Protective Effects And Mechanism Of Tigit Signaling Pathway In Skin Allograft Rejection In Mice

Objective: Tigit,defined as a co-inhibitory receptor,is expressed on lymphocytes.Tigit is extensively found in tumor infiltrating lymphocytes.When engaged with CD155 on tumor,Tigit signaling pathway is activated,leading to the immune tolerance of the tumor.In autoimmune diseases and chronic infection,inflammatory response and progression of these diseases would be relieved when Tigit signaling pathway is activated.However,the study of Tigit signaling pathway is poorly understood in graft rejection response.We hypothesize that the expression of Tigit might be up-regulated during graft rejection response.While the activation of Tigit signaling pathway would negatively regulate T lymphocyte function during activation and proliferation,causing a decrease of IFN-? in CD4+ T lymphocytes,which might cause relief of graft rejection and,as a result,protecting allograft from immune damage.Methods:(1).A MHC fully mis-matched allogenic BALB/c(H-2d)to C57BL/6(H-2b)skin allograft transplantation in mice was established.On day 7 post-transplantation,the expression of Tigit was detected on splenic lymphocytes by flow cytometry in two groups---allograft rejction group(graft rejection group),isogeneic graft control group(control group).The relative expression of Tigit m RNA in spleen of the above two groups was assayed by RT-PCR.(2).In vitro,splenic lymphocytes,in both graft rejection group and control group,were cultured for 24 hours in four following groups: blank group,CD155 group,PHA group,both CD155 and PHA group.Cell pellets and supernatant were collected separately for testing by flow cytometry and ELISA.Levels of IFN-? and IL-6 in the supernatant was determined.(3)Lymphocytes in the above two groups labeled with CFSE were co-cultured with mouse anti-CD3 and anti-CD28 antibodies for 72 hours,andwere divided into two groups,mouse anti-CD3 + anti-CD28 antibodies + recombinant CD155 protein or only mouse anti-CD3 + anti-CD28 antibodies added.The proliferation of lymphocytes was assayed by flow cytometry.(4).In vivo,a fully MHC-mismatched allogenic BALB/c(H-2d)to C57BL/6(H-2b)mouse skin transplantation was established.Mouse recombinant CD155 protein or PBS was respectively injected to CD155 treated group(CD155 group)and PBS group(PBS group)intraperitoneally to mice.Skin allografts were obtained post-operatively after a week.Grafts were stained with H&E and evaluated with rejecting degree.Splenic lymphocytes were assayed by flow cytometry to test the levels of IFN-?,CD25,CD69 in CD4+ T cells.Average survival time was recorded according to skin grafts totally rejected in two groups above.Results:(1).In mouse skin transplantation,the expression of Tigit was increased on CD4+ T cells and CD8+ T cells but decreased on NK cells(**P<0.01).When activated with PHA,CD4+ Tigit+ T cells showed less expression of IFN-?,CD25 and CD69 compared with CD4+ Tigit-T cells in both graft rejection group and control group(*P<0.05).The relative expression of Tigit m RNA in graft rejection group was elevated slightly compared with control group(*P<0.05).(2).In vitro,when recombinant CD155 protein was added to splenic lymphocytes activated by PHA,the levels of IFN-? and IL-6 in supernatant were relatively lower(***P<0.001)than the group with PHA added.By flow cytometry,levels of IFN-?,CD25 and CD69 in both CD155 and PHA group were decreased when compared with PHA added only(*P<0.05).(3).Proliferation assay was conducted with anti-CD3 e and CD28 cultured with CFSE-labeled splenic lymphocytes for 72 hours.When CD155 was added,there was only a slight decrease in proliferation(P>0.05).(4).In vivo,the percentage of IFN-?,CD25 and CD69 were proved lower when CD155 protein was injected(**P<0.01).H&E staining showed pathological injury in grafts was relieved in the CD155 treated group when compared with PBS group(*P<0.05).But the survival time of skin graft was not prolonged in both groups.Conclusions: The expression of Tigit was elevated in activated lymphocytes,both on CD4+ T cells and CD8+ T cells.Activating Tigit signal pathway could reduce the expression of IFN-? in CD4+ T cells effectively,suppresses the activation of CD4+ T cells effectively and relieve skin graft rejection and as a result,playing a protective role in skin allograft rejection in mice.