iNKT Cell Stimulation with Liposomal Alpha/Gal/Cer Amplifies Proliferation of Adoptively Transferred Regulatory T Cells In Vivo and Reduces GvHD

Cellular therapy with regulatory T cells (Tregs) has shown promising results for suppressing graft versus host diseases (GvHD) in animal models and phase I/II clinical trials. However, a paucity of Tregs in the peripheral blood makes it difficult to acquire sufficient numbers of cells and hampers further clinical applications. Invariant natural killer T (iNKT) cells are another compartment of regulatory cells that ameliorate GvHD by interacting with Tregs upon either transplantation with activation with α-galactosylceramide (αGalCer) or adoptive transfer of iNKT cells. The aim of this study was to explore the proliferation and anti-GvHD effect of Treg following stimulation of iNKT cells with αGalCer.We utilized a liposomal formulation of αGalCer that was found to have the potential of preventing GvHD in a recently published phase I/IIa clinical trial. Lethally (8.8Gy) irradiated BALB/c recipient mice were transferred with 5 x 10⁶T cell depleted bone marrow cells and 1 x 10⁶conventional CD4⁺and CD8⁺T cells (Tcons) from WT C57BL/6 donor mice, with or without 1 x 10⁵CD4⁺CD8^-CD25^hiFoxp3-GFP⁺cells (Tregs) that were freshly isolated from Foxp3-GFP⁺C57BL/6 mice. Immediately after cell injection, αGalCer-loaded or unloaded (Ctrl) liposomes were injected IV at 10 μg/kg. We utilized a subtherapeutic dose of Treg (1 x 10⁵) that did not provide survival benefit compared to those without Treg transfer. αGalCer injection alone did not result in improved survival, suggested that a single injection of αGalCer is not sufficiently potent to prevent GvHD. Mice transferred with a subtherapeutic dose of Treg and liposomal αGalCer injection showed significant improvement in survival.Tissue damage examined 10 days after BMT was less severe in the Treg plus αGalCer group compared to those of the other groups. We analyzed the Treg population among CD4⁺T cells in the spleen, superficial/mesenteric lymph nodes, and liver on day 10 following transplant. As αGalCer stimulation promoted both Tcon-derived (CD45.1⁺Foxp3⁺) and transferred (CD45.2⁺Foxp3-GFP⁺) Treg, the proportion of Treg in the mice treated with the combination of Treg plus αGalCer stimulation significantly increased in all organs compared to those of the other groups. We evaluated the transferred Tregs in vivo by bioluminescent imaging. Treg-signals in αGalCer treated mice did not show significant difference compared to those in Ctrl mice until day 7 and increased thereafter especially in the gastrointestinal tract and became significantly higher 2-3 weeks after BMT. Host iNKT cells rather than donor iNKT cells contributed more to GvHD suppression because survival benefit of αGalCer stimulation was not shown in the transplantation of WT C57BL/6 mice into Jα18^-/^- BALB/c mice, whereas it was in that from Jα18^-/^-C57BL/6 mice into WT BALB/c mice. In conclusion, liposomal αGalCer injection enhances Treg function through activation of host derived iNKT cells and could make Treg cell therapy more feasible and safer by reducing the requirement of Treg number.