Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells.

ABSTRACT

BACKGROUND: This study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. METHODS: Plasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-ß, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. RESULTS: Transduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-ß levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. CONCLUSIONS: Simultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.