Abstract
CML28 is a new tumor-associated antigen overexpressed in tumor cells, which is an attractive target for antigen-specific immunotherapy. In this study, we evaluated the possibility to induce CML28-specific cytotoxic T cells by cotransfection of CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector with dendritic cells in vitro. The full length CML28 cDNA was amplified from K562 by RT-PCR and cloned into a bicistronic vector pIRES2-EGFP to construct the CML28 nucleic acid vaccination. Dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMNCs) of HLA-A2+ healthy donor. Construct the recombinant plasmid psiRNA-hH1neo-SOCS1 encoding hairpin small interfering RNA (siRNA) targeting to suppressors of cytokine signaling 1 (SOCS1) sequences using a vector-base RNA interference technology. Cotransfect CML28 nucleic acid vaccination and recombinant siRNA vector psiRNA-hH1neo-SOCS1 into DCs by electroporation. Real-time RT-PCR was performed to validate the SCOS1 gene silencing efficacy. The expression products of CML28 nucleic acid vaccination, His-CML28 fusion protein and GFP protein, were measured by Western Blotting and fluorescence microscope respectively. Labeling autologous nonadherent fraction of PBMNCs as responder cells by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) firstly, and then coculture with cotransfected DCs for mixed lymphocyte reaction (MLR). Autogous T cell proliferative response of MLR was measured by FACS by detecting CFSE. Cotransfected DCs and the PBMNCs of a HLA-A2+ primary leukemia patient were used as target cells, untransfected DCs, K562 and HL60 were used as control. The standard 51Cr-release assay was performed to measure cytotoxicity of stimulated lyphocytes. CML28 nucleic acid vaccination could encode His-CML28 and GFP protein in DCs successfully. SOCS1-specific siRNA expression vector psiRNA-hH1neo-SOCS1 significantly suppress the expression of SOCS1 in DCs. Down regulation of SOCS1 resulted in higher expression level of CD80, CD86 and CD83 in cotranfected DCs and more rounds of cell division of responder cells in CFSE-MLR, which indicates SOCS1 gene silencing greatly contribute to maturation of DCs and enhance the the proliferative response of responder cells. CTLs induced by cotransfected DCs exhibit stronger CML28-specific cytotoxicity against HLA- matched target cells comparing to CTLs induced by CML28 nucleic acid vaccination transfected DCs only. SOCS1 gene silencing in DCs could greatly enhance the anti-tumor efficacy of CML28 nucleic acid vaccination. DCs cotransfected with CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector could effectively induce autologous CML28-specific cytotoxic T cells that lyse CML28 positive tumor cells in an antigen-specific and major histocompatibility complex (MHC) class I-restricted manner.
A development of the efficient siRNA-expression vector by the use of peptide-DNA conjugate
