Comparison Of The Treg-like Cells From Different Induction Methods In Various Aspects

ObjectiveTo creat the methods of obtaining Treg-like cells via Retrovirus mediated gene transduction and TGF-βinduction. To compare the yield efficiency and activity of Treg-like cells of two methods and explore its relative mechanisms of immunosuppression.Methods1.FOXP3 gene was reconstructed in retrovirus vector and transferred into PT67 by lipofectamine mediated method. Stable monoclonal cell line which secreting high titer of MSCV-FOXP3 were selected. To further enhance the titer of retrovirus, the viral supernatant was concentrated by centrifugation.2.The CD4+CD25-T cells were separated from PBMC by using MACS.The optimized methods were respectively applied in MSCV-FOXP3 transduction and TGF-βinduction to acquire Treg-like cells. Compare the higher yield efficiency of two methods and real-time quantitative PCR was used to detect the difference of FOXP3 mRNA expression.3.CFSE staining and Flow cytometry assay was used to detect the influence of nature Treg and Treg-like cells on CD4+T proliferation. ModFit software was used to analysis the dynamic model of CD4+T proliferation.4. The level of IL-10 and TGF-βsecreted from Treg-like cells was assayed by ELISA method. To compare the mRNA level difference of granzyme A and granzyme B which express from Treg-like cells, real-time quantitative PCR was used to observe them.Result1.The recombinant plasmid pMSCV-FOXP3 was constructed and transferred into PT67. The stable retrovirus-producing cell line which produces high titer retrovirus was selected. The titer of MSCV-FOXP3 can reach to 1.2×107cfu/ml by concentration.2.The purity of CD4+CD25+ and CD4+CD25-T cells were 90.82% and 92.46% respectively identified by flow cytometry. The vitality of isolated T cells was above 90% by Trypan blue exclusion test. When the viral titer was 1×107cfu/ml, The Treg-like cells attained the highest yield efficiency of 49.12%.In the other method, while TGF-B was 5ng/ml and time at 6th days, the induction of the Treg-like cells gain the highest yield efficiency of 41.08%.The yield efficiency of the Treg-like cells in MSCV-FOXP3 transduction group was higher than it in TGF-βinduction group (P<0.05).The mRNA level of FOXP3 in MSCV-FOXP3 transduction group is high than it inTGF-βinduction group (P<0.05).3.Compared with control group, the nTreg and Treg-like cells significantly inhibited the proliferation of autologous CD4+T cells (P<0.05).4. The level of IL-10 was up-regulated significantly in the two groups of Treg-like cells compared with control group (P<0.05).There was no statistic difference of secrecting TGF-βin the two groups of Treg-like cells compared with control group (P>0.05).The mRNA level of Granzyme B in TGF-βinduction group of Treg-like cells was higher than its in control group (P<0.05).ConclusionThe Treg-like cells having the ability of immunosupression can be obtained with the method of MSCV-FOXP3 transduction and the method of TGF-βinduction, While the higher yield efficiency was obtained with the method of MSCV-FOXP3 transduction and the ability of immunosupression of Treg-like cells with the method of MSCV-FOXP3 transduction was higher than the latter. IL-10 may join the proess of fuctioning immunosupression in two groups, and granzyme B maybe one factor of fuctioning immunosupression in Treg-like cells of TGF-βinduction group.