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BackgroundImmunotherapy as a new tumor treatment method, especially dendritic cells combined with tumor-associated antigen (TAA) cancer treatment, has become the focus of this field. Human telomerase reverse transcriptase has been identified as the best TAA, it is highly expressed in most, more than 85%, tumor cell ,while normal adult cells are mostly negative. Expression of hTERT leads to telomeres lengthening and cell immortalization, whereas degradation of hTERT by proteasomes prompts telomere shortening,cancer cell senescence(ageing) and apoptosis. Previous studies have shown that sufficient hTERT antigen presentation is achievable using APC. Using of synthetic hTERT peptide to pulse the dendritics cells, it appear to be effective at inducing anti-tumor immune responses in vitro.The further study shows, the single synthetic epitope peptide based on the hTERT gene sequence can arouse strongly anti-tumor effect in vitro, but weak effect in vivo. It may be due to ,under normal physiological conditions , the strongly arousing of immune response needs the DCs present more epitope peptides , not only one, and exist the synergy among those peptide.So the design of multi-epitope peptides has become the trend. But a simple mixture of a variety of single-epitope peptide, can not also solve the problem of poor antigenicity.ObjectiveBased on relevant theory,using the tandem dendritic multiple antigenic hTERT epitope peptides with four brounchs through artificial solid-phase synthesis contained I540,V461 and L766 to pulse the myeloid dendritic cells inducing specific anti-tumor effects of homologous lymphocytes, to explore a better treatment for tumor immunotherapy.MethodsSolid-phase artificial synthesis of the tandem dendritic multiple antigenic hTERT epitope peptides(MAP) with four brounchs and the three separate peptides of each branches, consisting of I540,V461 and L766. Immunofluorescence detect the expression of hTERT of tumor cell; Selecting the appropriate blood type ,not only HLA-A2+ but HLA-DR4 or DR11 OR DR15 positive, by PCR-SSP; magnetic activated cell sorting MDC, nylon wool purified T cells, both MDC and LC was cultured with serum-free medium; ELISA test the IL-12p70 secretion of MDC and the TNF-a, INF-r secretion of lymphocyte at different time points in the culture process; Related phenotype of MDC and LC were detected by flow cytometry ;the killing effect of effector T cells on HLA-A2+ tumor cell of A549, MDA-MB-231 and SW480 were, also, detected by FCM.ResultsAll the peptides through the Solid phase synthesis has the satisfactory purity; All the three kinds of tumor cells expressed hTERT, and the expression in nucleus more than the cytoplasm, analyzed by immunofluorescence; MDC has the high purity using magnetic activated cell sorting; The MDCs stimulated by the MAP and simplely mixed peptide seperately both can arous the special immuno response of autologous lymphocytes on tumor HLA-A2+ cells, and the MAP’s group has the stronger killing effect.ConclusionFirstly, it has confirmed that the two groups of MDC pulsed by MAP’s and simplely mixed hTERT epitope peptide through the Solid phase synthesis contained I540,V461 and L766, both can both can arous the special immuno response of autologous lymphocytes on HLA-A2+ tumor cells. Secondly, MAP’s group has the stronger killing effect than the mixed group. Thirdly, because of the high efficiency of antigen presenting, MDC can be used as the carrier of specific tumor vaccines. Lastly, to improve safety of the future clinical DC vaccines, using the serum-free culture methods would be a perfect choice.
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