lost in metastatic transition. These data highlight the power of phylogenetic disease tracking and functional T cell profiling to understand mechanisms of immune escape during combination therapies.

BACKGROUND:Type 1 diabetes remains an important health-care problem, with no disease-modifying therapies available in people with recent-onset, clinical type 1 diabetes. Adaptive trial designs, allowing faster evaluation of treatment modalities, remain underexplored in this stage of the disease. We aimed to identify the minimum effective dose of antithymocyte globulin (ATG) in people aged 5-25 years with recent-onset, clinical type 1 diabetes. METHODS:MELD-ATG was a phase 2, double-blind, randomised, placebo-controlled, multi-arm, adaptive dose-ranging, parallel-cohort trial done in 14 accredited trial centres in eight countries (the UK, Denmark, Germany, Finland, Italy, Belgium, Austria, and Slovenia). Participants aged 5-25 years, diagnosed with clinical, stage 3 type 1 diabetes 3-9 weeks before treatment, with random C-peptide concentrations 0·2 nmol/L or more and at least one diabetes-related autoantibody (GADA, IA-2A, or ZnT8) were randomly assigned by a web-based randomisation system into seven consecutive cohorts receiving placebo, 2·5 mg/kg ATG, 1·5 mg/kg ATG, 0·5 mg/kg ATG, or 0·1 mg/kg ATG. Participants in cohort 1 were randomly assigned 1:1:1:1:1, participants in cohorts 2 and 3 were randomly assigned 1:1:1:1, and participants in cohorts 4-7 were randomly assigned 1:1:1. All cohorts included one placebo group and one 2·5 mg/kg ATG group. The other groups were assigned to ATG doses that were determined based on accruing data and the decision of the dose determining committee. The trial cohorts were stratified by age group (5-9 years, 10-17 years, and 18-25 years) with block sizes varying by cohort. Concealment lists, outlining the treatment allocation, were only available for the pharmacists; participants and study teams were masked to treatment allocation. ATG was administered by an intravenous infusion over 2 consecutive days. The primary outcome was the area under the curve (AUC) of the stimulated C-peptide concentration during a 2-h mixed-meal tolerance test at 12 months measured as ln(AUC C-peptide + 1). Conditional on finding a statistically significant difference at p<0·05 for 2·5 mg/kg ATG versus placebo, the minimum effective dose of ATG was determined. All randomly assigned participants were included in the primary analysis. All participants who received the study drug were included in the safety analysis. The trial was registered at ClinicalTrials.gov (NCT04509791) and is completed. FINDINGS:Between Nov 24, 2020, and Dec 13, 2023, 152 people were recruited and screened, 117 of whom were randomly assigned (placebo n=31, 0·1 mg/kg ATG n=6, 0·5 mg/kg ATG n=35, 1·5 mg/kg ATG n=12, and 2·5 mg/kg n=33). 54 (46%) of 117 participants were male and 63 (54%) were female. Participants were mainly European. The 0·1 mg/kg dose and the 1·5 mg/kg dose were progressively dropped from the study. At 12 months, the mean ln(AUC C-peptide + 1) was 0·411 nmol/L per min (SD 0·032) in the placebo group and 0·535 nmol/L per min (0·032) in the 2·5 mg/kg ATG group. The mean difference in the ln(AUC C-peptide + 1) between 2·5 mg/kg ATG and placebo was 0·124 nmol/L per min (95% CI 0·043-0·205; p=0·0028). At 12 months, the mean ln(AUC C-peptide + 1) in the 0·5 mg/kg ATG group, the remaining middle dose, was 0·513 nmol/L per min (SD 0·032), with a mean baseline-adjusted difference from placebo of 0·102 nmol/L per min (95% CI 0·021-0·183; p=0·014). Cytokine release syndrome occurred in 11 (33%) of 33 participants in the 2·5 mg/kg ATG group, eight (24%) of 34 in the 0·5mg/kg ATG group, and no participants in the placebo group. Serum sickness occurred in 27 (82%) participants in the 2·5 mg/kg ATG group, 11 (32%) in the 0·5 mg/kg ATG group, and no participants in the placebo group. There were no deaths related to adverse events. INTERPRETATION:In young people with recent-onset, clinical type 1 diabetes, 2·5 mg/kg and 0·5 mg/kg ATG reduced loss of β-cell function, showing the potential of an affordable, repurposed agent, ATG, in a low and safe dose, as a disease-modifying agent in this population. FUNDING:The European Union's Innovative Medicines Initiative 2 Joint Undertaking INNODIA.

Background Type 1 diabetes is an autoimmune disease affecting over 400,000 children and adults in the United Kingdom for which currently the only available therapy is insulin. Objective(s) To determine the efficacy and safety of the monoclonal antibody ustekinumab targeting the interleukin 12/interleukin 23 immune pathway that generates T helper 1/T helper 17 T cells to slow down the autoimmune process and preserve beta cell production in type 1 diabetes. Design Randomised, double-blind, placebo-controlled, parallel-group phase II trial. Setting Paediatric and young adult diabetes clinics across 16 sites in the United Kingdom. Participants Newly diagnosed with type 1 diabetes and aged 12–18 years. Eligibility criteria Type 1 diabetes confirmed by islet autoantibody testing, within 100 days of first insulin injection, and with residual beta cell function (stimulated C-peptide level > 0.2 nmol/l). Interventions Ustekinumab at the highest approved doses or control (saline) subcutaneously at weeks 0, 4 and 12 and subsequently every 8 weeks to week 44 (seven doses). Main outcome measures Preservation of Mixed Meal Tolerance Test stimulated 2-hour insulin C-peptide area under the curve at week 52 as compared to control (saline) treatment by analysis of covariance adjusted for baseline parameters. Randomisation 2 : 1 Remote computerised randomisation with minimisation by age and baseline C-peptide groups. Blinding Blinding of participants, investigators, laboratory and trial staff. Numbers randomised Seventy-two participants were randomised, 60% male, 18% aged 16–18 years. Recruitment Two hundred and eight potentially eligible patients were approached, and 88 patients were screened. Four participants were lost to follow-up (6%). Four participants withdrew from the treatment but attended the primary end-point assessment. Numbers analysed Six participants were missing baseline data for the primary analysis. The final analysable sample was n = 62. Outcome Ustekinumab was associated with a 49% higher endogenous stimulated insulin production than control at week 52 after adjustments for baseline factors [geometric ratio of ustekinumab to control was 1.49 (95% confidence interval 1.08 to 2.06; p = 0.02)]. Secondary analyses showed no difference in C-peptide at week 28 suggesting that the effect was ‘late’ or ‘delayed’. Ancillary analysis showed a significant reduction in activated T helper 17.1 T cells ( p < 0.001) in the treatment group which was associated with C-peptide preservation from week 28 to week 52. Harms No severe adverse events were reported and there were no differences between ustekinumab and control groups in the proportion of participants overall experiencing mild (87% vs. 88%) or moderate (32% vs. 32%) events. Limitations Sensitivity analysis showed the primary end point to be robust to exclusion of small numbers of participants with some protocol deviations and extreme values in key covariates, but not to imputation of all missing data. Conclusions Ustekinumab appears to slow down the autoimmune process providing the first clinical trial evidence that interleukin 17-secreting T cells play a pathogenic role in type 1 diabetes. Alone, it is insufficient to halt the autoimmune process. Future work Replication of this result is ongoing in a trial with a similar design in Canada. If confirmed, consideration may be given to testing other drugs targeting the interleukin 17 pathway, using ustekinumab in combination with other agents or using it earlier in the disease pathway (preclinical disease) since it is so well tolerated and simple to use. Study registration Current Controlled Trials ISRCTN14274380. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 16/36/01) and is published in full in Efficacy and Mechanism Evaluation ; Vol. 12, No. 1. See the NIHR Funding and Awards website for further award information.

BACKGROUND:Amyotrophic lateral sclerosis (ALS) is a life-threatening disease characterised by progressive loss of motor neurons with few therapeutic options. The MIROCALS study tested the hypothesis that low-dose interleukin-2 (IL-2LD) improves survival and function in ALS. METHODS:In this randomised, double-blind, placebo-controlled trial, male and female riluzole-naive participants, with either a possible, laboratory-supported probable, probable, or definite ALS diagnosis (revised El Escorial criteria), aged 18-76 years, with symptom duration of 24 months or fewer, and slow vital capacity of 70% or more, underwent a riluzole-only 12-18 week run-in period before randomisation in a 1:1 ratio to either 2 million international units (MIU) IL-2LD or placebo by subcutaneous injection daily for 5 days every 28 days over 18 months. The primary endpoint was survival at 640 days (21 months). Secondary outcomes included safety, ALS Functional Rating Scale-Revised (ALSFRS-R) score, and biomarker measurements including regulatory T-cells (Tregs), cerebrospinal fluid (CSF)-phosphorylated-neurofilament heavy-chain (CSF-pNFH), and plasma and CSF-chemokine ligand 2 (CCL2). The primary endpoint analysis used unadjusted log-rank and Cox's model adjusted analyses using pre-defined prognostic covariates to control for the disease and treatment response heterogeneity. The study was 80% powered to detect a two-fold decrease in the risk of death by the log-rank test in the intention-to-treat (ITT) population, including all randomly allocated participants. MIROCALS is registered with ClinicalTrials.gov (NCT03039673) and is complete. FINDINGS:From June 19, 2017, to Oct 16, 2019, 304 participants were screened, of whom 220 (72%) met all criteria for random allocation after the 12-to-18-week run-in period on riluzole. 136 (62%) of participants were male and 84 participants (38%) were female. 25 (11%) of the 220 randomly allocated participants were defined as having possible ALS under El Escorial criteria. At the cutoff date there was no loss to follow-up, and all 220 patients who were randomly allocated were documented as either deceased (90 [41%]) or alive (130 [59%]), so all participants were included in the ITT and safety populations. The primary endpoint unadjusted analysis showed a non-significant 19% decrease in risk of death with IL-2LD (hazard ratio 0·81 [95% CI 0·54-1·22], p=0·33), failing to demonstrate the expected two-fold decrease in risk of death. The analysis of the primary endpoint adjusted on prognostic covariates, all measured at time of random allocation, showed a significant decrease of the risk of death with IL-2LD (0·32 [0·14-0·73], p=0·007), with a significant treatment by CSF-pNFH interaction (1·0003 [1·0001-1·0005], p=0·001). IL-2LD was safe, and significantly increased Tregs and decreased plasma-CCL2 at all timepoints. Stratification on CSF-pNFH levels measured at random allocation showed that IL-2LD was associated with a significant 48% decrease in risk of death (0·52 [0·30-0·89], p=0·016) in the 70% of the population with low (750-3700 pg/mL) CSF-pNFH levels, while in the 21% with high levels (>3700 pg/mL), there was no significant difference (1·37 [0·68-2·75], p=0·38). INTERPRETATION:With this treatment schedule, IL-2LD resulted in a non-significant reduction in mortality in the primary unadjusted analysis. However, the difference between the results of unadjusted and adjusted analyses of the primary endpoint emphasises the importance of controlling for disease heterogeneity in ALS randomised controlled trials. The decrease in risk of death achieved by IL-2LD therapy in the trial population with low CSF-pNFH levels requires further investigation of the potential benefit of this therapy in ALS. FUNDING:European Commission H2020 Programme; French Health Ministry PHRC2014; and Motor Neurone Disease Association.

SARS-CoV-2 immunity and innate immune training may influence influenza vaccine immunogenicity. We investigated this in India. Adult volunteers with hybrid SARS-CoV-2 immunity were administered FluarixTM Tetra (GlaxoSmithKlein) 2022/2023 NH Vaccine in 2022. Significant induction of hemagglutinin inhibition-specific antibodies and polyfunctional central memory CD4+ T-cells (TCM) were observed 1-week post-vaccination with variable induction of CD8+T-cell and innate effectors. Vaccination also expanded Flu-specific regulatory T-cells (Treg), which negatively correlated with CD4 responses, highlighting vaccine immunogenicity may be subject to Treg dampening. FluarixTM did not boost SARS-CoV-2 immunity. However, SARS-CoV-2-specific T-cell responses correlated positively with vaccine-induced T-cell responses. We evaluated trained immunity post-COVID-19 as a potential regulatory mechanism linking SARS-CoV-2 and heterologous vaccine immunogenicity. We observed, elevated frequencies of basal bacterial Lipopolysaccharide (LPS)-induced IL-6+IL1β+HLA-DR+CD14+CD16- frequencies post-COVID-19 correlated positively with vaccine-induced Fluarix-specific CD4 T-cell frequencies. Our study highlights a potential positive role for COVID-19-driven immune imprinting on heterologous vaccine immunogenicity in a post-COVID-19 era.

Viral infections in the first year of life are associated with islet autoimmunity and type 1 diabetes risk. The Anti-Viral Action against Type 1 Diabetes Autoimmunity (AVAnT1A)- study is a clinical phase IV investigator initiated, randomised, controlled, multicentre, primary prevention trial conducted to determine whether vaccination against COVID-19 from 6 months of age reduces the cumulative incidence of islet autoantibodies or type 1 diabetes in children with elevated genetic risk. Additionally, it investigates the role of viral infections in the etiology of islet autoimmunity by intense surveillance within the first two years of life. Infants aged 3.00-4.00 months from Germany, Belgium, UK and Sweden are eligible if they have a >10 % expected risk to develop islet autoantibodies by age 6 years as determined by HLA DR/DQ genotype, polygenic risk score and family history of type 1 diabetes. A total of 2252 eligible children are randomized 1:1 to COVID-19 vaccine (Comirnaty (R) 3 mu g Omicron XBB.1.5 or future new variants) or placebo (0.9% Sodium Chloride) administered three times. Children are followed until the minimum age of 2.5 years and maximum age of 6 years. The intervention is accompanied by analyses of immune and metabolic parameters to determine changes induced by viral infections and to investigate mechanisms by which viral infection may lead to islet autoimmunity. The Sponsor is the Klinikum rechts der Isar, Technical University Munich. The study was approved by Clinical Trials Information System (CTIS, EU Trial number: 2023-507348-35-00) and by Integrated Research Application System (IRAS, IRAS-ID: 1009668).

Abstract Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with hyperglycaemia, chronic inflammation and adverse health outcomes. Regulatory T cells (Tregs) are thought to contribute to GDM due to their role in suppressing inflammation. However, it remains unclear whether specific Treg subsets are impaired in patients with GDM. To investigate transcriptional variation in GDM Tregs, we applied single-cell RNA sequencing to Tregs isolated from the blood of 13 healthy pregnant women and 10 patients with GDM. We identified naive and effector Treg subsets, none of which significantly differ in the proportion of cells captured from GDM and controls. We report a naive Treg subset with reduced expression of AP-1 transcription factor subunits in GDM, including JUN, FOS, and EGR1, and an effector Treg subset with increased signalling of angiogenesis marker genes. Genes dysregulated in GDM Tregs independently predicted GDM status in pseudobulk and whole blood mRNA from independent cohorts. Remarkably, TXNIP, which regulates glucose levels, emerged as the most reliable standalone predictor in bulk mRNA (minimum AUC 0.7) equivalent to using body mass index (AUC 0.72) in our cohort. This study uncovers a disrupted molecular pathway in Treg cell subsets from GDM patients and proposes a panel of genes with translational potential as early disease biomarkers.