Effect Of LFA-1 Gene Deletion On The Differentiation And Functional Regulation Of Th17 Cells Induced By Mice Na(?)ve T Cells In Vitro

Aims:The aims of study were to observe the effects of LFA-1 gene deletion on the differentiation and functional regulation of T help cell 17(Th17)induced by mice na(?)ve T cell in vitro,and to explore the role in the pathogenesis of inflammatory bowel disease(IBD).Methods:1.The LFA-1-/-gene knockout mice were breed and reproduced in SPF condition and the genome DNA from the mice tails was extracted for genotype identification by PCR.2.The mice were divided into experimental group(LFA-1-/-)and control group(wild C57/B6 mice).The spleen was taken out in a sterile environment and measured them respectively.CD4+CD62L+ na(?)ve T cells were separated from PBMC by magnetic activated cell sorting,and then analyzed the purity by flow cytometry.The na(?)ve T cells were co-cultured in 96-well microplate coated with anti-CD3mAb and anti-CD28 mAb in different stimulating conditions for Thl7 cells differentiation(blank group,TGF-β 3ng/mlgroup,TGF-β3ng/ml+IL-6 40ng/ml group,TGF-β3ng/ml+IL-6 40ng/ml +IL-23 30ng/ml group).After co-cultured for 72-96 hours,each group cells was collected for analyzing the ratio of Th17 and surface markers including CD25,CD69,CD62L and CD45RB by flow cytometry.The expressions of ROR-yt mRNA and IL-17 mRNA were measured by qRT-PCR and the level of IL-17 in cultures was detected by ELISA.3.The cells of each group cultured in TGF-β+IL-6 +IL-23 stimulating conditions on day 1,day 3 and day 7 were separated and measured their apoptosis by Annexin V flow Cytometry Assay Kits.4.The mRNA expressions of IL-6,CSF2 and CCR6 were measured by qRT-PCR after na(?)ve T cells cultured in TGF-P+IL6 +IL23 stimulating conditions for 72 hours.Results:1.The LFA-1 gene knockout homozygous genotype mice were acquired successfully.2.The spleen of LFA-1-/-mice was significantly longer than that of wild mice(control group)in the same week,but the number of CD4 + T cell was lower than that of control group.3.The purity of CD4+CD62L+ na(?)ve T cells separated by magnetic activated cell sorting was satisfied for further study.Different differentiation ratio of Th17 was obtained from mice na(?)ve T cells in different culture conditions in vitro.The differentiation of Th17 cells could be induced by low-dose TGF-β with IL-6 in culture system,which was increased obviously when adding IL-23.Co-cultured at low-dose TGF-β,IL-6 and IL-23 for 72～96 hours,the number of Th17 cells was higher and expression of ROR-yt mRNA,IL-17 mRNA and IL-17A induced by na(?)ve T cells were increasing in LFA-1-/-group than that in control group.4.After na(?)ve T cells cultured at low-dose TGF-p,IL-6 and IL-23 for 96 hours,the expression of CD25 and CD69 was up-regulating while CD62L was down-regulating,and CD45RB was no any change.Compared with LFA-1-/-group mice,the ratio of surface markers changed in control group was higher.5.The number of apoptosis cell on day 1,and day 3 of LFA-1-/-group were higher than that of control group.But on day 7,there was no difference between two groups.6.After na(?)ve T cells of two group was co-cultured at low-dose TGF-β,IL-6 and IL-23 for 72-96 hours,not only the mRNA expression of ROR-yt and IL-17 was up-regulated,the mRNA expression of cytokine such as IL-6,CSF2(also called GM-CSF)and CCR6 also up-regulated.There was more obvious in LFA-1-/-group than that in control group.Conclusions:Deficiency of the LFA-1 gene could affect the differentiation of Th17 cells induced by mice na(?)ve T cells in vitro.However,more study is necessary to investigate the pathway involved in inflammatory phenotype induced by LFA-1 gene.