Induction And Identification Of Antigen-specific CTL In CIK-DC Co-culture System

CIK (cytokine induced killer) is a group of heterogeneous cells, with T cells as its main component. It has high cytotoxicity and replication activity and may be used for tumor immunotherapy. DC (dendritic cell) showing high antigen-presenting ability, can inhibit tumor escape from immune recognition and has been used to prepare tumor vaccine. Recently, many researches have demonstrated that CIK co-cultured with DC pulsed with peptide (CIK-DC co-culture system, or DCIK-P cell) showed a much higher killer activity against the human cells expressing related antigen, indicating the specificity of the killer activity and the induction of CTL (Cytotoxic T Lymphocyte).Breast cancer is one of women malignant tumors. To date, it has been mainly treated through specific regulation of host immunoresponse and many efforts has been focused on the development of tumor vaccines based on the receptor of the epidermal growth factor Her2 that is frequently expressed in breast tumor cells. However, there has been no investigations into the induction of CTL using CIK-DC co-culture system with Her2 as the pulsing antigen peptide of DC in China.The aim of the present investigation is to establish CIK-DCco-culture system with Her2 as the pulsing antigen peptide of DC, induce Her2-specific CTL and provide a technical data for the development of breast tumor vaccine using Her2.Three parts of work is carried out: (1) CIK and DC were first induced and isolated from human peripheral blood; CIK and DC pulsed with antigen Her2 were then co-cultured to prepare CIK-DC co-culture system(DCIK-P). (2) Using DCIK-P as effect cells and breast tumor cells as target cells, the antigen-specicfic cytotoxicity of DCIK-P was analysed by cell viability and cytotoxicity assay. (3) CTL produced in CIK-DC co-culture system was identified using enzyme-linked immunospot assay (ELISPOT). (4) Induction conditions including antigen pulsing duration, antigen density, rate of effect cells/target cells and antigen pulsing times, were optimized.Cell viability and cytotoxicity assay showed that the killer activity of DCIK-P cells was greatly enhanced against breast tumor cell strains with high expressing level of Her2, whereas no increased killer activity of DCIK-P cells was found against those strains with low Her2 expressing level or without the expression of Her2.ELISPOT assay indicated that the frequency of CTL cells produced under the induction of breast tumor strains with high expressing level of Her2 was prominently higher than those with low expressing level of the antigen. This result conforms the specific cytotoxity of DCIK-P cellsagainst breast tumor cells.The present study investigated the optimal method to induce CTL cells and established a CIK-DC co-culture system (DCIK-P) which could efficiently and specifically kill tumor cells expressing related antigen. The present results can provide tumor immunological data and experimental techniques for the development of breast tumor vaccines.