The Role And Mechanisms Of KLF4 In Regulating Small Intil Resident Memory CD4~+ T Cells

Background and objective:Oral vaccination is one of the most important precaution methods to protect people from suffering from digestive tract borne diseases.The mechanism of vaccines is to induce the formation of memory cells which can respond rapidly to the same antigens when they invade again.Tissue resident memory T cells（Trms）are a subset of memory T cells,which are characterized as a non-circulating subset retained in specific tissue sites.Due to the characters of responding most immediately to local pathogens in situ,Trms are getting more and more attention and also become the important targets of oral vaccines.However,the mechanisms of maintenance of Trms in tissues are still not very clear so far.On the other hand,KLF4（Krüppel-like factor 4）is an important transcriptional factor,which was first known as an important stemness factor by inducing fibroblasts into pluripotent stem cells.With more and more studies,other functions of KLF4 have been gradually excavated such as regulating many biological processes like cell cycle,cell proliferation,differentiation and apoptosis and also playing different roles in different diseases.Moreover,KLF4,as a gut-enriched factor,has not been reported to have relationships with CD4⁺Trms in small intestine up to now.So,this study is aiming to explore the role and mechanisms of KLF4 in regulating small intestinal resident memory CD4⁺T cells.We hope this study can discover some elements which can regulate the maintenance of Trms and provide some new insights in the development of vaccines and diagnosis or therapy of Trms-involved diseases.Methods:1.The oral vaccination model was established by gavaging mice with dmLT（double mutant E.coli heat labile）.Mice at different timepoints were sacrificed（Day14,30,60,90,150,300,350）and cells from small intestine lamina propria（si LP）and spleens/mesentery lymph nodes（SPL/MLN）were extracted and dmLT-specific CD4⁺T cells were enriched by MHC class II tetramers with immunomagnetic beads.The cells were stained by fluorescence-labeling antibodies and the phenotypes were observed by flow cytometry.2.The dmLT-specific CD4⁺T cells from si LP and SPL/MLN at day14 and day60⁺were isolated and sent to RNA Sequencing.Based on 4groups（Gut eff,Gut mem,Spl eff,Spl mem）,RNA-Seq results were analyzed to investigate the key genes of defining the tissue resident memory CD4⁺T cells.3.According to the result of Step 2,Klf4 may be the key gene.We specifically knocked out the Klf4 gene from CD4⁺T cells by crossing CD4-cre⁺mice with Klf4^fl/flmice and gavaged both KO mice(CD4-cre⁺Klf4^fl/fl)and WT mice(Klf4^fl/fl)mice with dmLT.The cells from si LP and SPL/MLN of both WT and KO mice at Day14 and Day60 were extracted and stained as mentioned above,and the phenotypes were observed and compared by flow cytometry.4.The dmLT-specific CD4⁺T cells from si LP of WT and KO mice at Day14 and Day60 were isolated and sent to RNA Sequencing.Based on 4 groups（D14WT,D14KO,D60WT,D60KO）,RNA-Seq analysis were performed to excavate the molecular mechanisms of KLF4 in regulating CD4⁺Trms.5.Na（?）ve CD4⁺T cells were isolated from WT mice and were induced to differentiate into Th1,Th2,and Th17 subsets in vitro by different cytokines such as IL-12,IL-4,TGF-βand IL-6.Klf4 mRNA relative amount in different cell subsets was then tested by q PCR.Results:1.Oral vaccination with dmLT can lead to the long-lived CD4⁺Trms cells.The dmLT-specific CD4⁺Trms had the phenotype of CD69⁺CD103-.Th1 and Th17 were the major subsets of the CD4⁺T cells induced by dmLT,but not Th2 or Treg.2.In the RNA-Seq results,GSEA results showed the genome of Gut mem cells were highly enriched in the“Trm”gene set.Among the Differential Gene Expression analysis of Gut vs Spl,Mem vs Eff,Gut mem vs Spl mem,and Gut mem vs Gut eff,Klf4 is one of the most up-regulated genes in all the former groups of 4 comparisons,so Klf4 might be the key gene.3.Compared to WT mice,the number of dmLT-specific CD4⁺T cells was decreased in si LP of Klf4 KO mice both at Day14 and Day60,but the decrease was more obvious at Day60.And no differences were seen in SPL/MLN between WT and KO mice.CD69 expression was also decreased in the si LP CD4⁺Trms of KO mice at Day60.In addition,Th17 characteristic transcriptional factor,RORγT,was decreased in si LP dmLT-specific CD4⁺Trms of KO mice at Day60,and cytokine IL-17expression in the CD4⁺T cells was also decreased in KO mice.No differences were observed in Th1 related molecules T-BET and IFNγbetween WT and KO mice.4.In the RNA-Seq results,GSEA results showed the genome of D60WT was still enriched in the“Trm”gene set,and the genome of D60KO was highly enriched in the“Glucose metabolism”gene set.Differential Expression Gene analysis of D60WT vs D60KO showed the genes such as Malt1,Prdm1,and Klf4 were up-regulated in D60WT,whereas genes such as Mxra8,Mcm3 were up-regulated in D60KO.Genes which were differentially expressed in D60WT and D60KO were not differentially expressed between D14WT and D14KO.Moreover,some important genes related to lipid metabolism,anti-apoptosis and Trms were up in D60WT;whereas some important genes related to glucose metabolism,pro-apoptosis and cell cycle were up in D60KO as well as D14WT and D14KO.GO/KEGG analysis showed the up-regulated genes in D60KO were mainly involved in the pathways of cell cycle,DNA replication,DNA helicase activity,glycolysis,and tricarboxylic acid cycle.5.The relative amount of Klf4 mRNA in Th17 cells induced by TGF-βand IL-6 was higher than Th1 cells induced by IL-12 and Th2cells induced by IL-4.Conclusions:KLF4 can positively regulate the maintenance of small intestinal resident memory CD4⁺T cells,and the mechanisms might be involved in the pathways of reducing cell proliferation and suppressing cell apoptosis.In addition,the cytokines such as TGF-β,IL-6,IL-12 and IL-4 might be related to the expression of Klf4.