The killing effect of NY-ESO-1-sensitzed dendritic cells-induced cellular toxic lymphocyte on human retinoblastoma cell

Abstract

Background Cell immunologic therapy for retinoblastoma （RB）is becoming a hot research topic. Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors. However,its effect on RB has not been investigated. Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes （CTL）induced by the cancer-testis antigen, NY-ESO-l-sensitized dendritic cells （DCs）, on human RB. Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI. Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1. The expression of NY-ESO-1 protein in human RB cells strain, HXO-RB44,was detected by immunofluoreseence and Western blot. Monoeytes wereisolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 x 107/ml. DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4 （ rhIL-4 ）. The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes. The resultant CTL were used as effector ceils. The growth of the CTL was detected by MTT assay. The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay. Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-lwas conforms with the sequence of the NY-ESO-1 gene. The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot. DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs. These DCs had high expression of surface molecules such as HLA-DR （42. 1% ） , CD80 （54.2%） , CD83 （39.7%） and CD86 （94.8%）. The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells. CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs, as shown by MTT asssay（ P〈0.05 ）. The anti-tumor activity was highest when the ratio of effector to target was 75 ： 1 （ P〈0. 05 ）. Conclusions DCs transfected by the recombinant plasmid pDC316/ NY-ESO-1 can induce the proliferation of allogenic CTLs, which showed a specific anti-tunmr effect against HXORB44 cells. These results present a new type of immunotherapy for the treatment of RB. Key words: Retinoblastoma; Ddendritic cell; Cytotoxic T lymphocyte; NY-ESO-1