Administration Of α-GalCer After Allogeneic Bone Marrow Transplantation Enhances Immune Reconstitution Without Aggravating Acute Graft-versus-host Disease

Objective:research on immune reconstitution and acute graft-versus-host disease(aGVHD) after hemopoietic stem cell transplantation(HSCT) is the hot in area.NKT cells are a distinct subset of T lymphocytes,as one kind of new regulatory cells.α-GalCer(α-galactosylceramide) can recognize and activate NKT cells,so induces NKT cells rapidly secrete large amounts of IFN-γ「and IL-4.We investigate whetherα-GalCer could enhance immune reconstitution without aggravating aGVHD in an experimental model of HSCT,and futher explore the mechanism.Methods:1.C57BL/6 mice were as donors,and BALB/c mice as recipients,two HSCT model with different severity of aGVHD were estabilished. 2.In HSCT model with lighter aGVHD,mice of experimental group were injected intraperitoneally withα-GalCer immediately after HSCT,while mice of control group received the diluent only.Splenocyte counts,CD3~+,CD4~+,CD8~+,B220~+, CD40~+,CD86~+,CD11c~+ and CD80~+ cell relative counts,donor chimerism, thymocyte counts,thymocyte constitute and hemological reconstitution were compared at the same time post transplantation between two groups,and we performed peripheral blood mobilized stem cell colony forming unit(CFU) assays in methylcellulose in vitro and lymphocyte proliferation assay in vitro and in vivo.3.In two HSCT model,Survival,clinical and pathological aGVHD,and donor chimerism were evaluated between two groups of each HSCT model,and serum 6 hours post transplantation and splenocyte cultural supernant day 27 post transplantation were examined by ELISA for cytokines of IL-4,IL-2,1L-10 and IFN-γ,and migration assays spleen,thymus,periphral blood and LN monocyte were performed in vitro and in vivo.Results:1.Two HSCT models with different severity of aGVHD were estabilished.2.In HSCT model of 1×10~7 splenocyte,on day 70 post transplantation,splenocyte counts,CD3~+,CD4~+,CD8~+,B220~+,CD40~+ and CD86~+ cell relative counts were obviously higher in experimental group than in control group.At the same time thymocyte counts and thymocyte constitute were no different between two groups,and both were obviously lower than normal mice.On day 7,donor chimerism was higher in experimental group than in control group.On day 3,hemological reconstitution was faster in experimental group than in control group,and CFU number per 10~3 CD34~+ cell was higher inα-GalCer culture group than in DMSO culture group.Lymphocyte proliferation assay in vitro and in vivo both showed thatα-GalCer promoted T cells and B cells proliferation.3.In two HSCT model,mice in experimental group both survived longer than in control group,and clinical and pathological aGVHD were lighter.4. In HSCT model of 1×10~7 splenocyte,IL-4 level of serum 6 hours post transplantation were no different among experimental group mice,normal mice injected withα-GalCer and mice irradiated and injected withα-GalCer,and all were higher than mice irradiated and injected with DMSO;IFN-γlevel were no different between experimental group mice and normal mice injected withα-GalCer,both were a litter higher than mice irradiated and injected with DMSO and obviously lower than normal mice injected withα-GalCer.On day 27 post transplantation,IL-4 and IL-10 level of splenocyte cultural supernant in experimental group were higher than in control group,while IFN-γand IL-2 level were lower.5.Migration assays in vitro and in vivo showed thatα-GalCer caused moncyte,CD3~+,CD4~+ and CD8~+ cell egress from thymus,spleen and peripheral blood to LN.Conclusions:1.Two HSCT models with different severity of aGVHD are established.2.α-GalCer accelerates immune reconstitution of T cells and B cells,and simultaneously enhances expression of the second stimulation signal CD86 and CD40.3.Acceleration of immune reconstitution byα-GalCer is correlated with enhancing early engraftment,hemopoietic stem cell proliferation and differation and proliferation of T cells and B cells byα-GalCer,which is independent of improving thymus.4.α-GalCer reduces aGVHD,which does not at the cost of preventing or impairing engraftment.One mechanism is that stimulation of host NKT cells through administration ofα-GalCer regulates acute aGVHD by inducing Th2 polarization of donor T cells.The other mechanism is thatα-GalCer causes T cells egress from thymus,spleen and peripheral blood to LN,which reduces T cells in circulating blood and prevents T cells from infiltrating into targets organ.