LEF-1 Assists TCF-1 In Determining The Function Of Tfh Cells During Protein Immunization

The humoral immune response mediated by B cells is an important means for the body to control the invasion of external antigens.So far,most of the effective vaccines exert their preventive function by inducing the generation and differentiation of memory B cells and long-lived plasma cells.Memory B cells and long-lived plasma cells are generated by antigen stimulation,germinal center（GC）formation,somatic hypermutation,and antibody class switch.In process,follicular helper T cells（Tfh）is necessary.The main surface marker of Tfh cells is chemokine receptor CXCR5.Tfh cells are recruited to B cell follicles,where CXCL13,a CXCR5 ligand,is produced constitutively.Homed Tfh cells interact with cognate B cells and exert the function of helping B cells.T cell factor 1（TCF-1）and lymphoid enhancer factor 1（LEF-1）are encoded by Tcf7 and Lef1 genes,respectively,and are members of the TCF/LEF transcription factor family.TCF-1and LEF-1 show high expression in various subpopulation from the early stage of T cell development to differentiation of CD4 and CD8.Both TCF-1 and LEF-1 have a high mobility group（HMG）DNA-binding domain at the C-terminus,which is highly conserved,and aβ-Catenin-binding domain（CBD）,thereby participating in the regulation of the canonical Wnt signaling pathway.In the presence of Wnt protein,freeβ-Catenin enters the nucleus,binds to TCF/LEF,and forms aβ-Catenin-TCF/LEF complex,which controls the expressions of target genes under the synergistic effect of other co-activators,ultimately regulating the development,differentiation,and apoptosis of embryos and cells.There are many studies on the regulation of Tfh cells by TCF-1 and LEF-1 in viral infection models,however,whether TCF-1 and LEF-1 play an important role in Tfh differentiation and function during protein immunization remains largely unknown.This project focuses on the regulation of Tfh differentiation and function by TCF-1 and LEF-1 during protein immunization,and makes the following three conclusions:1.TCF-1 is required for the activation of CD4⁺T cells during protein immunization.In consideration of the key role of TCF-1 in thymocyte development,we crossed the mice expressing a Tamoxifen-sensitive estrogen receptor fused with Cre（ERT2-Cre）with Tcf7^fl/flLef1^fl/flmice to generate ERT2-Cre⁺Tcf7^fl/flLef1^fl/+（hereafter designated as TKO）,ERT2-Cre⁺Tcf7^fl/flLef1^fl/fl（DKO）,ERT2-Cre⁺Tcf7^fl/+Lef1^fl/fl（LKO）,and ERT2-Cre⁺Tcf7^fl/+Lef1^fl/+（Ctrl）mice.At day 5 after Tamoxifen treatment of 5 succssive days,the four groups of micewere intraperitoneally（i.p.）immunized withthe mixture of4-Hydroxy-3-nitrophenylacetyl（NP）hapten conjugated to Keyhole Limpet Hemocyanin（NP-KLH）and aluminum adjuvant,and the splenocytes from immunized mice were analyzed at day 7 after immunization.We found that theproportions of CD44⁺in CD4⁺T cells from TKO and DKO mice were both obviously decreased compared with that from Ctrl mice,whereas LKO mice exhibited a comparable level.Meanwhile,there werenot any appreciable differencesof Tfh（CD44⁺CXCR5⁺）and Th2（CD44⁺CXCR5^–）cells among them.Moreover,the four groups of mice were respectively immunized i.p.with the mixture of NP conjugated to chicken ovalbumin（NP-OVA）and aluminum adjuvant,as well as subcutaneously（s.c.）with the mixture of NP conjugated tochickenγglobulin（NP-CGG）and complete Freund’s adjuvant,and similar phenomena were observed at day 7 after immunization,namely diminished proportions of CD44⁺in CD4⁺T cells from corresponding spleens and inguinal lymph nodes（i LNs）of TKO and DKO mice.Meanwhile,we noted that Tfh frequencies in CD44⁺CD4⁺cells from NP-OVA-immunized TKO and DKO mice as well as Tfh frequencies and numbers from NP-CGG-immunized DKO mice wereslightly decreased,however,the reduction was far less than that in viral infection models.Collectively,these findings demonstrated that TCF-1 was required for the activation of CD4⁺T cells,however,unlike viral infection,TCF-1 only played a small role in the differentiation of Tfh cells during protein immunization.2.LEF-1 assists TCF-1 in regulating B-cell response during protein immunization.We also examined B-cell response at day 7 after NP-KLH immunization andnoted that thefrequencies of both GC B cells（Fas⁺GL7⁺）in Ig D^–B220⁺cells and plasma cells(CD138⁺B220^low)in splenic lymphocytes were obviously decreased from TKO and DKO mice relative to Ctrl and LKO mice,though the numbers of GC B and plasma cells in TKO mice were not diminished.Similarly,in i LN lymphocytes at day 7 after NP-CGG immunization,we observed that the frequencies and numbers of GC B cells were obviously decreased in TKO and DKO mice relative to Ctrl and LKO mice,and meanwhile only DKO mice exhibited diminised frequencies and numbers of plasma cells.Next,the B-cell response was analyzed at day 7 after NP-OVA immunization.Compared with those from Ctrl and LKO mice,the frequencies and numbers of GC B cells in Ig D^–B220⁺cells from TKO and DKO mice were diminished,though the decreases of the frequencies and numbers of plasma cells in splenic lymphocytes only appeared in TKO mice.Furthermore,we titrated the anti-NP-CGG antibody in mouse serum at day 28 after NP-CGGimmunization by ELISA and observed that the titers of antigen-specific antibody in TKO and DKO sera were significantly lower than those in Ctrl and LKOsera.Taken together,these findings demonstrated that LEF-1 assisted TCF-1 in regulating the differentiation and function of B cells during protein immunization.3.The defects of B-cell response are not attributed to direct TCF-1 and LEF-1deficiencies in B cells during protein immunization.To be noted,the excisions of Tcf7 and Lef1 in B cells were also induced by Tamoxifen treatment.We thus detected the expressions of TCF-1 and LEF-1 in Tfh,Th2,and B cells in wild-type mice at day 7 after NP-KLH immunization.Obviously,TCF-1 and LEF-1 were expressed in Tfh and Th2 cells and the expression levels in Tfh cells were higher than those in Th2 cells,whereas the expressions of TCF-1 and LEF-1 in B cells were almost undetectable.To confirm the notion that LEF-1 assisted TCF-1 in determining the function of Tfh cells during protein immunization,rather than the differentiation and function of B cells directly,we adoptively co-transferred wild-type（CD45.1）and Ctrl or DKO（CD45.2）splenocytes at a3.5:1 ratio into lethally irradiated recipient mice（CD45.1 and CD45.2）,in which thefunction of Ctrl or DKO Tfh cells was negligible to B cells.At day 7 after NP-KLH immunization,we observed that there were not any appreciable differencesof GC B and plasma cells in the recipients receiving Ctrl or DKO splenocytes.Collectively,these results demonstrated that the B-cell response defects were not attributed to the B cells inherent lacking TCF-1 and LEF-1during protein immunization.In summary,TCF-1 is required for the activation of CD4⁺T cells during protein immunization.Unlike viral infection,TCF-1 only plays a negligiblerole in Tfh differentiation,however,LEF-1 assists TCF-1 in determining the function of Tfh cells during protein immunization.Our findings provide important insights into the roles of TCF-1 and LEF-1 in Tfh differentiation and function during protein immunization.