Objective: To prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression.
Methods: The bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA.
Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells.
Conclusions: The genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.