Tim-1 blockade with RMT1-10 increases T regulatory cells and prolongs the survival of high-risk corneal allografts in mice

Abstract

Anti-Tim-1 monoclonal antibody (mAb) RMT1-10 is effective in promoting allograft survival through blocking Tim-1. However, its role in corneal transplantation is unclear. This study aims to evaluate the effect of RMT1-10 on high-risk corneal transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and intraperitoneally injected with RMT1-10 or isotype IgG. The transparency of corneal graft was evaluated by slit lamp biomicroscopy. Flow cytometry was used to determine the phenotype of CD4(+) T cells, including CD154, Tim-3, CD25 and Foxp3, and to analyze the proliferation capacity of CD4(+) T cells and the suppressive capacity of T regulatory (Treg) cells. The levels of interferon-gamma (IFN-γ), IL-4 and transforming growth factor-beta1 (TGF-β1) were investigated by intracellular staining and/or ELISA assay. The delayed-type hypersensitivity (DTH) response was evaluated by ear swelling assay. RMT1-10 therapy delayed the onset of rejection and significantly prolonged the survival of corneal allograft. In RMT1-10 treated mice, percentages of CD4(+)CD154(+) cells and CD4(+)Tim-3(+) cells were significantly decreased while the frequency of CD4(+)CD25(+)Foxp3(+) Treg cells was significantly up-regulated, compared with those of isotype IgG treated mice. And, in vitro proliferation of CD4(+) T cells was significantly inhibited by RMT1-10. In addition, percentage of intracellular expression of IFN-γ and IL-4 in CD4(+) T cells isolated from RMT1-10 treated mice was significantly reduced. After co-culturing with RMT1-10 in vitro, CD4(+) T cells produced significantly decreased levels of IFN-γ and IL-4 and significantly increased levels of TGF-β1. Furthermore, RMT1-10 inhibited DTH response of recipient mice and enhanced the suppressive capacity of Treg cells isolated from RMT1-10 treated mice. Our data indicate that Tim-1 blockade with RMT1-10 could suppress immunological rejection and prolong the survival of corneal allograft through regulating T cell responses.

Keywords: T regulatory cells; Tim-1; high-risk corneal allograft rejection; mice.