Mechanism Of Action Of TSC1 Of Dendritic Cells On Antigen-specific Initial T Cell Activation And Proliferation

Background:Dentritic cells are pivotal cells which can induce T cells activate and proliferate.Tuberous sclerosis complex 1(Tscl)is a crucial upstream negative regulator of mammalian target of rapamycin(mTOR).Recent studies mostly highlight the role of Tsc1 in the development of DCs,but whether or how in vivo Tsc1 is directly regulate the function of mature DCs is still not very clear.Objectives:This study focused on exploring how Tscl expressed by DCs regulate T cell activation and proliferation.Methods:(1)Tsc1f/f mice were crossed with CD11cCre transgenic mice to generate CD11cCreTsc1f/f(KO mice)and Tsclf/f mice(WT mice).CD 11 cCreTsc1f/f mice were crossed with Raptorrf/f mice to generate CD11cCreTsc1f/f Raptorf/+ mice.We tested Tsc1 gene's knocking out at the genetic level through ordinary PCR and protein level through westernblot.(2)Activation of T cells and expression of Ki67 and cytokines were detected by flow cytometry.(3)Proliferation of T cells were detected by flow cytometry with or without blocking Nrpl after utilizing the in vitro DCs naive T-cell co-culture system.;expression of Nrpl were detected by flow cytometry.(4)The signaling pathway alterations were explored by flow cytometry.Results:(1)Tscl gene were knocked out successfully at the genetic level and protein level.(2)After Tscl knocking out,activation,proliferation and cytokines of KO mice splenic CD4+T cells and CD8+T cells were increased.(3)After Tscl were deleted,naive T-cell proliferation induced by KO DCs and the expression of Nrp1 by KO DCs were increased;blocking Nrpl,the enhanced priming of naive CD4+ T cells by KO DCs were partly dampened.(4)In KO mice DCs,the activation of S6K and PPAR-? were obviously increased;in CD11cCreTsc1f/f Raptor f/+ DCs,the expression of Nrp1 and the activation of T cells were largely rescued,the expression of mTORCl were decreased and inhibiting PPAR-?,the expression of Nrpl were decreased in KO DCs.Conclusion:(1)We successfully generated a mouse line with Tscl conditionally depleted in DCs.(2)Tscl in DCs prevents T cells from activating spontaneously in vivo.(3)Tscl represses Nrpl in DCs to prevent naive T-cell from proliferating.(4)Tscl can represse Nrpl expression through inhibiting mTORC1-PPAR-? pathway.Objective:To explore the mechanisms modulating the function of dendritic cells(DCs)and suppressing the T cell activation and proliferation by transforming growth factor-(3(TGF-?).Methods:Mouse splenic DCs were purified with CD11c+ immunomagnetic beads,and the purity of isolated DCs were examined by flow cytometry.After stimulation with TGF-?,gene chip detected DCs gene expression then real-time PCR determined the mRNA levels of variable genes in microarray.The activation and proliferation of CD4+ T cells which were co-cultured with DCs after stimulation with TGF-? were detected by flow cytometry.Results:The purity of DCs reached over 95%after isolation.TGF-? down-regulated the expression of cell surface markers CD53?CD69?CD33?CD74 and CD93;decreased the expression of chemokines Ccl3?Ccl5?Ccl9?Ccl6?Ccl17?Cxcl/10?Ccl22?Ccl4?Ccr7?Ccl2?Cxcl9 and Ccr7;reduced the expression of inflammatory cytokines IL-2ra?IL12-rb2?IL-15ra?IL-lb and IL-15.In addition,DCs mediated activation and proliferation of CD4+ T cells were suppressed.Conclusion:TGF-? suppressed the expression of DCs surface markers,chemokines,inflammatory cytomines and suppressed the proliferation and activation of CD4+T cells.