Ny-eso-1Positive Human Retinoblastoma Cell Was Killed By NY-ESO-1-Impulsed Dendritic Cells Induced CTL

Objectives:Present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocyte (CTL), which induced by cancer testis antigen NY-ESO-1-impulsed dendritic cells (DCs), against human retinoblastoma (RB)Method:1. We constructed a type of pDC316/NY-ESO-1recombinant plasmid,at the same time,its expression was detected in cultured eukaryocyte.2. Peripheral blood mononuclear cells(PBMC) was seperated from60ml peripheral blood of healthy donor by Ficoll density-gradient centrifugation. DCs were induced with interleukin-4(IL-4) and granulocyte/macrophage colony-stimulating factor(GM-CSF) from human PBMC. The recombinant plasmid pDC316/NY-ESO-1was transfected into DCs.Then this kind of DCs were co-culture with T lymphocytes,and the resultant CTLs was increased. 3. HXO-RB44cell lines was specially killed by CTLs.The proliferation of the CTL and the effect of the killing tumor cell were test by MTT.Result:1. The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-1is fully consistent with the sequence of NY-ESO-1gene.Restriction endonuclease digestion result was the same with expectation.2. NY-ESO-1gene was detected in the HXO-RB44cell line, and the expression of in was fairly strong by Western blot and Immunoflourescence.3. DCs were successfully induced with IL-4and GM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1was successfully transferred into DCs.This kind of DCs which were transferred had high expression of surface molecule such as HLA-DR(42.1%)、 CD80(54.2%)、CD83(39.7%) and CD86(94.8%).Praliferative capacity of CTL which induced by DCs-impulsed with NY-ESO-1is stronger than the CTL induced by DCs which was not transfected by NY-ESO-1plasmid.4. CTL induced by DCs-impulsed with NY-ESO-1,CTL induced by DCs without NY-ESO-1and T lymphocytes without DCs inducing are separately co-culture with HXO-RB44cell line.CTL induced by DCs group had stronger cytotoxic effect against HXO-RB44cells compared with un-impulsed DCs group and no-DCs group(P<0.05).The anti-tumor activity was highest when the ratio of effector:target was75:1(P<0.05).ConclusionThis study approved that NY-ESO-1gene express fairly strongly in the HXO-RB44cell line.NY-ESO-1is the most immunogenic CT antigen, meanwhile it is one of the most immunugenic tumor antigens.DCs transferred by the recombinant plasmid pDC316/NY-ESO-1can induce proliferation of allogenic CTLs,which show specific anti-tumor effect against HXO-RB44cells.As a basic study, it still needs a lot of research and improvement before gene therapy for retinoblastoma been applied in clinical work...