Cloning, Characterization Of Human IL-12 And Functional Studies On IL-12 Engineered Human Dendritic Cells.

Interleukin 12 (IL-12), naturally produced by antigen presenting cells (APCs), has been demonstrated to have strong immunological effects in inducing the secretion of IFN-γ, facilitating the development of Th1 cells, and in enhancing CTL response against microorganisms as well as tumor cells. Dendritic cells (DCs) were shown to be the most efficient APCs. The main aim of the present study was to evaluate the importance of IL-12 provided in the context of DC in its antigen presenting functions, and to establish the rationale in developing dendritic cells in sufficient quantity loaded with IL-12 and relevant epitopes to serve as a broad spectrum therapeutic vaccine against cancer.By using RT-PCR, cDNA of the p40 subunit of IL-12 was amplified from mRNA extracted from the cord blood dendritic cells of Beijing newborn ' s. The cDNA was directly cloned into the TA system of InVitrogen. Sequencing data from two independent clones demonstrated the complete open reading frame of p40 cDNA having high homology with those reported but being unique at codon 210, which is GCC (Ala). For functional assessment, a recombinant bicistronic retroviral vector expressing both p40 and p35 (from ATCC) was constructed. After transduction into a hepatocellular carcinoma (HCC) cell line and selection, its supernatant demonstrated clearly detectable IL-12 by ELISA and also potent stimulatory activity on lymphoblasts. Western Blot analysis showed the specific band. On the basis of the structural and functional findings presented above, it is suggested that the human IL-12 p40 gene might be polymorphic.Using plate-coated stem cell factor (SCF) to adsorb the hematopoietic stem cells, more than 80% of which were CD34 positive. These cells were...