Introduction:

Regulatory T cell (Treg) therapy has been extensively studied as a strategy to induce immune tolerance in various types of organ transplantation. However, only a few studies have evaluated the therapeutic effects of locally administered Tregs or compared outcomes based on different routes of administration. In this study, we investigated how the route of Treg delivery influences the local immune microenvironment surrounding the graft in a subcutaneous islet transplantation model using syngeneic mice.

Methods:

Following our previously established protocol, neovascularization was induced in the subcutaneous dorsal region of recipient C57BL/6 mice two weeks before transplantation to prepare the transplant bed. A total of 500 syngeneic islets were subcutaneously transplanted. On the day of transplantation, 1.0 × 10⁶ syngeneic Tregs were administered either intravenously via the tail vein (systemic group: B) or co-transplanted locally around the graft (co-transplant group: C). Graft outcomes and the local immune microenvironment were evaluated and compared between the two delivery routes.

Results:

Treg-treated groups showed improved graft survival rates compared to the control group (Group A: 64%, Group B: 92.3%, Group C: 100%), and the time to graft engraftment was significantly reduced (median: Group A, 22 days; Group B, 23 days; Group C, 11 days; A vs. C: P = 0.0042, B vs. C: P = 0.0049; Steel-Dwass test). On day 5 post-transplantation, RNA-seq analysis of the tissue surrounding the graft identified 150 differentially expressed genes between the control and co-transplant groups, including 42 immune-related genes. In the co-transplant group, the expression of immunoregulatory antigen-presenting cell–associated genes (Siglec15, Cd209e, and Clec4b1) was upregulated, while genes associated with inflammatory T cells (Trav11d, Trav6-5, and Lat), IFN-γ responses (Gbp5, Gbp10, Igtp, and Iigp1), and innate immunity (Defb1 and Klra9) were downregulated. In the immunoglobulin gene set, natural antibody–like clones, such as Ighv1-34, Igkv8-30, and Ighv1-69, were upregulated, whereas Igkv6-20, Igkv4-90, and Ighv1-81 were downregulated. Additionally, TCRβ variable region genes (Trbv20, Trbv2, and Trbv1) were upregulated, while TCRα variable region genes (Trav11d and Trav6-5) were downregulated, suggesting selective reshaping of the T-cell receptor repertoire.

Conclusions:

Local Treg administration suppressed the expression of genes involved in inflammation and innate immune activation while inducing transcriptional changes associated with the re-establishment of immune homeostasis. These modifications to the local immune microenvironment may have contributed to enhanced graft engraftment.