Study On The Mechanism Of FGL2 In The T Cell Response And The Cooperative Role Of TCF-1 And LET-1 In Regulating T_FH Cell Function

Follicular helper T cells(T_FH cells),a special subset of CD4+ helper T cells,plays critical roles in the establishment of germinal center?GC?reaction,the selection of GC B cells bearing high-affinity antigen receptors,and the differentiation of selected cells into memory B cells and long-lived plasma cells.T_FH differentiation is a multi-stage,multi-factor process including Bcl6,identified as a “master regulator”,and other transcription factor,such as TCF-1,IRF4,Batf,Ascl2,and STATs.Fibrinogen-like protein 2?FGL2?,also named Fibroleukin,can be expressed as a membrane-associated protein?mFGL2?and secreted protein?sFGL2?.mFGL2 can proteolyticly convert from prothrombin into thrombin.sFGL2 can inhibit the proliferation and function of CD8+ T cells in HBV patients,and inhibit the proliferation of T cells though keeping the suppression of Regulatory T cells?TReg cells?in vitro experience.However,the role of FGL2 in the T cell response in vivo remains unclear.T cell factor 1?TCF-1,encoded by Tcf7?and lymphoid enhancer-binding factor 1?LEF-1,encoded by Lef1?,the downstream effector transcription factors of the canonical Wnt pathway,have been known to be critical for the thymocyte development,differentiation of T cells,and maintenance of memory CD8+ T cells.LEF-1 and TCF-1 play a key role in the CD4-versus-CD8 lineage choice during the process of CD4+CD8+ double positive stage.In the mature CD8+ T cells,TCF-1 maintains the persistence of memory CD8+ T cells,and LEF-1,combined with TCF-1,cooperatively regulate the generation of memory precursors and protective memory CD8+ T cells.In the mature CD4+ T cells,TCF-1 promotes the differentiation of TH2 cells by up-regulated GATA-3.TCF-1 restricts the expression of IL-17 A during the developing thymocytes and activated CD4+ T cells.In addition,upon acute LCMV infection,TCF-1 initiates the differentiation of T_FH cells by directly regulated Bcl6-Blimp1 axis.However,during protein immunization,the role of TCF-1 and LEF-1 for the differentiation and function of T_FH cells remain unclear.Therefore,this research focus on the effect of FGL2 in T cell response in vivo and the role of TCF-1 and LEF-1 in the differentiation and function of T_FH cells during protein immunization.Main contents1.The effect of FGL2 in T cell response in vivo during acute virus infectionFirstly,we detected the induction of FGL2 expression after LCMV infection.On the day of 3,5,and 8 after LCMV infection,the FGL2 expression is 65%,111%,and 151% more than the na?ve mice.To verify the role of up-regulation FGL2 during the T cell response,we infected the Fgl2 gene knockout mice and C57BL/6J mice with LCMV,and detected the proliferation,differentiation and function of T cells on the day 8 after infection.We also transferred SMARTA or P14 cells into Fgl2-/-and C57BL/6J mice,and detected the proliferation,differentiation and function of T cells to investigate the extrinsic role of FGL2.Our results indicated that the up-regulation of FGL2 was dispensable for T cell response in vivo during acute viral infection.2.TCF-1 and LEF-1 cooperatively regulate the function of T_FH cells during protein immunizationWe mixed the NP-CGG or GST-GP66-77 with Freund's adjuvant,and immunized the Lef1fl/+Tcf7fl/+?Ctrl?,Lef1-/-Tcf7-/-?DKO?,Lef1-/-Tcf7fl/+?LKO?,and Lef1fl/+Tcf7-/-?TKO?groups respectively,whose genes were induced ablated by Tamoxifen.8 days after immunization,we detected the frequency of T_FH and GC B cells in lymph node.Our results indicated that there were no difference in the frequency of T_FH cells in four groups?p>0.05?during NP-CGG or GST-GP66-77 protein immunization.However,compared with the Ctrl group,the frequency of GC B cells showed 40% off in TKO group and 80% off in DKO group after NP-CGG protein immunization?p<0.001?.We think that there are two reasons for the decrease of frequency of GC B cells.Firstly,TCF-1 and LEF-1 have an important role in B-cell response,the ablation of TCF-1 and LEF-1 in B cells,also induced by Tamoxifen,may result in the deficiency of GC B cells in DKO mice group;secondly,TCF-1 and LEF-1 cooperatively regulate the function of T_FH cell,which cause the decrease of GC B cells.Considering the absent expression of TCF-1 in B cells,we first plan to adoptively transfer SMARTA cells or B1-8Hi cells,which specially recognizes GP66-77 or NP epitode from GST-GP66-77 or NP-CGG protein,respectively,into the four group mice in vivo and co-culture the sorted T_FH cells from four group mice and B cells from WT mice in vitro to investigate the function and mechanism of TCF-1 and LEF-1 in the formation and differentiation of GC B cells.If these two experiments will show the similar function of T_FH cells in four group mice,we will cross CD19-Cre or AID-cre with Tcf7fl/flLef1fl/fl mice to investigate the function and mechanism of TCF-1 and LEF-1 in B-cell response.ConclusionWe verify the up-regulation of FGL2 expression during LCMV infection,and the up-regulation of FGL2 is dispensable for T cell response in vivo during acute viral infection.After protein immunization,the frequency of T_FH cells in four group mice show similar,while the frequency of GC B cells show 40% off in TKO group and 80% off in DKO group,compared with the Ctrl group.To investigate the function and molecular mechanism of TCF-1 and LEF-1 in the formation and differentiation of GC B cells,the adoptive transfer experiments in vivo and co-culture experiments in vitro will be tested in the future.