The Study In The Molecular Regulation And Stability Of BCL6 During Follicular Helper T Cell Differentiation

Follicular helper T(Tfh)cells is an important CD4+T cell subset.They mainly provide help for Germinal Center(GC)formation,sustain the clonal selection of GC B cells,thus providing help for the production of high-affinity antibodies,and are essential for maintaining effective humoral response.Independent of other CD4+T cell subsets,such as Thl,Th2 or Th17 cells,Tfh cells have their specific lineage-defining factor,Bcl6.Previous studies demonstrate that Bcl6-deficient T cells lose the ability to differentiate into Tfh cells and forced expression of Bcl6 in CD4+T cells significantly promotes Tfh differentiation.Bcl6 is now mainly recognized as a gene repressor,as it promotes Tfh differentiation mainly through repression of other subsets-associated genes.Despite its critical roles in Tfh differentiation,the expression level of Bcl6 is not homogeneous and is strictly regulated during the whole process.Previous studies have revealed many factors participating in Tfh differentiation,including cytokines,co-stimulatory molecules,transcriptional factors and other factor-modifying enzymes or chaperone molecules.However,roles played by most of these molecules in Tfh differentiation finally affect differentiation through Bcl6.In this paper,our research involves the regulation in Bcl6 and consists of two parts.During Tfh differentiation,IL6 stimulation induces activation of STAT3 and subsequent expression of Bcl6 expression at the early phase and IL2 induces activation of STAT5 and inhibits Bcl6 expression.In this project,we studied the role of another extracelluar molecule,Extracellular Matrix protein 1(ECM1),in Tfh differentiation and antibody production.Previous study has demonstrated that ECM1 could induce Klf2 expression by inhibiting IL2 signaling.However,IL2-STAT5 and Klf2 mediate two important negative signaling pathways in Tfh differentiation.Then what is the role of ECM1 in Tfh cell differentiation?Firstly,we observed enhanced phosphorylation of STAT5(p-STAT5)in Ecm1-/-Tfh-like cells.Then we immunized Ecm1-/-and WT mice with complete Freund's adjuvant(CFA)-emulsified KLH and found that the development of Tfh and GC B were significantly decreased in Ecm1-/-mice compared with WT mice.Moreover,impaired Tfh and GC B development could be rescued by inhibition of IL2 signaling.Then we found that ECM1 promoted Tfh differentiation in a partially autocrine manner with mixed bone marrow chimera analysis and conditional knockout mouse model(Ecm1f/fCD4-Cre+/-).Thereafter,we found that ECM1 expression in Tfh cells was induced by IL6/IL21-STAT3 pathway.Most importantly,we found that administration of exogenous ECM1 could enhance Tfh differentiation,GC development and production of influenza-specific neutralizing antibodies.Our research identified the first non-cytokine secretory molecule in promoting Tfh differentiation in mice,enriched the regulation pathway antagonizing IL2 signaling during Tfh differentiation and provide a new potential molecule targeting Tfh response in anti-virus strategy.In the second part,we aimed to identify deubiquitinating(DUB)enzymes stabilizing BCL6 in Tfh differentiation.Ubiquitination and deubiquitination are two important ways of post-translational modification in most of biological processes.The inconsistency between the transcripts level and protein level of Bcl6 occurs in Tfh differentiation,implying the critical role of posttranslational modification in Bcl6 regulation.In this study,we firstly identified several DUB molecules stabilizing BCL6 in 293T cells by co-expression of the two molecules.Then we identified several Bcl6-interacting DUBs in 293T cells through co-immunoprecipitation.Tfh-like cells are in-vitro activated T cells under stimulation of IL6 and IL21,which has the most similar gene expression pattern with in-vivo differentied Tfh cells.Next step,we identified Usp21 and Usp37,which could stabilize BCL6 protein level in Tfh-like cells when overexpressed in these cells by retrovirus transduction,from BCL6-interacting DUBs in 293T cells.To study whether the two molecules were required for Tfh differentiation,we generated T cell-conditional knock out mice,Usp21f/fCD4-Cre+/-and Usp37f/fCD4-Cre+/-.Then we analyzed Tfh differentiation in the influenza virus infection model and adoptive transfer model with OTII and Smarta system.However,we didn't observe the impaired Tfh differentiation in Usp21-or Usp37-deficient mice.Correspondingly,the reduction in GC B cell development or IL21 production were not observed.Our data indicate that although overexpression of Usp21 or Usp37 could enhance B16 in vitro,they are not necessary for the BCL6 regulation and Tfh differentiation in vivo.