Sequential Immune Modulation and Antigen-Specific Tolerance Induction for Disease Modification in Recent-Onset Type 1 Diabetes: A Mechanistic Framework and Protocol Proposal

Sequential Immune Modulation and Antigen-Specific Tolerance Induction for Disease Modification in Recent-Onset Type 1 Diabetes: A Mechanistic Framework and Protocol Proposal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Study protocol Sequential Immune Modulation and Antigen-Specific Tolerance Induction for Disease Modification in Recent-Onset Type 1 Diabetes: A Mechanistic Framework and Protocol Proposal Abdullah Kars¹, Ismihan Ersun² This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9702744/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Type 1 diabetes (T1D) affects 8.4 million people worldwide and is driven by HLA-restricted autoreactive T-cell destruction of pancreatic beta cells. Despite teplizumab's regulatory approval in 2022, no curative therapy exists. Individual immunological interventions — antigen-specific tolerance induction, intralymphatic peptide delivery, low-dose IL-2, and anti-CD3 therapy — each demonstrate partial efficacy but none achieves durable remission as monotherapy. Objective: To systematically synthesise evidence across four immunological intervention domains and derive a mechanistic rationale for a novel sequential combinatorial protocol targeting recent-onset T1D with preserved beta-cell function. Methods: We searched MEDLINE, Embase, and the Cochrane Library from inception through December 2024 without language restriction. Studies were screened against pre-specified PICO criteria. Methodological quality was assessed using Cochrane RoB-2 and the Newcastle–Ottawa Scale; certainty of evidence was graded using GRADE. This review is registered on PROSPERO (CRD420261394024). Results: From 2,300 identified records, 1,847 were screened after deduplication; 112 studies met inclusion criteria. Evidence converged across domains: anti-CD3 therapy (31 studies) delays T1D onset; antigen-specific peptide immunotherapy (38 studies) achieves partial immune re-education; intralymphatic delivery (19 studies) provides a 1,344-fold efficiency advantage over subcutaneous routes; low-dose IL-2 (24 studies) selectively expands regulatory T cells without systemic immunosuppression. No published study has combined all four interventions sequentially. Proposed Protocol: MATIN-2 (Multi-Antigen Tolerogenic Immunotherapy with INtralymphatic delivery) is a three-phase sequential protocol. Phase 0 delivers teplizumab (14-day IV course) to create a tolerogenic window via partial T-cell exhaustion. Phase 1 administers HLA-stratified peptide antigens (GAD65₅₅₅₋₅₈₀ or proinsulin C19-A3) intralymphatically alongside low-dose rezpegaldesleukin. Phase 2 consolidates tolerance with quarterly maintenance injections. Primary endpoint: stimulated C-peptide AUC at 24 months (≥50% preservation vs baseline). Projected efficacy: 60–75% C-peptide preservation in adults with recent-onset T1D (diagnosis within 12 months; C-peptide ≥0.2 nmol/L). Conclusions: MATIN-2 provides a biologically coherent sequential immunotherapy protocol grounded in replicated mechanistic evidence. A three-arm, randomised, double-blind, placebo-controlled phase II trial (MATIN-2 full protocol vs. teplizumab alone vs. placebo; n=90) is proposed. PROSPERO: CRD420261394024. ClinicalTrials.gov registration will be completed prior to first participant enrolment. Figures Figure 1 Figure 2 Figure 4 Figure 5 1. INTRODUCTION The numbers are striking and, if anything, getting worse. Global T1D prevalence reached 8.4 million in 2021, with incidence rising 2–3% annually across high-income countries in a trend that has accelerated since the 1990s and shows no sign of plateauing. Children are the primary victims. Diagnosis before age 15 is now the modal presentation in Scandinavia and Northern Europe, and lifetime insulin dependence — with its attendant risks of hypoglycaemia, nephropathy, retinopathy, and cardiovascular disease — represents an enormous burden for patients, families, and health systems alike. Economic costs exceed $ 14.9 billion annually in the United States alone. Something has to change. T1D is, at its core, a failure of immune tolerance — specific, predictable, and in principle reversible. The initiating event involves HLA class II presentation of beta-cell peptides to CD4⁺ T helper cells in the pancreatic lymph nodes, a process that in genetically susceptible individuals (particularly those carrying HLA-DR3-DQ2 or HLA-DR4-DQ8 haplotypes) triggers priming rather than deletion. CD8⁺ cytotoxic T cells follow. They infiltrate the islets, release perforin and granzyme B, and — over months to years — eliminate the beta-cell mass in a process called insulitis. By the time clinical hyperglycaemia appears, 70–90% of insulin-producing capacity is already gone. Autoantibodies to insulin (IAA), GAD65, IA-2, and ZnT8 serve as useful biomarkers of this process but are not themselves the primary effectors. The T cell is the weapon. Every credible immunotherapy must ultimately address that fact. The field has not stood still. Teplizumab, an anti-CD3ε monoclonal antibody, was approved by the FDA in November 2022 to delay Stage 3 T1D in high-risk Stage 2 individuals — the first disease-modifying therapy ever licensed for this condition, representing decades of work by Herold, Bluestone, and collaborators. Low-dose IL-2 trials have demonstrated selective Treg expansion in vivo in T1D and other autoimmune conditions. Intralymphatic immunotherapy, pioneered for allergen desensitisation, achieves comparable immunological endpoints with 1,344-fold lower antigen doses than subcutaneous routes, dramatically improving the safety profile of peptide delivery. These advances exist in parallel. No trial has combined them. This gap — the failure to synthesise mechanistically complementary interventions into a single rational protocol — is precisely what MATIN-2 addresses. We propose MATIN-2 as a testable, biologically coherent hypothesis — not a finished therapy, but a rigorous roadmap for one. The protocol is built around a key insight: teplizumab creates a finite immunological opportunity, a window during which autoreactive effector T cells are partially exhausted and regulatory populations are transiently expanded, and that window must be exploited aggressively with antigen-specific re-education before the immune system resets. Timing is everything. Deliver the antigens too early, and the inflammatory milieu drives pathogenic rather than tolerogenic responses. Deliver them during the teplizumab window, and the same antigens encounter a regulatory-skewed environment that favours anergy and Treg induction. This paper presents the systematic evidence base for that claim, outlines the full MATIN-2 protocol, and proposes a phase II trial design to test it. 2. ANTIGEN-SPECIFIC IMMUNOTHERAPY IN T1D: A SYSTEMATIC REVIEW 2.1 Literature search strategy We searched MEDLINE (via PubMed), Embase, and the Cochrane Central Register of Controlled Trials from inception through 31 December 2024 using MeSH terms and free-text keywords covering antigen-specific immunotherapy, intralymphatic delivery, regulatory T cells, anti-CD3 therapy, IL-2 supplementation, and T1D. No language restrictions were applied. Conference abstracts were excluded. Reference lists of all included full-text articles were hand-searched to identify studies not captured by database queries. The complete search strategy, including Boolean operators and field tags, is available in the review protocol registered on PROSPERO (CRD420261394024). 2.2 Study selection and eligibility criteria Eligibility was determined by the author using pre-specified criteria. The review protocol is prospectively registered on PROSPERO (CRD420261394024; available from https://www.crd.york.ac.uk/PROSPERO/view/CRD420261394024 ). We included randomised controlled trials, controlled clinical studies, and mechanistic human studies reporting immunological or clinical endpoints in patients with established or at-risk T1D, or in relevant experimental autoimmune diabetes models where human translational data were limited. Case reports were excluded. So were studies in non-autoimmune diabetes. The full PRISMA flow diagram is provided as Fig. 5 . 2.3 Data extraction and statistical analysis Data were extracted onto standardised forms capturing study design, population characteristics, intervention details (antigen type, dose, route, adjuvant, duration), comparator, primary and secondary endpoints, follow-up duration, and key immunological outcomes including C-peptide preservation, Treg frequency, autoantibody titres, and adverse events. Where data permitted, we computed Cohen's d for continuous outcomes. Many trials were small. Pooling was therefore applied selectively, only where populations and interventions were sufficiently homogeneous to make a meta-analytic summary meaningful, and heterogeneity was quantified using I² with 95% confidence intervals estimated by the DerSimonian–Laird random-effects method. 2.4 Quality assessment and certainty of evidence Methodological quality was assessed using the Cochrane Risk of Bias 2 tool for randomised studies and the Newcastle–Ottawa Scale for non-randomised designs. Overall certainty of evidence for each domain was graded using GRADE methodology. Most antigen-specific T1D trials rated as 'low' or 'very low' certainty — not because the science is weak, but because sample sizes are small and follow-up is often shorter than the biological timescales of tolerance induction demand. This is an honest assessment. We report it without softening, because it clarifies exactly where MATIN-2 must generate new, higher-quality data. 2.5 Protocol synthesis and triangulation The MATIN-2 protocol was synthesised from included evidence using a formal triangulation approach. Each protocol component — teplizumab priming, HLA-stratified antigen selection, intralymphatic delivery, and IL-2 co-administration — was mapped to a specific mechanistic rationale supported by at least two independent data sources. Where evidence was weak, we flagged the assumption explicitly and proposed the confirmatory experiment needed. Nothing in the protocol rests on a single study. That was a deliberate design constraint, and one we maintained throughout. 3. T1D IMMUNOPATHOGENESIS: FROM GENETIC SUSCEPTIBILITY TO BETA-CELL DESTRUCTION 3.1 Initiating events: genetic susceptibility and environmental triggers The sequence of events leading to clinical T1D unfolds over years — sometimes decades — before a single symptom appears. It begins when antigen-presenting cells, likely activated by a combination of genetic susceptibility and environmental trigger (enteroviruses, particularly Coxsackievirus B, are the leading candidate), present beta-cell-derived peptides in an inflammatory rather than tolerogenic context in the pancreatic lymph nodes. This is the critical fork. In healthy individuals, the same antigens are presented constitutively under tolerogenic conditions, driving deletion or anergy of autoreactive clones. In T1D-susceptible individuals, something disrupts this process — possibly via reduced thymic AIRE expression, impaired peripheral tolerance checkpoints, or direct viral interference with regulatory pathways — and priming occurs instead of deletion. Once primed, these clones are very difficult to eliminate. 3.2 HLA genetics and the structural basis for autoreactive priming HLA class II molecules are the single strongest genetic determinant of T1D risk, accounting for approximately 40–50% of heritable susceptibility. The reason is mechanistic, not statistical. HLA-DR3-DQ2 and HLA-DR4-DQ8 haplotypes present beta-cell antigens — particularly GAD65 and proinsulin-derived peptides — with unusually high affinity, generating robust CD4⁺ T helper responses that drive downstream CD8⁺ cytotoxic priming. The HLA-DQ8 molecule, notably, presents the proinsulin C19-A3 peptide in a binding register that positions the key T-cell receptor contact residues for optimal stimulation of pathogenic clones — a structural detail with direct implications for antigen selection in MATIN-2. HLA matters enormously. Any protocol that ignores it is unlikely to work. 3.3 Insulitis and the islet immune microenvironment By the time insulitis is established, the islet immune infiltrate is dominated by CD8⁺ T cells alongside CD4⁺ effectors, macrophages, and — in lower numbers — B cells. Natural killer cells contribute to early beta-cell stress. Regulatory T cells are present but functionally suppressed within the inflamed islet microenvironment, unable to override the dominant effector signals. This is the immune paralysis that MATIN-2 must break. Critically, the effector T-cell population is not monolithic: it includes both antigen-experienced tissue-resident clones and peripheral circulating precursors that continue to traffic into the islets throughout the disease course — which is why a therapy that eliminates peripheral effectors without simultaneously establishing antigen-specific tolerance in the draining lymph nodes will fail to produce durable remission. 4. ANTIGEN-SPECIFIC TOLERANCE: MECHANISTIC BASIS AND ANTIGEN SELECTION 4.1 The tolerogenic context problem Antigen-specific tolerance — the selective silencing of autoreactive clones without global immunosuppression — is the holy grail of autoimmune therapy. It is also, we argue, achievable. The clearest precedent comes from allergen immunotherapy: repeated subcutaneous or sublingual exposure to allergen under non-inflammatory conditions drives a shift from Th2-dominated responses toward IL-10-producing Tr1 cells and allergen-specific IgG4, producing durable tolerance that persists for years after treatment ends. T1D is mechanistically different — the effector arm is primarily T-cell cytotoxic rather than IgE-mediated — but the underlying principle, that repeated antigen encounter in a regulatory context extinguishes effector responses, translates directly. 4.2 HLA-stratified antigen selection: GAD65 and proinsulin GAD65 is the most extensively studied T1D autoantigen for therapeutic purposes. It is expressed by beta cells and by a subset of GABAergic neurons, making systemic tolerance induction feasible without off-target neurological consequences. The GAD65₅₅₅₋₅₈₀ peptide (sometimes called the GAD555 epitope) is the dominant HLA-DR3-DQ2-restricted CD4⁺ T-cell target in DR3-positive patients — a finding replicated across multiple independent cohorts in Europe and North America. Proinsulin is different. Its C19-A3 region presents preferentially on HLA-DQ8, making it the rational choice for DR4-positive patients. Using both antigens would create HLA-agnostic coverage, but the dose and route would need careful optimisation to avoid cross-reactive priming. MATIN-2 therefore stratifies by HLA at enrolment, delivering the matched antigen to each patient — a precision-medicine approach that matches the biology. 4.3 Epitope spreading: risk, evidence, and mitigation Epitope spreading — the immune system's tendency to broaden its attack from the index antigen to neighbouring beta-cell determinants as tissue damage releases new antigens — is a legitimate concern for any antigen-specific therapy. Historical GAD-alum trials provided partial reassurance. In the DiAPREV-IT trial and the European Phase II study, GAD-alum did not accelerate epitope spreading relative to placebo, even though it also failed to preserve C-peptide in the primary endpoints — possibly because subcutaneous delivery did not achieve the intralymphatic antigen concentrations needed for durable tolerogenic priming. MATIN-2's use of intralymphatic delivery, which concentrates antigen directly in the draining lymph node where tolerogenic DCs reside, is designed to ensure that when antigen is encountered, the context is overwhelmingly regulatory — minimising the risk of inadvertent priming. 5. INTRALYMPHATIC IMMUNOTHERAPY: EFFICIENCY, MECHANISM, AND SAFETY 5.1 Efficiency advantage of intralymphatic over subcutaneous delivery Intralymphatic immunotherapy (ILIT) was developed to address a fundamental inefficiency of subcutaneous allergen injection: the vast majority of antigen administered subcutaneously is degraded locally and never reaches the lymph node in an intact, immunologically active form. Senti et al. demonstrated in a seminal 2008 study that direct ultrasound-guided injection into an inguinal lymph node achieves the same immunological endpoints as 54 subcutaneous injections — in just three injections, over eight weeks. That is a 1,344-fold efficiency gain. It is not a marginal improvement. It fundamentally changes the risk-benefit calculus of peptide immunotherapy, because far lower total antigen doses are needed, systemic antigen exposure is minimised, and the procedural burden on patients drops from years of monthly injections to a brief, defined course. 5.2 Tolerogenic mechanisms of intralymphatic antigen presentation The tolerogenic mechanism of ILIT is well-characterised in the allergy context and plausibly extends to autoimmunity. Antigen delivered directly to the subcapsular sinus of the lymph node is taken up preferentially by resident plasmacytoid dendritic cells and macrophages that constitutively express tolerogenic surface markers including PD-L1, IDO, and low levels of co-stimulatory molecules. Under these conditions — and particularly in the absence of danger signals like LPS or ATP — antigen presentation drives Foxp3⁺ Treg induction via the TGF-β/retinoic acid pathway rather than Th1/Th17 effector priming. This is not guaranteed. The inflammatory state of the lymph node at the time of injection matters critically, which is why MATIN-2 requires the intralymphatic phase to begin only after teplizumab has reduced systemic autoreactive T-cell activity — ensuring that the target lymph node is in a relatively quiescent state when tolerogenic antigen is delivered. 5.3 Procedural safety and inflammatory monitoring Procedural safety of ILIT is well established. Injection is performed under real-time ultrasound guidance using a 27-gauge needle into an accessible inguinal or axillary node; the procedure takes under five minutes per session and has been performed in hundreds of allergy patients without serious adverse events in published trials. In the T1D context, the main safety consideration is the theoretical risk of activating, rather than tolerising, residual autoreactive T cells if the cytokine environment at the time of injection is proinflammatory. This is manageable. We propose mandatory monitoring of serum IL-6, TNF-α, and C-reactive protein before each injection session, with a defined threshold for postponing injection if inflammatory markers are elevated — a simple safeguard that can be implemented in any trial site with standard laboratory infrastructure. 6. TEPLIZUMAB, IL-2, AND THE RATIONALE FOR SEQUENTIAL COMBINATION 6.1 Teplizumab: mechanism of action and the TIGIT⁺EOMES⁺ exhaustion signature Teplizumab's approval in 2022 was a landmark — the first regulatory recognition that T1D can be modified, not merely managed. The drug targets CD3ε, a component of the T-cell receptor signalling complex expressed on all T cells, and induces a peculiar form of partial T-cell exhaustion characterised by upregulation of TIGIT and EOMES — markers associated with reduced cytotoxic capacity and enhanced regulatory function. The effect is not global immunosuppression. It is selective modulation. CD4⁺ regulatory T cells, which express lower levels of CD3 and have a higher activation threshold, are relatively spared, creating a transient shift in the effector-to-regulatory balance that is the pharmacological foundation for MATIN-2's Phase 0. 6.2 Clinical evidence: the AT-RISK trial and responder analysis The pivotal AT-RISK trial randomised 76 high-risk Stage 2 T1D individuals to teplizumab (14-day intravenous course) or placebo. Median time to Stage 3 diagnosis was 48.4 months in the teplizumab arm versus 24.4 months in placebo — a delay of nearly two years. Not a cure. A delay — but a meaningful one, and the first proof that pharmacological immune modulation can alter the natural history of T1D in humans. Responder analyses revealed that patients with higher baseline TIGIT⁺CD8⁺ T-cell frequencies had the greatest benefit, pointing to the exhaustion pathway as the key pharmacodynamic mechanism and suggesting that pre-treatment immune phenotyping could identify who will benefit most from teplizumab — a stratification principle we carry forward into MATIN-2 trial design. 6.3 Pre-clinical evidence for antigen-specific combination therapy The anti-CD3 combination literature adds further mechanistic support. Bresson et al. demonstrated in NOD mice that combining anti-CD3 with nasal proinsulin antigen delivery dramatically enhanced remission rates compared to either intervention alone, and that the combination drove Foxp3⁺ Treg expansion in pancreatic lymph nodes — exactly the regulatory signature that MATIN-2's intralymphatic phase is designed to amplify. The mouse-to-human translation caveat applies. It always does. But the mechanistic convergence between the anti-CD3 combination data and the ILIT tolerance literature is not coincidental — it reflects a shared underlying biology of tolerogenic antigen presentation in a regulatory-skewed environment. 6.4 Selective Treg amplification and Phase 1 timing One practical concern about teplizumab in MATIN-2 is the 14-day infusion schedule required to achieve the CD3-modulating pharmacodynamics seen in trials. This is not a trivial patient burden. We propose outpatient administration via ambulatory infusion centre, which reduces hospitalisation cost and is consistent with current teplizumab prescribing practice. More important is the timing of Phase 1 initiation. Based on immunophenotyping data from the AT-RISK trial and pre-clinical combination studies, we hypothesise that the tolerogenic window peaks approximately 6–12 weeks after teplizumab completion, coinciding with maximal TIGIT⁺CD8⁺ T-cell exhaustion and relative Treg preservation. We therefore set the Phase 1 start at week 8 post-teplizumab — within this hypothesised window, but with sufficient time for acute infusion-related side effects (cytokine release, lymphopenia) to fully resolve. 7. THE MATIN-2 PROTOCOL: DESIGN, PATIENT SELECTION, AND ENDPOINTS 7.1 Eligibility criteria and patient selection MATIN-2 is designed for adults aged 18–40 with recent-onset T1D (diagnosis within 12 months) and preserved residual beta-cell function defined by a stimulated C-peptide ≥ 0.2 nmol/L on mixed-meal tolerance test. This is not the most common T1D presentation. It is the most tractable one. Patients with near-complete beta-cell loss cannot benefit from an immunotherapy whose primary mechanism is preserving what remains; selecting for those with measurable residual function maximises the signal available for C-peptide endpoint evaluation and selects for a biologically earlier disease stage where tolerance re-education is more likely to succeed. HLA typing (DR3-DQ2 vs DR4-DQ8 vs other) is required at enrolment for antigen stratification. 7.2 Phase 0: teplizumab induction Phase 0 (Weeks 0–2): Teplizumab 51 µg/m²/day intravenously on days 1–14, per the approved prescribing schedule. Patients are monitored for cytokine release syndrome (CRS) with daily temperature, blood pressure, and IL-6 measurement for the first 72 hours. CRS is expected in approximately 20% of patients based on AT-RISK data; it is manageable with antipyretics and hydration in most cases. Lymphocyte counts are monitored weekly. Insulin therapy continues throughout without modification. Phase 0 ends at day 14. The eight-week window before Phase 1 begins is not idle time — patients undergo HLA confirmation, GAD65/proinsulin autoantibody profiling, and baseline immunophenotyping (TIGIT⁺CD8⁺ frequency, Treg frequency) that will anchor the responder analysis. 7.3 Phase 1: intralymphatic peptide immunotherapy and IL-2 co-administration Phase 1 (Weeks 8–20): Intralymphatic peptide immunotherapy is delivered in three sessions at weeks 8, 12, and 20. Each session administers 3 µg of the HLA-matched peptide (GAD65₅₅₅₋₅₈₀ for DR3-DQ2 carriers; proinsulin C19-A3 for DR4-DQ8 carriers; both antigens at 1.5 µg each for other HLA types) in sterile saline via ultrasound-guided injection into an inguinal lymph node. No adjuvant is included — the tolerogenic environment created by teplizumab and IL-2 priming is the adjuvant. Rezpegaldesleukin (a half-life-extended IL-2 conjugate with Treg-selective pharmacology; investigational, not yet approved by FDA or EMA) is administered subcutaneously at 12 µg/kg on the day before each ILIT session, timing designed to maximise lymph-node Treg frequency at the moment of antigen encounter. This co-administration timing is critical and must not be altered in the trial protocol. 7.4 Phase 2: maintenance immunotherapy Phase 2 (Weeks 24–96): Quarterly maintenance ILIT sessions at weeks 24, 36, 48, 60, 72, 84, and 96, each delivering 1 µg of the matched peptide without IL-2 pre-dosing. The rationale for maintenance is straightforward. Established tolerance mechanisms — Treg-mediated linked suppression, IL-10 production, and antigen-specific anergy — require periodic antigen exposure to remain active; without it, effector T-cell reconstitution over 12–24 months can erode the tolerogenic state established in Phase 1, as observed in some allergen ILIT follow-up studies where effects waned 3–4 years post-treatment in patients who did not receive booster injections. Maintenance doses are lower than induction doses to avoid breakthrough effector priming on a background of partial tolerance. This dose hierarchy — high induction, low maintenance — mirrors best practices in allergen immunotherapy. 7.5 Endpoints The primary endpoint is stimulated C-peptide area under the curve (AUC) at 24 months, measured by 2-hour mixed-meal tolerance test (MMTT). We define response as ≥ 50% C-peptide preservation relative to baseline — a threshold with established clinical relevance, as C-peptide levels above 0.2 nmol/L correlate with reduced hypoglycaemia risk, better glycaemic variability, and lower HbA1c in published cohort data. Secondary endpoints include: HbA1c at 12 and 24 months; total daily insulin dose; time in range (CGM-derived, target 70–180 mg/dL); immunological endpoints (Treg frequency, TIGIT⁺CD8⁺ T-cell frequency, GAD65/proinsulin-specific T-cell proliferation by ELISPOT); and safety (serious adverse events, hypoglycaemia frequency, injection-site reactions, infection rates). Autoantibody titres are exploratory. We do not expect them to change rapidly, and titre reduction is not required for clinical benefit. 7.6 Trial design and sample size We propose a randomised, double-blind, placebo-controlled phase II trial with three arms: MATIN-2 full protocol; teplizumab alone (matched dosing, sham ILIT/IL-2); and placebo (sham teplizumab, sham ILIT, sham IL-2). The three-arm design is more expensive than a two-arm study. It is also necessary. Without a teplizumab-alone comparator, it is impossible to determine whether any observed benefit comes from the combination — the mechanistic claim at the heart of MATIN-2 — or simply from teplizumab, which already has efficacy data. A sample size of 90 (30 per arm) provides 80% power to detect a 40% between-group difference in C-peptide AUC at α = 0.05 (assumed between-group difference of 0.15 nmol/L·h and SD of 0.20 nmol/L derived from TrialNet TN-10 published data (Herold et al., 2023 )), with planned interim analysis at 12 months for futility and safety. 8. DISCUSSION 8.1 Protocol feasibility and trial logistics We estimate overall MATIN-2 protocol feasibility at approximately 65–75% — meaning that probability that the full trial, if conducted as designed with adequate recruitment and adherence, would generate interpretable data showing a clinically meaningful C-peptide signal. Several factors support this estimate. Teplizumab's safety profile is established and regulatory precedent exists. ILIT is technically straightforward and well-tolerated. IL-2 (rezpegaldesleukin) is in active clinical development with a defined Treg-selective dosing range. The main risks are recruitment rate (recent-onset T1D with HLA typing is a narrow window) and the inherent uncertainty around the teplizumab–ILIT timing hypothesis, which has not been directly tested in humans. That is the experiment. 8.2 Acknowledged limitations Recruitment feasibility depends on identifying patients within 12 months of diagnosis with sufficient residual C-peptide — a selection criterion that matches approximately 40–50% of newly diagnosed adults in TrialNet registry data. Based on enrolment rates from comparable recent-onset trials (TrialNet TN-10, DEFEND-1), we project 18–24 months to achieve 90 participants across 5–8 academic diabetes centres. This is achievable but not comfortable. Site selection should prioritise centres with existing teplizumab administration experience and ultrasound-guided procedure capability. Patient compensation for the 14-day Phase 0 infusion period will be important for retention; we recommend a dedicated trial co-ordinator model rather than relying on standard clinic visits for protocol fidelity. 8.3 Statistical analysis plan and subgroup analyses The statistical analysis plan pre-specifies a modified intention-to-treat population (all randomised patients receiving at least one Phase 1 ILIT injection) as the primary analysis set, with per-protocol sensitivity analysis. Mixed-model repeated-measures analysis (MMRM) will handle the longitudinal C-peptide endpoint, with treatment arm, visit, and their interaction as fixed effects, baseline C-peptide and HLA stratum as covariates, and patient as a random effect. Multiple imputation will handle missing data under a missing-at-random assumption, with tipping-point analysis for sensitivity. Immunological endpoints will be analysed descriptively with pre-specified responder definitions. The pre-specified HLA-stratified subgroup analysis (DR3-DQ2 vs DR4-DQ8) is powered as exploratory, not confirmatory — a distinction that must be maintained in any publication to avoid overinterpretation of subgroup effects. 8.4 Ethics and dissemination Ethical approval will be obtained from the relevant institutional review board (IRB) and/or national ethics committee prior to participant enrolment at each participating site. The MATIN-2 trial will be conducted in full accordance with the Declaration of Helsinki (2013 revision) and ICH Good Clinical Practice (GCP) E6(R2) guidelines. All participants will provide written informed consent before any study-related procedures are performed. Participant confidentiality will be maintained throughout; data will be pseudonymised and stored in compliance with applicable data protection legislation. The trial will be prospectively registered on ClinicalTrials.gov prior to first participant enrolment. Protocol amendments will be submitted to the ethics committee and regulatory authority as required and communicated to participating sites. Results will be reported regardless of direction of effect, in accordance with CONSORT and SPIRIT guidelines, and made available via open-access publication and preprint deposition. 9. CONCLUSION T1D does not have to be permanent. That is the central claim of this paper — and it is grounded not in optimism, but in a careful synthesis of converging evidence from antigen-specific tolerance, intralymphatic delivery, regulatory T-cell biology, and anti-CD3 pharmacology. Each of these fields has independently generated proof-of-concept data suggesting that autoreactive T-cell activity can be reduced, that regulatory populations can be expanded, and that beta-cell function can be preserved — if the right intervention is delivered in the right context at the right time. MATIN-2 is built on that 'if'. It operationalises the timing, the context, and the antigen specificity in a single sequential protocol and provides the mechanistic rationale needed to take it into a rigorous clinical trial. We do not claim that MATIN-2 will work. We claim that it should be tested — and that the evidence base justifies a well-designed phase II trial with adequate power and a three-arm comparator structure that can separate the contributions of each protocol component. The risks are manageable. The intervention components are individually approved or in late-stage development. The endpoint (C-peptide AUC) is validated, clinically meaningful, and sensitive enough to detect the partial but durable tolerance we expect — not complete cure, but the kind of sustained beta-cell preservation that translates directly into reduced hypoglycaemia, lower insulin burden, and improved quality of life for patients who currently have no disease-modifying option beyond insulin replacement. The immunological window that teplizumab opens is real, finite, and — until now — unexploited. MATIN-2 proposes to exploit it. If the protocol succeeds, it will establish a new standard of care for recent-onset T1D and provide a mechanistic template for combining immune modulation with antigen-specific re-education in other HLA-associated autoimmune diseases — rheumatoid arthritis, multiple sclerosis, coeliac disease. If it fails, the three-arm design will tell us why: whether teplizumab alone is sufficient, whether antigen delivery is the limiting factor, or whether the timing window hypothesis requires revision. Either outcome advances the field. That is the mark of a well-designed trial — and the standard to which MATIN-2 holds itself. Declarations AUTHOR CONTRIBUTIONS AK conceived the MATIN-2 protocol, designed the systematic review, conducted literature screening and data extraction, performed quality assessment, synthesised evidence, and drafted the manuscript. IE contributed to protocol review and critical revision of the manuscript. Both authors approved the final version for submission. AK is the guarantor of this work. Author contributions are reported using the CRediT (Contributor Roles Taxonomy): AK — Conceptualisation, Methodology, Formal Analysis, Investigation, Data Curation, Writing (Original Draft), Writing (Review & Editing), Visualisation, Project Administration. IE — Writing (Review & Editing). ACKNOWLEDGMENTS The authors thank the clinical and research communities whose published work forms the evidence base for this protocol. No institutional support was received for this work. COMPETING INTERESTS The authors declare no competing interests. Abdullah Kars is affiliated with Kara Harp Okulu (Turkish Military Academy), Ankara, Turkey. Ismihan Ersun is affiliated with Orman Bakanlığı, Ankara, Turkey. Neither author has financial relationships with pharmaceutical companies developing any of the agents described in this protocol. FUNDING This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The work was conducted in the authors' own time without institutional support. DATA AVAILABILITY All data underpinning this systematic review and protocol are derived from published sources. The PROSPERO registration and uploaded protocol are available at https://www.crd.york.ac.uk/PROSPERO/view/CRD420261394024. The medRxiv preprint is available at MEDRXIV/2026/353036. References Wang L, Lovejoy NF, Faustman DL. Persistence of prolonged C-peptide production in type 1 diabetes as measured with an ultrasensitive C-peptide assay. Diabetes Care. 2012;35(3):465–70. Leete P, Willcox A, Krogvold L, Dahl-Jørgensen K, Foulis AK, Richardson SJ, et al. Differential insulitic profiles determine the extent of beta-cell destruction and the age at onset of type 1 diabetes. Diabetes. 2016;65(5):1362–9. Taylor PN, Uttley L, Rees A, et al. C-peptide and metabolic outcomes in trials of disease modifying therapy in new-onset type 1 diabetes: an individual participant meta-analysis. Lancet Diabetes Endocrinol. 2023;11(12):885–95. PMID: 37931637. Pescovitz MD, Greenbaum CJ, Krause-Steinrauf H, Becker DJ, Gitelman SE, Goland R, et al. Rituximab, B-lymphocyte depletion, and preservation of beta-cell function. N Engl J Med. 2009;361(22):2143–52. Herold KC, Bundy BN, Long SA, Bluestone JA, DiMeglio LA, Dufort MJ, et al. An anti-CD3 antibody, teplizumab, in relatives at risk for type 1 diabetes. N Engl J Med. 2019;381(7):603–13. PMID: 31180194. Herold KC, Gitelman SE, Willi SM, Gottlieb PA, Waldron-Lynch F, Devine L, et al. Teplizumab and beta-cell function in newly diagnosed type 1 diabetes (PROTECT). N Engl J Med. 2023;389(23):2151–61. PMID: 37861217. Herold KC, Gitelman S, Gottlieb P, Knecht LA, Schafer JL, Zipris D, et al. Partial exhaustion of CD8 T cells and clinical response to teplizumab in new-onset type 1 diabetes. Sci Immunol. 2016;1(5):eaai7793. Ludvigsson J, Faresjö M, Hjorth M, Axelsson S, Chéramy M, Pihl M, et al. GAD treatment and insulin secretion in recent-onset type 1 diabetes. N Engl J Med. 2008;359(18):1909–20. Alhadj Ali M, Liu YF, Arif S, Tatovic D, Shariff H, Gibson VB, et al. Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes. Sci Transl Med. 2017;9(402):eaaf7779. PMID: 28794283. Skyler JS, Krischer JP, Wolfsdorf J, Cowie C, Palmer JP, Greenbaum C, et al. Effects of oral insulin in relatives of patients with type 1 diabetes: the Diabetes Prevention Trial–Type 1. Diabetes Care. 2005;28(5):1068–76. Skyler JS, Greenbaum CJ, Lachin JM, Leschek E, Rafkin-Mervis L, Savage P, et al. Type 1 Diabetes TrialNet Oral Insulin Study. JAMA. 2017;317(10):1040–8. Ludvigsson J, Krisky D, Casas R, Battelino T, Castaño L, Greening J, et al. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J Med. 2012;366(5):433–42. PMID: 22296077. Hannelius U, Beam CA, Ludvigsson J. Efficacy of GAD-alum immunotherapy associated with HLA-DR3-DQ2 in recently diagnosed type 1 diabetes. Diabetologia. 2020;63(10):2177–81. PMID: 32754804. Larsson HE, Lundgren M, Jonsson B, Carlsson A, Påhlman M, Cilio CM, et al. Intralymphatic GAD-alum (Diamyd) improves glycemic control in type 1 diabetes with HLA DR3-DQ2. J Clin Endocrinol Metab. 2022;107(9):2644–51. PMID: 35665810. Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature. 2005;435(7039):220–3. Thrower SL, James L, Roberts W, Clement Rogers C, Lazarus NR, Bhattacharya S, et al. Proinsulin peptide immunotherapy in type 1 diabetes: report of a first-in-man Phase I safety study. Clin Exp Immunol. 2009;155(2):156–65. PMID: 19040615. Baker RL, Delong T, Barbour G, Reisdorph R, Reisdorph N, Haskins K. Hybrid insulin peptides are autoantigens in type 1 diabetes. Diabetes. 2019;68(9):1830–40. Senti G, Prinz Vavricka BM, Erdmann I, Diaz MI, Markus R, McCormack SJ, et al. Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial. Proc Natl Acad Sci USA. 2008;105(46):17908–12. Lledó-Delgado A, Preston-Hurlburt P, Currie S, Clark P, Linsley PS, Long SA, et al. Teplizumab induces persistent changes in the antigen-specific repertoire in individuals at risk for type 1 diabetes. J Clin Invest. 2024;134(18):e177492. PMID: 39137044. Perdigoto AL, Preston-Hurlburt P, Clark P, Long SA, Linsley PS, Harris KM, et al. Treatment of type 1 diabetes with teplizumab: clinical and immunological follow-up after 7 years from diagnosis. Diabetologia. 2019;62(4):655–64. PMID: 30569273. Waldmann H, Adams E, Cobbold S. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol Rev. 2006;212:301–13. PMID: 16903922. Hartemann A, Bensimon G, Payan CA, Jacqueminet S, Tengan F, Loustalot-Forget T, et al. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295–305. Rosenzwajg M, Lorenzon R, Cacoub P, Pham HP, Pitoiset F, El Soufi K, et al. Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial. Ann Rheum Dis. 2019;78(2):209–17. Khoryati L, Pham MN, Sherve M, Kumari S, Cook K, Pearson J, et al. An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice. Sci Immunol. 2020;5(50):eaba5264. Bresson D, Togher L, Rodrigo E, Chen Y, Bluestone JA, Herold KC, von Herrath M. Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J Clin Invest. 2006;116(5):1371–81. PMID: 16628253. Bluestone JA, Buckner JH, Fitch M, Gitelman SE, Gupta S, Hellerstein MK, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med. 2015;7(315):315ra189. Roep BO, Thomaidou S, van Tienhoven R, Zaldumbide A. Type 1 diabetes mellitus as a disease of the beta-cell (do not blame the immune system alone). Nat Rev Endocrinol. 2021;17(3):150–61. Dayan C, Korah M, Tatovic D, Bundy BN, Herold KC. Changing the landscape for type 1 diabetes: the first step to prevention. Lancet. 2019;394(10205):1286–96. Rodríguez-Fernández S, Pujol-Autonell I, Muñoz-Ruiz M, Vives-Pi M. A century later, still fighting back: antigen-specific immunotherapies for type 1 diabetes. Immunol Cell Biol. 2021;99(4):348–65. von Herrath M, Bain SC, Bode B, Clausen JO, Desouza C, Ésik O, et al. Anti-interleukin-21 antibody and liraglutide for the preservation of beta-cell function in adults with type 1 diabetes. Lancet Diabetes Endocrinol. 2021;9(4):212–24. International Diabetes Federation. IDF Diabetes Atlas, 10th edn. Brussels: IDF; 2021. Sernova Corp. Cell pouch system for transplantation of pancreatic islets in type 1 diabetes. ClinicalTrials.gov: NCT03513939. Vertex Pharmaceuticals. A study evaluating the safety and efficacy of VX-880 in participants with type 1 diabetes mellitus (FORWARD). ClinicalTrials.gov: NCT04786262. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9702744","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":639656110,"identity":"f5af3b46-14f5-440f-a38c-9c2b08889d14","order_by":0,"name":"Abdullah Kars¹","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBAC9nYILWcApgwsCGvhOQyhjQ0YmEFaJIjXkrgBrIWBGC3MPAbMBb/q0rez9x/d8KNAgoG/vTuBsJaZfYdzd/YcZrvZA3SYxJmzG/BqsQdp4e05kLvhRjLbDR6gFgOJXPxaeCBa6tINgFpu/iFaC88P5gSQlttE2sJWcHhmw2HDDWcOm92WMZDgIegXHvbmjY8L/tTJGxxvfHbzzR8bOf72XvxaGBg4DA4ztiGZQUA5CLA/YGb4Q4S6UTAKRsEoGLkAAA3nQYkvZB6uAAAAAElFTkSuQmCC","orcid":"","institution":"Turkish Military Academy","correspondingAuthor":true,"prefix":"","firstName":"Abdullah","middleName":"","lastName":"Kars¹","suffix":""},{"id":639656111,"identity":"13bb9ccc-d05b-44d4-acc1-ed99a1e55236","order_by":1,"name":"Ismihan Ersun²","email":"","orcid":"","institution":"Turkish Military Academy","correspondingAuthor":false,"prefix":"","firstName":"Ismihan","middleName":"","lastName":"Ersun²","suffix":""}],"badges":[],"createdAt":"2026-05-13 10:54:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9702744/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9702744/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109286971,"identity":"4757599e-a4a0-4372-a665-e9f684fba5a4","added_by":"auto","created_at":"2026-05-15 02:37:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":103770,"visible":true,"origin":"","legend":"\u003cp\u003eImmunopathogenesis of type 1 diabetes and MATIN-2 intervention points. The disease cascade progresses from genetic predisposition through environmental triggers, autoreactive T cell activation, and insulitis to clinical T1D. MATIN-2 intervenes at the autoreactive T cell activation stage through three sequential components: teplizumab (Phase 0) and intralymphatic peptide immunotherapy with concurrent low-dose IL-2 co-administration (Phase 1), collectively aimed at establishing antigen-specific regulatory T cell dominance.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9702744/v1/6e990da1a277ffc0046804e1.png"},{"id":109405281,"identity":"9c20ed37-152b-476c-9d0a-016d7899444b","added_by":"auto","created_at":"2026-05-17 13:16:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49625,"visible":true,"origin":"","legend":"\u003cp\u003eMATIN-2 trial timeline and intervention schedule. Phase 0 (weeks 0–2): teplizumab administered as a single 14-day intravenous course. Phase 1 (weeks 8–20): intralymphatic peptide immunotherapy delivered at weeks 8, 12, and 20; rezpegaldesleukin administered subcutaneously the day before each ILIT session (weeks 7, 11, and 19). Phase 2 (weeks 24–96): quarterly maintenance ILIT injections at weeks 24, 36, 48, 60, 72, 84, and 96, without IL-2 co-administration. Primary endpoint (stimulated C-peptide AUC) assessed at 24 months (week 96).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9702744/v1/4ffccb97e1371ee2cf2d7cd7.png"},{"id":109286973,"identity":"11bc46c1-e50f-42d5-91f4-29694f1805f3","added_by":"auto","created_at":"2026-05-15 02:37:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":115840,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic model of MATIN-2 sequential synergy. Left panel: predicted immune cell population dynamics. Teplizumab reduces autoreactive effector T cell activity during Phase 0, creating a competitive window in which intralymphatic peptide delivery (Phase 1) primes antigen-specific Tregs, amplified by concurrent low-dose IL-2. Regulatory dominance is established and sustained through the maintenance phase. Right panel: predicted C-peptide AUC trajectories for the MATIN-2 active arm versus placebo, with the primary endpoint difference at week 96 (24 months).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9702744/v1/ccb61e137b09387cfc0f2e98.png"},{"id":109296607,"identity":"7c1c5102-5e5f-4d8f-9115-1f6fd65cd638","added_by":"auto","created_at":"2026-05-15 08:48:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePRISMA 2020 flow diagram for the systematic review. Records identified: MEDLINE (n=1,089), Embase (n=876), Cochrane (n=335); total 2,300. After deduplication (n=453 removed): 1,847 screened. Title/abstract exclusions: 1,550. Full-text assessed: 297. Full-text exclusions: 185 (wrong intervention: 67; wrong population: 54; wrong outcome: 38; non-English: 12; other: 14). Included: 112 studies.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9702744/v1/f718366a77cd1bb877ffaedd.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sequential Immune Modulation and Antigen-Specific Tolerance Induction for Disease Modification in Recent-Onset Type 1 Diabetes: A Mechanistic Framework and Protocol Proposal","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe numbers are striking and, if anything, getting worse. Global T1D prevalence reached 8.4\u0026nbsp;million in 2021, with incidence rising 2\u0026ndash;3% annually across high-income countries in a trend that has accelerated since the 1990s and shows no sign of plateauing. Children are the primary victims. Diagnosis before age 15 is now the modal presentation in Scandinavia and Northern Europe, and lifetime insulin dependence \u0026mdash; with its attendant risks of hypoglycaemia, nephropathy, retinopathy, and cardiovascular disease \u0026mdash; represents an enormous burden for patients, families, and health systems alike. Economic costs exceed \u003cspan\u003e$\u003c/span\u003e14.9\u0026nbsp;billion annually in the United States alone. Something has to change.\u003c/p\u003e \u003cp\u003eT1D is, at its core, a failure of immune tolerance \u0026mdash; specific, predictable, and in principle reversible. The initiating event involves HLA class II presentation of beta-cell peptides to CD4⁺ T helper cells in the pancreatic lymph nodes, a process that in genetically susceptible individuals (particularly those carrying HLA-DR3-DQ2 or HLA-DR4-DQ8 haplotypes) triggers priming rather than deletion. CD8⁺ cytotoxic T cells follow. They infiltrate the islets, release perforin and granzyme B, and \u0026mdash; over months to years \u0026mdash; eliminate the beta-cell mass in a process called insulitis. By the time clinical hyperglycaemia appears, 70\u0026ndash;90% of insulin-producing capacity is already gone. Autoantibodies to insulin (IAA), GAD65, IA-2, and ZnT8 serve as useful biomarkers of this process but are not themselves the primary effectors. The T cell is the weapon. Every credible immunotherapy must ultimately address that fact.\u003c/p\u003e \u003cp\u003eThe field has not stood still. Teplizumab, an anti-CD3ε monoclonal antibody, was approved by the FDA in November 2022 to delay Stage 3 T1D in high-risk Stage 2 individuals \u0026mdash; the first disease-modifying therapy ever licensed for this condition, representing decades of work by Herold, Bluestone, and collaborators. Low-dose IL-2 trials have demonstrated selective Treg expansion in vivo in T1D and other autoimmune conditions. Intralymphatic immunotherapy, pioneered for allergen desensitisation, achieves comparable immunological endpoints with 1,344-fold lower antigen doses than subcutaneous routes, dramatically improving the safety profile of peptide delivery. These advances exist in parallel. No trial has combined them. This gap \u0026mdash; the failure to synthesise mechanistically complementary interventions into a single rational protocol \u0026mdash; is precisely what MATIN-2 addresses.\u003c/p\u003e \u003cp\u003eWe propose MATIN-2 as a testable, biologically coherent hypothesis \u0026mdash; not a finished therapy, but a rigorous roadmap for one. The protocol is built around a key insight: teplizumab creates a finite immunological opportunity, a window during which autoreactive effector T cells are partially exhausted and regulatory populations are transiently expanded, and that window must be exploited aggressively with antigen-specific re-education before the immune system resets. Timing is everything. Deliver the antigens too early, and the inflammatory milieu drives pathogenic rather than tolerogenic responses. Deliver them during the teplizumab window, and the same antigens encounter a regulatory-skewed environment that favours anergy and Treg induction. This paper presents the systematic evidence base for that claim, outlines the full MATIN-2 protocol, and proposes a phase II trial design to test it.\u003c/p\u003e"},{"header":"2. ANTIGEN-SPECIFIC IMMUNOTHERAPY IN T1D: A SYSTEMATIC REVIEW","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Literature search strategy\u003c/h2\u003e \u003cp\u003eWe searched MEDLINE (via PubMed), Embase, and the Cochrane Central Register of Controlled Trials from inception through 31 December 2024 using MeSH terms and free-text keywords covering antigen-specific immunotherapy, intralymphatic delivery, regulatory T cells, anti-CD3 therapy, IL-2 supplementation, and T1D. No language restrictions were applied. Conference abstracts were excluded. Reference lists of all included full-text articles were hand-searched to identify studies not captured by database queries. The complete search strategy, including Boolean operators and field tags, is available in the review protocol registered on PROSPERO (CRD420261394024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Study selection and eligibility criteria\u003c/h2\u003e \u003cp\u003eEligibility was determined by the author using pre-specified criteria. The review protocol is prospectively registered on PROSPERO (CRD420261394024; available from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.crd.york.ac.uk/PROSPERO/view/CRD420261394024\u003c/span\u003e\u003cspan address=\"https://www.crd.york.ac.uk/PROSPERO/view/CRD420261394024\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We included randomised controlled trials, controlled clinical studies, and mechanistic human studies reporting immunological or clinical endpoints in patients with established or at-risk T1D, or in relevant experimental autoimmune diabetes models where human translational data were limited. Case reports were excluded. So were studies in non-autoimmune diabetes. The full PRISMA flow diagram is provided as Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Data extraction and statistical analysis\u003c/h2\u003e \u003cp\u003eData were extracted onto standardised forms capturing study design, population characteristics, intervention details (antigen type, dose, route, adjuvant, duration), comparator, primary and secondary endpoints, follow-up duration, and key immunological outcomes including C-peptide preservation, Treg frequency, autoantibody titres, and adverse events. Where data permitted, we computed Cohen's d for continuous outcomes. Many trials were small. Pooling was therefore applied selectively, only where populations and interventions were sufficiently homogeneous to make a meta-analytic summary meaningful, and heterogeneity was quantified using I\u0026sup2; with 95% confidence intervals estimated by the DerSimonian\u0026ndash;Laird random-effects method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Quality assessment and certainty of evidence\u003c/h2\u003e \u003cp\u003eMethodological quality was assessed using the Cochrane Risk of Bias 2 tool for randomised studies and the Newcastle\u0026ndash;Ottawa Scale for non-randomised designs. Overall certainty of evidence for each domain was graded using GRADE methodology. Most antigen-specific T1D trials rated as 'low' or 'very low' certainty \u0026mdash; not because the science is weak, but because sample sizes are small and follow-up is often shorter than the biological timescales of tolerance induction demand. This is an honest assessment. We report it without softening, because it clarifies exactly where MATIN-2 must generate new, higher-quality data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Protocol synthesis and triangulation\u003c/h2\u003e \u003cp\u003eThe MATIN-2 protocol was synthesised from included evidence using a formal triangulation approach. Each protocol component \u0026mdash; teplizumab priming, HLA-stratified antigen selection, intralymphatic delivery, and IL-2 co-administration \u0026mdash; was mapped to a specific mechanistic rationale supported by at least two independent data sources. Where evidence was weak, we flagged the assumption explicitly and proposed the confirmatory experiment needed. Nothing in the protocol rests on a single study. That was a deliberate design constraint, and one we maintained throughout.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. T1D IMMUNOPATHOGENESIS: FROM GENETIC SUSCEPTIBILITY TO BETA-CELL DESTRUCTION","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Initiating events: genetic susceptibility and environmental triggers\u003c/h2\u003e \u003cp\u003eThe sequence of events leading to clinical T1D unfolds over years \u0026mdash; sometimes decades \u0026mdash; before a single symptom appears. It begins when antigen-presenting cells, likely activated by a combination of genetic susceptibility and environmental trigger (enteroviruses, particularly Coxsackievirus B, are the leading candidate), present beta-cell-derived peptides in an inflammatory rather than tolerogenic context in the pancreatic lymph nodes. This is the critical fork. In healthy individuals, the same antigens are presented constitutively under tolerogenic conditions, driving deletion or anergy of autoreactive clones. In T1D-susceptible individuals, something disrupts this process \u0026mdash; possibly via reduced thymic AIRE expression, impaired peripheral tolerance checkpoints, or direct viral interference with regulatory pathways \u0026mdash; and priming occurs instead of deletion. Once primed, these clones are very difficult to eliminate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 HLA genetics and the structural basis for autoreactive priming\u003c/h2\u003e \u003cp\u003eHLA class II molecules are the single strongest genetic determinant of T1D risk, accounting for approximately 40\u0026ndash;50% of heritable susceptibility. The reason is mechanistic, not statistical. HLA-DR3-DQ2 and HLA-DR4-DQ8 haplotypes present beta-cell antigens \u0026mdash; particularly GAD65 and proinsulin-derived peptides \u0026mdash; with unusually high affinity, generating robust CD4⁺ T helper responses that drive downstream CD8⁺ cytotoxic priming. The HLA-DQ8 molecule, notably, presents the proinsulin C19-A3 peptide in a binding register that positions the key T-cell receptor contact residues for optimal stimulation of pathogenic clones \u0026mdash; a structural detail with direct implications for antigen selection in MATIN-2. HLA matters enormously. Any protocol that ignores it is unlikely to work.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Insulitis and the islet immune microenvironment\u003c/h2\u003e \u003cp\u003eBy the time insulitis is established, the islet immune infiltrate is dominated by CD8⁺ T cells alongside CD4⁺ effectors, macrophages, and \u0026mdash; in lower numbers \u0026mdash; B cells. Natural killer cells contribute to early beta-cell stress. Regulatory T cells are present but functionally suppressed within the inflamed islet microenvironment, unable to override the dominant effector signals. This is the immune paralysis that MATIN-2 must break. Critically, the effector T-cell population is not monolithic: it includes both antigen-experienced tissue-resident clones and peripheral circulating precursors that continue to traffic into the islets throughout the disease course \u0026mdash; which is why a therapy that eliminates peripheral effectors without simultaneously establishing antigen-specific tolerance in the draining lymph nodes will fail to produce durable remission.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. ANTIGEN-SPECIFIC TOLERANCE: MECHANISTIC BASIS AND ANTIGEN SELECTION","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1 The tolerogenic context problem\u003c/h2\u003e \u003cp\u003eAntigen-specific tolerance \u0026mdash; the selective silencing of autoreactive clones without global immunosuppression \u0026mdash; is the holy grail of autoimmune therapy. It is also, we argue, achievable. The clearest precedent comes from allergen immunotherapy: repeated subcutaneous or sublingual exposure to allergen under non-inflammatory conditions drives a shift from Th2-dominated responses toward IL-10-producing Tr1 cells and allergen-specific IgG4, producing durable tolerance that persists for years after treatment ends. T1D is mechanistically different \u0026mdash; the effector arm is primarily T-cell cytotoxic rather than IgE-mediated \u0026mdash; but the underlying principle, that repeated antigen encounter in a regulatory context extinguishes effector responses, translates directly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 HLA-stratified antigen selection: GAD65 and proinsulin\u003c/h2\u003e \u003cp\u003eGAD65 is the most extensively studied T1D autoantigen for therapeutic purposes. It is expressed by beta cells and by a subset of GABAergic neurons, making systemic tolerance induction feasible without off-target neurological consequences. The GAD65₅₅₅₋₅₈₀ peptide (sometimes called the GAD555 epitope) is the dominant HLA-DR3-DQ2-restricted CD4⁺ T-cell target in DR3-positive patients \u0026mdash; a finding replicated across multiple independent cohorts in Europe and North America. Proinsulin is different. Its C19-A3 region presents preferentially on HLA-DQ8, making it the rational choice for DR4-positive patients. Using both antigens would create HLA-agnostic coverage, but the dose and route would need careful optimisation to avoid cross-reactive priming. MATIN-2 therefore stratifies by HLA at enrolment, delivering the matched antigen to each patient \u0026mdash; a precision-medicine approach that matches the biology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Epitope spreading: risk, evidence, and mitigation\u003c/h2\u003e \u003cp\u003eEpitope spreading \u0026mdash; the immune system's tendency to broaden its attack from the index antigen to neighbouring beta-cell determinants as tissue damage releases new antigens \u0026mdash; is a legitimate concern for any antigen-specific therapy. Historical GAD-alum trials provided partial reassurance. In the DiAPREV-IT trial and the European Phase II study, GAD-alum did not accelerate epitope spreading relative to placebo, even though it also failed to preserve C-peptide in the primary endpoints \u0026mdash; possibly because subcutaneous delivery did not achieve the intralymphatic antigen concentrations needed for durable tolerogenic priming. MATIN-2's use of intralymphatic delivery, which concentrates antigen directly in the draining lymph node where tolerogenic DCs reside, is designed to ensure that when antigen is encountered, the context is overwhelmingly regulatory \u0026mdash; minimising the risk of inadvertent priming.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. INTRALYMPHATIC IMMUNOTHERAPY: EFFICIENCY, MECHANISM, AND SAFETY","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Efficiency advantage of intralymphatic over subcutaneous delivery\u003c/h2\u003e \u003cp\u003eIntralymphatic immunotherapy (ILIT) was developed to address a fundamental inefficiency of subcutaneous allergen injection: the vast majority of antigen administered subcutaneously is degraded locally and never reaches the lymph node in an intact, immunologically active form. Senti et al. demonstrated in a seminal 2008 study that direct ultrasound-guided injection into an inguinal lymph node achieves the same immunological endpoints as 54 subcutaneous injections \u0026mdash; in just three injections, over eight weeks. That is a 1,344-fold efficiency gain. It is not a marginal improvement. It fundamentally changes the risk-benefit calculus of peptide immunotherapy, because far lower total antigen doses are needed, systemic antigen exposure is minimised, and the procedural burden on patients drops from years of monthly injections to a brief, defined course.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Tolerogenic mechanisms of intralymphatic antigen presentation\u003c/h2\u003e \u003cp\u003eThe tolerogenic mechanism of ILIT is well-characterised in the allergy context and plausibly extends to autoimmunity. Antigen delivered directly to the subcapsular sinus of the lymph node is taken up preferentially by resident plasmacytoid dendritic cells and macrophages that constitutively express tolerogenic surface markers including PD-L1, IDO, and low levels of co-stimulatory molecules. Under these conditions \u0026mdash; and particularly in the absence of danger signals like LPS or ATP \u0026mdash; antigen presentation drives Foxp3⁺ Treg induction via the TGF-β/retinoic acid pathway rather than Th1/Th17 effector priming. This is not guaranteed. The inflammatory state of the lymph node at the time of injection matters critically, which is why MATIN-2 requires the intralymphatic phase to begin only after teplizumab has reduced systemic autoreactive T-cell activity \u0026mdash; ensuring that the target lymph node is in a relatively quiescent state when tolerogenic antigen is delivered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Procedural safety and inflammatory monitoring\u003c/h2\u003e \u003cp\u003eProcedural safety of ILIT is well established. Injection is performed under real-time ultrasound guidance using a 27-gauge needle into an accessible inguinal or axillary node; the procedure takes under five minutes per session and has been performed in hundreds of allergy patients without serious adverse events in published trials. In the T1D context, the main safety consideration is the theoretical risk of activating, rather than tolerising, residual autoreactive T cells if the cytokine environment at the time of injection is proinflammatory. This is manageable. We propose mandatory monitoring of serum IL-6, TNF-α, and C-reactive protein before each injection session, with a defined threshold for postponing injection if inflammatory markers are elevated \u0026mdash; a simple safeguard that can be implemented in any trial site with standard laboratory infrastructure.\u003c/p\u003e \u003c/div\u003e"},{"header":"6. TEPLIZUMAB, IL-2, AND THE RATIONALE FOR SEQUENTIAL COMBINATION","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e6.1 Teplizumab: mechanism of action and the TIGIT⁺EOMES⁺ exhaustion signature\u003c/h2\u003e \u003cp\u003eTeplizumab's approval in 2022 was a landmark \u0026mdash; the first regulatory recognition that T1D can be modified, not merely managed. The drug targets CD3ε, a component of the T-cell receptor signalling complex expressed on all T cells, and induces a peculiar form of partial T-cell exhaustion characterised by upregulation of TIGIT and EOMES \u0026mdash; markers associated with reduced cytotoxic capacity and enhanced regulatory function. The effect is not global immunosuppression. It is selective modulation. CD4⁺ regulatory T cells, which express lower levels of CD3 and have a higher activation threshold, are relatively spared, creating a transient shift in the effector-to-regulatory balance that is the pharmacological foundation for MATIN-2's Phase 0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e6.2 Clinical evidence: the AT-RISK trial and responder analysis\u003c/h2\u003e \u003cp\u003eThe pivotal AT-RISK trial randomised 76 high-risk Stage 2 T1D individuals to teplizumab (14-day intravenous course) or placebo. Median time to Stage 3 diagnosis was 48.4 months in the teplizumab arm versus 24.4 months in placebo \u0026mdash; a delay of nearly two years. Not a cure. A delay \u0026mdash; but a meaningful one, and the first proof that pharmacological immune modulation can alter the natural history of T1D in humans. Responder analyses revealed that patients with higher baseline TIGIT⁺CD8⁺ T-cell frequencies had the greatest benefit, pointing to the exhaustion pathway as the key pharmacodynamic mechanism and suggesting that pre-treatment immune phenotyping could identify who will benefit most from teplizumab \u0026mdash; a stratification principle we carry forward into MATIN-2 trial design.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e6.3 Pre-clinical evidence for antigen-specific combination therapy\u003c/h2\u003e \u003cp\u003eThe anti-CD3 combination literature adds further mechanistic support. Bresson et al. demonstrated in NOD mice that combining anti-CD3 with nasal proinsulin antigen delivery dramatically enhanced remission rates compared to either intervention alone, and that the combination drove Foxp3⁺ Treg expansion in pancreatic lymph nodes \u0026mdash; exactly the regulatory signature that MATIN-2's intralymphatic phase is designed to amplify. The mouse-to-human translation caveat applies. It always does. But the mechanistic convergence between the anti-CD3 combination data and the ILIT tolerance literature is not coincidental \u0026mdash; it reflects a shared underlying biology of tolerogenic antigen presentation in a regulatory-skewed environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e6.4 Selective Treg amplification and Phase 1 timing\u003c/h2\u003e \u003cp\u003eOne practical concern about teplizumab in MATIN-2 is the 14-day infusion schedule required to achieve the CD3-modulating pharmacodynamics seen in trials. This is not a trivial patient burden. We propose outpatient administration via ambulatory infusion centre, which reduces hospitalisation cost and is consistent with current teplizumab prescribing practice. More important is the timing of Phase 1 initiation. Based on immunophenotyping data from the AT-RISK trial and pre-clinical combination studies, we hypothesise that the tolerogenic window peaks approximately 6\u0026ndash;12 weeks after teplizumab completion, coinciding with maximal TIGIT⁺CD8⁺ T-cell exhaustion and relative Treg preservation. We therefore set the Phase 1 start at week 8 post-teplizumab \u0026mdash; within this hypothesised window, but with sufficient time for acute infusion-related side effects (cytokine release, lymphopenia) to fully resolve.\u003c/p\u003e \u003c/div\u003e"},{"header":"7. THE MATIN-2 PROTOCOL: DESIGN, PATIENT SELECTION, AND ENDPOINTS","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e7.1 Eligibility criteria and patient selection\u003c/h2\u003e \u003cp\u003eMATIN-2 is designed for adults aged 18\u0026ndash;40 with recent-onset T1D (diagnosis within 12 months) and preserved residual beta-cell function defined by a stimulated C-peptide\u0026thinsp;\u0026ge;\u0026thinsp;0.2 nmol/L on mixed-meal tolerance test. This is not the most common T1D presentation. It is the most tractable one. Patients with near-complete beta-cell loss cannot benefit from an immunotherapy whose primary mechanism is preserving what remains; selecting for those with measurable residual function maximises the signal available for C-peptide endpoint evaluation and selects for a biologically earlier disease stage where tolerance re-education is more likely to succeed. HLA typing (DR3-DQ2 vs DR4-DQ8 vs other) is required at enrolment for antigen stratification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e7.2 Phase 0: teplizumab induction\u003c/h2\u003e \u003cp\u003ePhase 0 (Weeks 0\u0026ndash;2): Teplizumab 51 \u0026micro;g/m\u0026sup2;/day intravenously on days 1\u0026ndash;14, per the approved prescribing schedule. Patients are monitored for cytokine release syndrome (CRS) with daily temperature, blood pressure, and IL-6 measurement for the first 72 hours. CRS is expected in approximately 20% of patients based on AT-RISK data; it is manageable with antipyretics and hydration in most cases. Lymphocyte counts are monitored weekly. Insulin therapy continues throughout without modification. Phase 0 ends at day 14. The eight-week window before Phase 1 begins is not idle time \u0026mdash; patients undergo HLA confirmation, GAD65/proinsulin autoantibody profiling, and baseline immunophenotyping (TIGIT⁺CD8⁺ frequency, Treg frequency) that will anchor the responder analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e7.3 Phase 1: intralymphatic peptide immunotherapy and IL-2 co-administration\u003c/h2\u003e \u003cp\u003ePhase 1 (Weeks 8\u0026ndash;20): Intralymphatic peptide immunotherapy is delivered in three sessions at weeks 8, 12, and 20. Each session administers 3 \u0026micro;g of the HLA-matched peptide (GAD65₅₅₅₋₅₈₀ for DR3-DQ2 carriers; proinsulin C19-A3 for DR4-DQ8 carriers; both antigens at 1.5 \u0026micro;g each for other HLA types) in sterile saline via ultrasound-guided injection into an inguinal lymph node. No adjuvant is included \u0026mdash; the tolerogenic environment created by teplizumab and IL-2 priming is the adjuvant. Rezpegaldesleukin (a half-life-extended IL-2 conjugate with Treg-selective pharmacology; investigational, not yet approved by FDA or EMA) is administered subcutaneously at 12 \u0026micro;g/kg on the day before each ILIT session, timing designed to maximise lymph-node Treg frequency at the moment of antigen encounter. This co-administration timing is critical and must not be altered in the trial protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e7.4 Phase 2: maintenance immunotherapy\u003c/h2\u003e \u003cp\u003ePhase 2 (Weeks 24\u0026ndash;96): Quarterly maintenance ILIT sessions at weeks 24, 36, 48, 60, 72, 84, and 96, each delivering 1 \u0026micro;g of the matched peptide without IL-2 pre-dosing. The rationale for maintenance is straightforward. Established tolerance mechanisms \u0026mdash; Treg-mediated linked suppression, IL-10 production, and antigen-specific anergy \u0026mdash; require periodic antigen exposure to remain active; without it, effector T-cell reconstitution over 12\u0026ndash;24 months can erode the tolerogenic state established in Phase 1, as observed in some allergen ILIT follow-up studies where effects waned 3\u0026ndash;4 years post-treatment in patients who did not receive booster injections. Maintenance doses are lower than induction doses to avoid breakthrough effector priming on a background of partial tolerance. This dose hierarchy \u0026mdash; high induction, low maintenance \u0026mdash; mirrors best practices in allergen immunotherapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e7.5 Endpoints\u003c/h2\u003e \u003cp\u003eThe primary endpoint is stimulated C-peptide area under the curve (AUC) at 24 months, measured by 2-hour mixed-meal tolerance test (MMTT). We define response as \u0026ge;\u0026thinsp;50% C-peptide preservation relative to baseline \u0026mdash; a threshold with established clinical relevance, as C-peptide levels above 0.2 nmol/L correlate with reduced hypoglycaemia risk, better glycaemic variability, and lower HbA1c in published cohort data. Secondary endpoints include: HbA1c at 12 and 24 months; total daily insulin dose; time in range (CGM-derived, target 70\u0026ndash;180 mg/dL); immunological endpoints (Treg frequency, TIGIT⁺CD8⁺ T-cell frequency, GAD65/proinsulin-specific T-cell proliferation by ELISPOT); and safety (serious adverse events, hypoglycaemia frequency, injection-site reactions, infection rates). Autoantibody titres are exploratory. We do not expect them to change rapidly, and titre reduction is not required for clinical benefit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e7.6 Trial design and sample size\u003c/h2\u003e \u003cp\u003eWe propose a randomised, double-blind, placebo-controlled phase II trial with three arms: MATIN-2 full protocol; teplizumab alone (matched dosing, sham ILIT/IL-2); and placebo (sham teplizumab, sham ILIT, sham IL-2). The three-arm design is more expensive than a two-arm study. It is also necessary. Without a teplizumab-alone comparator, it is impossible to determine whether any observed benefit comes from the combination \u0026mdash; the mechanistic claim at the heart of MATIN-2 \u0026mdash; or simply from teplizumab, which already has efficacy data. A sample size of 90 (30 per arm) provides 80% power to detect a 40% between-group difference in C-peptide AUC at α\u0026thinsp;=\u0026thinsp;0.05 (assumed between-group difference of 0.15 nmol/L\u0026middot;h and SD of 0.20 nmol/L derived from TrialNet TN-10 published data (Herold et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)), with planned interim analysis at 12 months for futility and safety.\u003c/p\u003e \u003c/div\u003e"},{"header":"8. DISCUSSION","content":"\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e8.1 Protocol feasibility and trial logistics\u003c/h2\u003e \u003cp\u003eWe estimate overall MATIN-2 protocol feasibility at approximately 65\u0026ndash;75% \u0026mdash; meaning that probability that the full trial, if conducted as designed with adequate recruitment and adherence, would generate interpretable data showing a clinically meaningful C-peptide signal. Several factors support this estimate. Teplizumab's safety profile is established and regulatory precedent exists. ILIT is technically straightforward and well-tolerated. IL-2 (rezpegaldesleukin) is in active clinical development with a defined Treg-selective dosing range. The main risks are recruitment rate (recent-onset T1D with HLA typing is a narrow window) and the inherent uncertainty around the teplizumab\u0026ndash;ILIT timing hypothesis, which has not been directly tested in humans. That is the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e8.2 Acknowledged limitations\u003c/h2\u003e \u003cp\u003eRecruitment feasibility depends on identifying patients within 12 months of diagnosis with sufficient residual C-peptide \u0026mdash; a selection criterion that matches approximately 40\u0026ndash;50% of newly diagnosed adults in TrialNet registry data. Based on enrolment rates from comparable recent-onset trials (TrialNet TN-10, DEFEND-1), we project 18\u0026ndash;24 months to achieve 90 participants across 5\u0026ndash;8 academic diabetes centres. This is achievable but not comfortable. Site selection should prioritise centres with existing teplizumab administration experience and ultrasound-guided procedure capability. Patient compensation for the 14-day Phase 0 infusion period will be important for retention; we recommend a dedicated trial co-ordinator model rather than relying on standard clinic visits for protocol fidelity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e8.3 Statistical analysis plan and subgroup analyses\u003c/h2\u003e \u003cp\u003eThe statistical analysis plan pre-specifies a modified intention-to-treat population (all randomised patients receiving at least one Phase 1 ILIT injection) as the primary analysis set, with per-protocol sensitivity analysis. Mixed-model repeated-measures analysis (MMRM) will handle the longitudinal C-peptide endpoint, with treatment arm, visit, and their interaction as fixed effects, baseline C-peptide and HLA stratum as covariates, and patient as a random effect. Multiple imputation will handle missing data under a missing-at-random assumption, with tipping-point analysis for sensitivity. Immunological endpoints will be analysed descriptively with pre-specified responder definitions. The pre-specified HLA-stratified subgroup analysis (DR3-DQ2 vs DR4-DQ8) is powered as exploratory, not confirmatory \u0026mdash; a distinction that must be maintained in any publication to avoid overinterpretation of subgroup effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e8.4 Ethics and dissemination\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003ewill be obtained from the relevant institutional review board (IRB) and/or national ethics committee prior to participant enrolment at each participating site. The MATIN-2 trial will be conducted in full accordance with the Declaration of Helsinki (2013 revision) and ICH Good Clinical Practice (GCP) E6(R2) guidelines. All participants will provide written informed consent before any study-related procedures are performed. Participant confidentiality will be maintained throughout; data will be pseudonymised and stored in compliance with applicable data protection legislation. The trial will be prospectively registered on ClinicalTrials.gov prior to first participant enrolment. Protocol amendments will be submitted to the ethics committee and regulatory authority as required and communicated to participating sites. Results will be reported regardless of direction of effect, in accordance with CONSORT and SPIRIT guidelines, and made available via open-access publication and preprint deposition.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"9. CONCLUSION","content":"\u003cp\u003eT1D does not have to be permanent. That is the central claim of this paper \u0026mdash; and it is grounded not in optimism, but in a careful synthesis of converging evidence from antigen-specific tolerance, intralymphatic delivery, regulatory T-cell biology, and anti-CD3 pharmacology. Each of these fields has independently generated proof-of-concept data suggesting that autoreactive T-cell activity can be reduced, that regulatory populations can be expanded, and that beta-cell function can be preserved \u0026mdash; if the right intervention is delivered in the right context at the right time. MATIN-2 is built on that 'if'. It operationalises the timing, the context, and the antigen specificity in a single sequential protocol and provides the mechanistic rationale needed to take it into a rigorous clinical trial.\u003c/p\u003e \u003cp\u003eWe do not claim that MATIN-2 will work. We claim that it should be tested \u0026mdash; and that the evidence base justifies a well-designed phase II trial with adequate power and a three-arm comparator structure that can separate the contributions of each protocol component. The risks are manageable. The intervention components are individually approved or in late-stage development. The endpoint (C-peptide AUC) is validated, clinically meaningful, and sensitive enough to detect the partial but durable tolerance we expect \u0026mdash; not complete cure, but the kind of sustained beta-cell preservation that translates directly into reduced hypoglycaemia, lower insulin burden, and improved quality of life for patients who currently have no disease-modifying option beyond insulin replacement.\u003c/p\u003e \u003cp\u003eThe immunological window that teplizumab opens is real, finite, and \u0026mdash; until now \u0026mdash; unexploited. MATIN-2 proposes to exploit it. If the protocol succeeds, it will establish a new standard of care for recent-onset T1D and provide a mechanistic template for combining immune modulation with antigen-specific re-education in other HLA-associated autoimmune diseases \u0026mdash; rheumatoid arthritis, multiple sclerosis, coeliac disease. If it fails, the three-arm design will tell us why: whether teplizumab alone is sufficient, whether antigen delivery is the limiting factor, or whether the timing window hypothesis requires revision. Either outcome advances the field. That is the mark of a well-designed trial \u0026mdash; and the standard to which MATIN-2 holds itself.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAK conceived the MATIN-2 protocol, designed the systematic review, conducted literature screening and data extraction, performed quality assessment, synthesised evidence, and drafted the manuscript. IE contributed to protocol review and critical revision of the manuscript. Both authors approved the final version for submission. AK is the guarantor of this work. Author contributions are reported using the CRediT (Contributor Roles Taxonomy): AK \u0026mdash; Conceptualisation, Methodology, Formal Analysis, Investigation, Data Curation, Writing (Original Draft), Writing (Review \u0026amp; Editing), Visualisation, Project Administration. IE \u0026mdash; Writing (Review \u0026amp; Editing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the clinical and research communities whose published work forms the evidence base for this protocol. No institutional support was received for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. Abdullah Kars is affiliated with Kara Harp Okulu (Turkish Military Academy), Ankara, Turkey. Ismihan Ersun is affiliated with Orman Bakanlığı, Ankara, Turkey. Neither author has financial relationships with pharmaceutical companies developing any of the agents described in this protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The work was conducted in the authors\u0026apos; own time without institutional support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data underpinning this systematic review and protocol are derived from published sources. The PROSPERO registration and uploaded protocol are available at https://www.crd.york.ac.uk/PROSPERO/view/CRD420261394024. The medRxiv preprint is available at MEDRXIV/2026/353036.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang L, Lovejoy NF, Faustman DL. Persistence of prolonged C-peptide production in type 1 diabetes as measured with an ultrasensitive C-peptide assay. Diabetes Care. 2012;35(3):465\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeete P, Willcox A, Krogvold L, Dahl-J\u0026oslash;rgensen K, Foulis AK, Richardson SJ, et al. Differential insulitic profiles determine the extent of beta-cell destruction and the age at onset of type 1 diabetes. Diabetes. 2016;65(5):1362\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor PN, Uttley L, Rees A, et al. C-peptide and metabolic outcomes in trials of disease modifying therapy in new-onset type 1 diabetes: an individual participant meta-analysis. Lancet Diabetes Endocrinol. 2023;11(12):885\u0026ndash;95. PMID: 37931637.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePescovitz MD, Greenbaum CJ, Krause-Steinrauf H, Becker DJ, Gitelman SE, Goland R, et al. Rituximab, B-lymphocyte depletion, and preservation of beta-cell function. N Engl J Med. 2009;361(22):2143\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerold KC, Bundy BN, Long SA, Bluestone JA, DiMeglio LA, Dufort MJ, et al. An anti-CD3 antibody, teplizumab, in relatives at risk for type 1 diabetes. N Engl J Med. 2019;381(7):603\u0026ndash;13. PMID: 31180194.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerold KC, Gitelman SE, Willi SM, Gottlieb PA, Waldron-Lynch F, Devine L, et al. Teplizumab and beta-cell function in newly diagnosed type 1 diabetes (PROTECT). N Engl J Med. 2023;389(23):2151\u0026ndash;61. PMID: 37861217.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerold KC, Gitelman S, Gottlieb P, Knecht LA, Schafer JL, Zipris D, et al. Partial exhaustion of CD8 T cells and clinical response to teplizumab in new-onset type 1 diabetes. Sci Immunol. 2016;1(5):eaai7793.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLudvigsson J, Faresj\u0026ouml; M, Hjorth M, Axelsson S, Ch\u0026eacute;ramy M, Pihl M, et al. GAD treatment and insulin secretion in recent-onset type 1 diabetes. N Engl J Med. 2008;359(18):1909\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhadj Ali M, Liu YF, Arif S, Tatovic D, Shariff H, Gibson VB, et al. Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes. Sci Transl Med. 2017;9(402):eaaf7779. PMID: 28794283.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkyler JS, Krischer JP, Wolfsdorf J, Cowie C, Palmer JP, Greenbaum C, et al. Effects of oral insulin in relatives of patients with type 1 diabetes: the Diabetes Prevention Trial\u0026ndash;Type 1. 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PMID: 30569273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaldmann H, Adams E, Cobbold S. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol Rev. 2006;212:301\u0026ndash;13. PMID: 16903922.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartemann A, Bensimon G, Payan CA, Jacqueminet S, Tengan F, Loustalot-Forget T, et al. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2013;1(4):295\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenzwajg M, Lorenzon R, Cacoub P, Pham HP, Pitoiset F, El Soufi K, et al. Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial. 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Sci Transl Med. 2015;7(315):315ra189.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoep BO, Thomaidou S, van Tienhoven R, Zaldumbide A. Type 1 diabetes mellitus as a disease of the beta-cell (do not blame the immune system alone). Nat Rev Endocrinol. 2021;17(3):150\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDayan C, Korah M, Tatovic D, Bundy BN, Herold KC. Changing the landscape for type 1 diabetes: the first step to prevention. Lancet. 2019;394(10205):1286\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Fern\u0026aacute;ndez S, Pujol-Autonell I, Mu\u0026ntilde;oz-Ruiz M, Vives-Pi M. A century later, still fighting back: antigen-specific immunotherapies for type 1 diabetes. Immunol Cell Biol. 2021;99(4):348\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon Herrath M, Bain SC, Bode B, Clausen JO, Desouza C, \u0026Eacute;sik O, et al. Anti-interleukin-21 antibody and liraglutide for the preservation of beta-cell function in adults with type 1 diabetes. Lancet Diabetes Endocrinol. 2021;9(4):212\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInternational Diabetes Federation. IDF Diabetes Atlas, 10th edn. Brussels: IDF; 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSernova Corp. Cell pouch system for transplantation of pancreatic islets in type 1 diabetes. ClinicalTrials.gov: NCT03513939.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVertex Pharmaceuticals. A study evaluating the safety and efficacy of VX-880 in participants with type 1 diabetes mellitus (FORWARD). ClinicalTrials.gov: NCT04786262.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9702744/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9702744/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Type 1 diabetes (T1D) affects 8.4 million people worldwide and is driven by HLA-restricted autoreactive T-cell destruction of pancreatic beta cells. Despite teplizumab's regulatory approval in 2022, no curative therapy exists. Individual immunological interventions — antigen-specific tolerance induction, intralymphatic peptide delivery, low-dose IL-2, and anti-CD3 therapy — each demonstrate partial efficacy but none achieves durable remission as monotherapy.\u003c/p\u003e\n\u003cp\u003eObjective: To systematically synthesise evidence across four immunological intervention domains and derive a mechanistic rationale for a novel sequential combinatorial protocol targeting recent-onset T1D with preserved beta-cell function.\u003c/p\u003e\n\u003cp\u003eMethods: We searched MEDLINE, Embase, and the Cochrane Library from inception through December 2024 without language restriction. Studies were screened against pre-specified PICO criteria. Methodological quality was assessed using Cochrane RoB-2 and the Newcastle–Ottawa Scale; certainty of evidence was graded using GRADE. This review is registered on PROSPERO (CRD420261394024).\u003c/p\u003e\n\u003cp\u003eResults: From 2,300 identified records, 1,847 were screened after deduplication; 112 studies met inclusion criteria. Evidence converged across domains: anti-CD3 therapy (31 studies) delays T1D onset; antigen-specific peptide immunotherapy (38 studies) achieves partial immune re-education; intralymphatic delivery (19 studies) provides a 1,344-fold efficiency advantage over subcutaneous routes; low-dose IL-2 (24 studies) selectively expands regulatory T cells without systemic immunosuppression. No published study has combined all four interventions sequentially.\u003c/p\u003e\n\u003cp\u003eProposed Protocol: MATIN-2 (Multi-Antigen Tolerogenic Immunotherapy with INtralymphatic delivery) is a three-phase sequential protocol. Phase 0 delivers teplizumab (14-day IV course) to create a tolerogenic window via partial T-cell exhaustion. Phase 1 administers HLA-stratified peptide antigens (GAD65₅₅₅₋₅₈₀ or proinsulin C19-A3) intralymphatically alongside low-dose rezpegaldesleukin. Phase 2 consolidates tolerance with quarterly maintenance injections. Primary endpoint: stimulated C-peptide AUC at 24 months (≥50% preservation vs baseline). Projected efficacy: 60–75% C-peptide preservation in adults with recent-onset T1D (diagnosis within 12 months; C-peptide ≥0.2 nmol/L).\u003c/p\u003e\n\u003cp\u003eConclusions: MATIN-2 provides a biologically coherent sequential immunotherapy protocol grounded in replicated mechanistic evidence. A three-arm, randomised, double-blind, placebo-controlled phase II trial (MATIN-2 full protocol vs. teplizumab alone vs. placebo; n=90) is proposed. PROSPERO: CRD420261394024. ClinicalTrials.gov registration will be completed prior to first participant enrolment.\u003c/p\u003e","manuscriptTitle":"Sequential Immune Modulation and Antigen-Specific Tolerance Induction for Disease Modification in Recent-Onset Type 1 Diabetes: A Mechanistic Framework and Protocol Proposal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 02:37:53","doi":"10.21203/rs.3.rs-9702744/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a1b202f-d0a0-49b9-9e29-d7e56207bd80","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"editorAssigned","content":"","date":"2026-05-15T06:19:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-15T06:19:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Trials","date":"2026-05-13T10:36:11+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T02:37:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 02:37:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9702744","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9702744","identity":"rs-9702744","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}