Differentiation Direction Of Thymus Gammadelta T Cells

IntroductionPluripotent lymphocytle stem cells transfer to thymic,gradually from the cortex into the the medulla in thymus.After negative selection and positive selection,T cells have become Progressive differentiation and maturation.T cell receptor have TCRαβand TCRγδ,αchain andβchain composed of TCRαβ,theγchain andδchain composed of TCRγδ.To date,about TCRγδT cells in the process of differentiation and maturation,physiological functions,as well as to identify the characteristics of antigen also do not know enough,but TCRαβcells as far from exhaustive.γδT cells mainly distributed in the skin and mucosal tissue and small quantity.But it has to resist against external pathogens,immune regulation and tumor immunity,and many other aspects plays an irreplaceable role.Therefore,γδT has also aroused the concern of many scholars.In recent years,studies have shown thatγδT cells development in two ways outside the thymus and thymus.Thymus development is based on the application of bone marrow stem cells in the thymus can differentiate intoγδT.Ciofani M et al study found a large number of thymus-derived signals and the highly conserved way to promote T cell differentiation in 2007,T cell development happen in an orderly manner based on the cascade.TCR gene order of the boot is TCR D(δ) -TCRG(γ) -TCRB (β)-TCRA(α).αβT cell differentiation can not be bypassed TCRD(δ)-TCRG(γ) gene expression directlyαβTCR.So wo are speculatedαβT thymocytes from the thymusγδT cells,thymocytesγδT is between the immune response and adaptive immune responses in the transition phase.Gestational age in 14.5d C57BL/6 mice for our the study,by using of mouse fetal thymus organ culture,FCSE Staining,Flow cytometry sorting and detected to observe development and differentiation direction of gammadelta T thymocytes.For the transition theory of the immune response to provide a basis.Materials and MethodsMaterials 1,AnimalC57BL/6 timed pregnant mice(SPF,8-10weeks old)were purchased from Chinese Academy of Medical Sciences Center,Beijing experimental animals(We usually place two female and one male mice in a cage in the evening(7-8 PM) and separate them in the morning(8-9 AM).Gestational days are tentatively designated by assigning the day at which mice are separated as 0.5 d and are confirmed on the day of experiment according to the size and many developmental features of fetuses).2,ReagentsAPC-anti-mouseCD4;PE-anti-mouseCD8;Cy5-anti-mouseCD3;CFSE;Goat-anti-m ouse TCRγ:IgG and Texa Red-anti-mouse;Goat-anti-mouse TCRβ:IgG;DMSO; Mtb-Ag;IL-2;Culture mediumâ… :RPMI1640 supplemented with 20%fetal calf serum (FCS);Culture mediumâ…¡:RPMI1640 supplemented with 20%adult mice serum(In addition to the different serum,the two other components of the same culture medium).Method1,method for Fetal thymus organ culture(FTOC) in vitro Millipore Will be placed in 24-well plates,theâ… group add in culture mediumâ… 1ml;Theâ…¡group add in culture mediumâ…¡1ml.Fetal thymus from timed 14.5d pregnant mice,try to randomize the way the lobes are placed.Two lobes from one fetus should be divided into different groups when multiple experimental groups are set up,four lobes are placed on each membrane.Ascertain that the lobes are placed at the interface between the membrane and air.Place the culture plate in a 37℃,5% CO₂ incubator.2,Determine the vivo of fetal mouse thymus organ peakγδT cells By using of Fluorescent antibody markers and Flow cytometry detected separately 12.5d,13.5d,14.5d,15.5d,16.5d C57BL/6 thymocytes.3,The concentration of stimulating factor and time of promote proliferationStimulating factor include Mtb-Ag and IL-2.Prepare a single-cell suspension of C57BL/6 mouse thymocytes.Cell wera cultured in two 96-well plates.Divided into experimental groups and control groups.Experimental groups inject Mtb-Ag of 10μl of 5μg/ml,7μg/ml,9μg/ml separately,and control groups inject PBS of 0.1M.Place the culture plate in a 37℃,5%CO₂ incubator.Continue to cultivate.The proliferation of thymus cell from C57BL / 6 were made by MTT colorimetric technique afte incubator for 3,6d.The best concentration of stimulating factors and single-cell suspension of C57BL/6 mouse thymocytes are together cultivated,to betecteγδT cell proliferation time.4,Sorting thymicγδT cell and CFSE stainingA single-cell suspension of C57BL/6 14.5d fetal mouse thymocytes and stimulating factor are together cultivated in 2 days,γ-chain fluorescent antibody marked for sorting.TheγδT cell sorting are sterilly staining with the best concentration of CFSE.5,γδT thymus cells differentiation whereTheγδT cell marked CFSE inject culture medium of fetal thymus lobes to cultivate.At the first 5,8 day,add the fluorescent anti-βchain antibodies,to detect respectively.Statistical analysisAll date are show as mean values±DS unless otherwise indicated.Comparison between groups was made using the one-way ANOVA analyse.Values of p < 0.05 were considered significant.ResultFTOC system used in the culture medium I may better close to pre-T cell development in vivo;Gestational age 14.5d thymocytesγδT cells of the total 15.14%, With the increase in gestational age fetal mice,thymusγδT cells in the gradual reduction;Mtb-Ag 7μg/ml and 5μg/ml,9μg/ml compared to promote the proliferation of mouse thymic lymphocytes has a clear role(p<0.05 );the peakγδT cells is the second day of cultured in vivo training when injected the best concentration of stimulating factors;Flow cytometry sortingγδT cell is 3%;the best best concentration of CFSE is 30μm;When 5th day and 8th day the differentiation rate of CFSE labeled cells are separately 8.12%,3.36%.DiscussionMolecular genetics studies have proved that when Pre-T cells enter the thymus.Its receptor gene rearrangement has not yet happened at embryonic period.The rearrangement of TCRγare up to high peak of the transcription at 14.5d fetal thymic. TCRβgene start rearrangements,TCRαgene start rearrangement after 2d.TCRγ,5 express mainly on immature T-cell in the fetal thymus,while TCRα,βexpress mainly in mature T cell membrane.T cells are in the immature and origina stage in 14.5d fetal thymus,so wo cultivate 14.5d fetal thymocytes in vitro.With extension of culture in vitro,T cells go through positive and negative selection.Continue to differentiate into CD4⁺CD8⁺ cells.And with prolonged cultivation further differente CD4⁺CD8^- and CD4^-CD8⁺ cells.A considerable number of T cell maturation to single positive cells after 8d.So 14.5d fetal thymocytes are the best starting time for study of thymus cells. Therefore we chose 14.5d fetal thymus organ technology used in vitro FTOC.We found that many CD4⁺CD8⁺ double-positive cells utput thymic in culture medium.Its mechanism is not clear.It provides a space micro-environment of the thymus for enteringγδT thymus cells of CFSE labeled.14.5d C57BL/6 fetal thymusγδT thymocytes are able to differentiate intoαβT thymocytes under appropriate culture.The differentiation rate of CFSE labeled cells are separately 8.12%[812/(1×10⁴个/ml×0.1ml)],3.36%[336/(1×10⁴个/ml×0.1ml)]on 5th day and 8th day.2007 scholars have found that the group of CD4⁺γδthymocytes represents transient and independent subsets that are never exported from thymus as TCRγδT cells.CD4⁺γδthymocytes are able to alter their phenotype to TCRαβthymocytes under appropriate culture conditions. For the transition of the immune response to provide a theory basis.Conclusion14.5d C57BL/6 fetal thymocytesγδT cells sorted and CFSE staining are able to alter their phenotype to TCRαβthymocytes in Fetal thymus organ culture(FTOC) in vitro.