Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation

Type 1 diabetes (T1D) is an autoimmune disease where the body's immune system attacks and destroys insulin-producing pancreatic islet cells. Current treatments focus on managing symptoms with insulin injections, but these don't address the underlying immunopathology and come with associated comorbidities. While islet transplantation can restore insulin production, it requires lifelong immunosuppression to prevent rejection. A promising alternative is inducing antigen-specific tolerance, selectively inhibiting harmful immune responses without compromising protective immunity. However, current methods struggle to precisely target and integrate immune signals, resulting in limited efficacy and systemic immunosuppression. This research investigates a novel intra-lymph node (iLN) injection strategy to address these limitations. The approach utilizes biodegradable microparticles (MPs) that deliver self-antigens and the immunomodulatory drug rapamycin directly to lymph nodes, creating a localized tolerogenic environment. This targeted delivery aims to promote antigen-specific tolerance, avoiding the widespread immunosuppression associated with systemic therapies.

Existing therapies for autoimmune diseases typically involve systemic immunosuppression, which isn't curative and has significant side effects. Even targeted therapies like anti-CD3 monoclonal antibodies, while showing promise in delaying disease progression in T1D, are not curative and lack complete specificity. Islet transplantation offers a potential cure but requires potent immunosuppression to prevent rejection. The concept of antigen-specific tolerance, where the immune system is trained to ignore specific self-antigens, is a promising alternative. Several strategies, focusing on generating regulatory T cells (Tregs), have been explored. These strategies often involve delivering autoantigens with regulatory cues to modulate T cell responses. However, achieving effective signal integration in lymph nodes, crucial for T cell fate determination, remains a major challenge. Recent studies have shown the potential of iLN injection of soluble vaccines; however, these signals are quickly cleared from lymph nodes. The current study uses a novel system using iLN injection of MPs, which are designed to slowly release antigens and regulatory cues, creating a local depot of immune signals in the lymph nodes.

The researchers synthesized poly(glycolide-co-lactide) (PLGA) MPs encapsulating rapamycin (Rapa) and three distinct peptide antigens: NRP-V7 (CD8-mediated T1D), p31 (CD4-mediated T1D), and Ea (alloantigen in islet transplantation). They characterized MP properties (Supplementary Table 1) and assessed their effects in vitro. In co-cultures of dendritic cells (DCs) and antigen-specific T cells, MPs containing both antigen and Rapa promoted Treg expansion and reduced inflammatory cytokine production (IFNγ, IL-17) compared to antigen-only MPs. In vivo studies involved two T1D models: an accelerated model using adoptive transfer of activated antigen-specific T cells, and a spontaneous model in NOD mice. They also used a full MHC-mismatched islet transplantation model in STZ-treated mice. Mice received a single iLN injection of MPs before or after disease induction. Blood glucose levels were monitored to assess disease progression and survival. Researchers analyzed lymph nodes for antigen-specific Treg expansion, mTOR activity (using ps6), antigen presentation (using Y-Ae mAb), and structural changes (laminin α4/α5 ratio) using flow cytometry and immunohistochemistry. The impact of APC migration on antigen presentation was investigated using FTY720, an S1P receptor antagonist. Finally, they examined the expression of memory markers (CD44, CD62L, CCR7, Bcl-2, CD127) on antigen-specific Tregs to assess the durability of tolerance.

In both T1D models and the islet transplantation model, MPs containing both antigen and Rapa significantly prevented or delayed disease onset, with single treatment sufficient for long-term protection in certain models. Antigen alone was insufficient; rapamycin alone provided limited, non-durable effect. Treatment expanded antigen-specific Tregs in both treated and untreated lymph nodes, suggesting systemic effects from localized treatment. The MPs were retained locally, but antigen presentation occurred in both treated and untreated lymph nodes, independent of APC migration, likely due to lymphatic drainage. Importantly, treatment led to structural changes in lymph nodes, characterized by an increased laminin α4:α5 ratio, associated with a tolerogenic environment. Antigen-specific Tregs in treated mice exhibited enhanced memory markers (CD44, CD62L, CCR7, Bcl-2, CD127), suggesting a more durable, long-lasting tolerogenic response. Rapamycin's role appeared to be in modulating mTOR signaling during T cell priming. The study also observed that while the antigen alone expanded antigen-specific T cells in both treated and untreated lymph nodes, the addition of Rapamycin was necessary to polarize these cells towards a Treg phenotype.

This study demonstrates the power of targeted lymph node manipulation to induce antigen-specific tolerance. The iLN delivery of MPs containing both antigen and Rapa offers several advantages over existing systemic therapies. The localized delivery minimizes systemic side effects, and the sustained release of both antigen and Rapamycin creates a local microenvironment that promotes durable tolerance. The expansion of antigen-specific Tregs in both treated and untreated lymph nodes suggests a systemic effect that extends beyond the injection site. The finding that antigen is presented in untreated lymph nodes independent of APC migration points to lymphatic drainage as a key mechanism for achieving systemic tolerance. The observed structural changes in the lymph nodes, and the increased expression of memory markers on Tregs indicate a mechanism leading to long-lasting protection from autoimmune attack.

This research successfully demonstrated that the iLN delivery of tolerogenic MPs, co-encapsulating self-antigens and rapamycin, is a highly effective strategy for inducing antigen-specific tolerance in pre-clinical models of T1D and islet transplantation. This approach offers a potential new therapeutic avenue with enhanced efficacy and reduced side effects compared to traditional systemic immunosuppressive treatments. Future studies could explore optimizing MP formulation, investigating various lymph node injection sites, and examining long-term effects in spontaneous T1D models to advance this approach toward clinical translation.

The study primarily used pre-clinical mouse models, and the findings need to be validated in human clinical trials. While the results demonstrate durable tolerance in some models, longer-term follow-up would strengthen the conclusions. The mechanism of action, specifically the interplay between antigen-specific Tregs and inflammatory T cells, requires further investigation. Finally, the optimal formulation and dosage of MPs may vary depending on the specific antigen and disease context.