Donor-Specific Tolerance Induction through Expansion of DCregs and Tregs by a Liposomal Formulation of KRN7000 (RGI-2001).

Pharmacological induction of donor-specific tolerance would provide significant benefits in both organ transplantation and bone marrow transplantation settings. We investigated the ability of alpha-galactosylceramide (α-GC) in inducing donor-specific tolerance when given in a liposomal formulation. α-GC is a ligand for CD1d molecules expressed on antigen-presenting cells. Upon presentation by CD1d to invariant natural killer T (iNKT) cells, α-GC induces the rapid release of Th1, Th2, or immune regulatory cytokines and initiation of multiple downstream cellular events such as T cell polarization and expansion of dendritic cell subsets. Previously, using RGI-2001 (a liposomal formulation of a synthetic derivative of α-GC, KRN7000), we have demonstrated that the activation of iNKT cells by RGI-2001 induces expansion of regulatory dendritic cells (DCreg) and subsequent generation of antigen-specific Foxp3⁺ regulatory T (Treg) cells in the presence of target antigens. In the present study, we examined the effects of RGI-2001 on immune responses against alloantigens using a murine in vivo experimental system. In brief, Balb/c (H-2d) recipients were primed with 5x10e6 C57BL/6 (H-2b) whole spleen cells (WSC) with varying doses of RGI-2001 (0.002 to 20 μg/mouse) given intravenously. Seven days later, WSC from the Balb/c recipients were examined for their cellular composition by FACS analysis. Subtle but reproducible dose-dependent increases were noted in the percentage of the Lin^negCD11c^intCD45RB⁺ dendritic cell (DC) population, known to be enriched for regulatory DC (DCreg). Since RGI-2001 induces an increase in the total spleen cells, the absolute DCreg cell numbers in RGI-2001-treated spleen increased in a statistically significant manner as compared with untreated controls. As for the percentages of CD4⁺Foxp3⁺ Treg cells, no apparent differences were observed. However, an analysis using the Ki67 cycling cell specific nuclear marker revealed a clear dose-dependent increase in the cycling cell fraction among CD4⁺Foxp3⁺ Treg cells. These results together confirmed that RGI-2001 induces expansion of DCregs and Tregs. Next, we investigated the effects of RGI-2001 on immune responses against the donor alloantigens. The WSC of the recipient mice were restimulated in vitro with the mitomycin C-treated, T cell depleted donor (C57BL/6) WSC, and the levels of proliferation were measured by MTT colorimetric assay (one-way MLR). It was found that RGI-2001 treatment reproducibly and significantly suppressed proliferation of host WSC in response to donor alloantigens. A dose of 2μg/mouse of RGI-2001 induced in average ~30% reduction in host WSC proliferation. IL-2 production was also reduced to ~50%, further indicating the suppressive effects of RGI-2001. Notably, it was confirmed that suppressive effect of RGI- 2001 was restricted to the responses towards donor specific alloantigens, as no suppression in proliferation nor IL-2 production was noted when a third party (C3H) WSC was used as the stimulators. Collectively, the results suggest that RGI-2001, when administered together with allogeneic donor cells, can induce donor specific tolerance by expanding DCregs and inducing antigen-specific Tregs. RGI-2001 may have a potential to be a novel therapy to prevent organ rejection as well as GvHD in bone marrow transplantation.