Regulation Of Xenogeneic Genetically-modified Pig Mesenchymal Stromal Cells To Human T-cell Immune Function

From previous studies,we found that pig mesenchymal stromal cells(pMSC)can be obtained efficiently from adipose tissue of ?1,3-galactosyltransferase gene knock-out pigs transgenic for human CD46(GTK.O/hCD46)and GTKO/CD46 pMSCs have been proved to possess low xenoantigenicity and good inhibitory effect on human T-cell xenoresponses.However,mechanism of GTKO/hCD46 pMSC exerting its immune regulation effect to human T cells is still unclear.Based on the literature and previous studies,we proposed the following hypothesis,that is GTKO/hCD46 pMSC may inhibit T cell immune response through 1)inducing T cell apoptosis;2)inhibiting T cell activation;3)inducing production of regulatory T cells(Treg).AIM and METHODS:In order to further evaluate the immune regulating effect of GTKO/hCD46 pMSC on CD4 + T and CD8 + T cells in vitro,and explore possible mechanism involved,experiment will be conducted from the following aspects:Part one:Isolation and culture of pMSC and porcine aortic endothelial cells(pAEC)which were used for experiment and identification of pMSC through stem cell molecular markers and differentiation induction.Part two:Mixed lymphocyte reaction(MLR)system was set up using purified CD4+T and CD8 + T cells as responder cells and using pMSC,pAEC or both as stimulator cells,respectively.T cell proliferation was assessed by detecting the(?)precursor of T cell DNA(3H-thymidine nucleoside)and result will be represented using number of counts per minute(CPM).In addition,two-step MLR system was built up to assess T cell immune status by removing the pMSC from coculture system.Part three:Activation,expression of granzyme B and induction of apoptosis or regulatory T cells(Treg)of human T cells were detected flow cytometry in order to explore how GTKO/hCD46 pMSC suppress T cell proliferation.Part four:Activation of T cell signal transduction and transcription activating factor 5(STAT5)was tested in order to ascertain that whether STAT5 plays a role in the immune regulation of GTKO/hCD46 pMSC on T cells.RESULTS:Part one:pMSC can be obtained effectively in large amounts from GTKO/hCD46 porcine adipose tissue in form of colony forming unit-fibroblastoid(CFU-F).pMSC can express typical stem cell molecular markers and display good potential to differentiate to adiposecytes,osteoblasts and chondrocytes.Part two:After co-culture with GTKO/hCD46 pMSC,proliferation of CD4 + T and CD8 + T response is as weak as spontaneous group(T cells culuture alone),respectively(P>0.05);GTKO/hCD46 pMSC can significantly inhibit T cell proliferation in dose dependent manner comparing with positive control(P<0.05),Two-step MLR shows that hCD4+T cells can still maintain state of low reaction to stimulator(GTKO pAEC),while CD8 + T cells can quickly restore immune response after removing GTKO/hCD46 pMSC from coculture system.Part three:1)GTKO/hCD46 pMSC can significantly reduce granular enzyme B expression levels of CD4 + T and CDS + T cells comparing with pAEC coculture group(P<0.05),while there was no significant increase of the proportion of CD4 +CD25 + Foxp3hi T cells among all CD4+T cells after coculturing with GTKO/hCD46 pMSC(P>0.05).There was no significant increase of the ratio of apoptosis T cells within all CD4+T cells after coculturing with GTKO/hCD46 pMSC(P>0.05).GTKO/hCD46 pMSC can significantly increase CD69 expression of T cell(P<0.01).without affecting CD25 or LAG-3 expression significantly(P>0.05).Part four:Cell signal transduction and transcription activating factor 5(STAT5)phosphorylation rate of T cells is significantly higher in GTKO/hCD46 pMSC coculture group than T cell culture alone group(P<0.05)CONCLUSION:1)GTKO/hCD46 pMSC can be obtained effectively in large amounts from GTKO/hCD46 porcine adipose tissue in form of colony forming unit-fibroblastoid;2)GTKO/CD46 pMSC can significantly inhibit CD4 + and CD8+T cell proliferation in dose dependent manner in vitro,and the inmmunoregulatory effects of MSC is more durable on CD4 + T than on CD8 + T cells.3)GTKO/CD46 pMSC exert T cell immune regulation effect without inducing Treg or apoptosis;4)GTKO/CD46 pMSC may modulate CD69 expression,which in turn affecting the STAT5 signaling pathway in downstream,and finally realize the T cell proliferation inhibition.