Graft-Specific Regulatory T Cells for Long-Lasting, Local Tolerance Induction

Abstract

Background: Solid organ transplantation is hindered by immune-mediated chronic graft dysfunction and the side effects of immunosuppressive therapy. Regulatory T cells (Tregs) are crucial for modulating immune responses post-transplantation; however, the transfer of polyspecific Tregs alone is insufficient to induce allotolerance in rodent models.

Methods: To enhance the efficacy of adoptive Treg therapy, we investigated different immune interventions in the recipients. By utilizing an immunogenic skin transplant model and existing transplantation medicine reagents, we facilitated the clinical translation of our findings. Specifically, antigen-specific Tregs were used.

Results: Our study demonstrated that combining the available induction therapies with drug-induced T-cell proliferation due to lymphopenia effectively increased the Treg/T effector ratios. This results in significant Treg accumulation within the graft, leading to long-term tolerance after the transfer of antigen-specific Tregs. Importantly, all the animals achieved operational tolerance, which boosted the presence of adoptively transferred Tregs within the graft.

Conclusions: This protocol offers a means to establish tolerance by utilizing antigen-specific Tregs. These results have promising implications for future trials involving adoptive Treg therapy in organ transplantation.

Keywords: alloantigen-specific; immune tolerance; regulatory T cells; solid organ transplantation.

Figure 1

Allospecific cTregs prolonged allograft survival. (A) Schematic structure of skin grafting in the lymphopenic model. All BALB/c-RAG1^−/− recipients were reconstituted with 5 × 10⁵ BALB/c CD4⁺CD45RB^hi cells with or without additional Tregs on day −1. On day 0, C57Bl/6 skin was grafted onto the recipients. (B) Survival of syngeneic grafts (n = 4), allogeneic grafts without Tregs (n = 12), allogeneic grafts with nTregs (n = 3), Thy1.1 control vector-transduced cells (cTeffs; n = 7), allospecific cTregs (n = 30), and allospecific F1 cTregs (n = 6). (C) IFN-γ recall ELISPOT assays were performed with responder splenocytes from naïve, tolerant, or graft-rejected mice mixed with irradiated stimulator cells (cells from either naïve C57Bl/6J or naïve third-party C3H/HeJ mice). (D) Survival of third-party grafts (n = 3) and allogeneic grafts without Tregs (n = 12; from (B)).

Figure 2

Local, but not systemic, tolerance restrained graft rejection. (A) H&E staining of explanted, tolerant syngeneic (left) or allogeneic skin with Tregs (right) or rejected allogeneic skin without adoptively transferred Tregs (middle). (B) IHC image of explanted, tolerant skin after 100 days (40×). Stainings for DAPI (white), CD4 (green), thy1.1 (red), and Foxp3 (blue) are shown. (C) The quantification of (B) is shown in these bar graphs. (D) Tolerant skin samples were regrafted onto reconstituted BALB/c-RAG1^−/− mice (re-Tx-tolerant skin, n = 2, p = 0.016) or reconstituted with tolerized splenocytes (n = 3, p = 0.38) before skin transplantation. (E) Flow cytometric analysis of T cells from inside the explanted graft.

Figure 3

Allospecific cTregs induced long-term tolerance in an immunocompetent skin transplant model after niche generation. (A) Schematic representation of skin grafting in an immunocompetent mouse model. (B) Effects of rapamycin (n = 7), anti-Thy1.2 antibodies (n = 9), rapamycin with anti-Thy1.2 antibodies (n = 12), and cTregs with rapamycin and anti-Thy1.2 antibodies (n = 5) on allogeneic graft survival in the immunocompetent skin Tx model.