| Objective: To explore the killing efficiency of EpCAM antigen-specificimmune effector cells cultured in vitro against EpCAMhighadenocarcinomacells. Compare the killing rates between EpCAM antigen-specific immuneeffectors and full antigens of EpCAMhighadenocarcinoma cells-specificimmune effectors to EpCAMhighadenocarcinoma cells, respectively. Comparethe killing rates of EpCAM antigen-specific immune effector cell toEpCAMhighand EpCAMlowadenocarcinoma cells, respectively.Methods: Cultured a variety of adenocarcinoma cell lines in vitro,detected the surface expression of EpCAM antigen in differentadenocarcinoma cell lines by flow cytometry, EpCAMhighLS174-T colonadenocarcinoma cells and EpCAMlowPaCa-2pancreatic cells were selected tobe target cells. Collected mononuclear cells (PBMCs) from peripheral bloodwhich acquired from healthy adults by density gradient centrifugation.Isolated lymphocytes and imDCs by adhesion method. Induction of Tlymphocytes into CIK cells and amplification of CIK cells in vitro. ImDCspushed with LS174-T cells lysate to generate LS174-T adenocarcinoma cellslysate mature DCs that named LS-mDCs. LS-mDCs and autologous CIK cellswere co-cultured in vitro, part of CIK cells turn to full antigens of LS174-Tcells-specific cytotoxic CTL after full antigens information of LS174-T cellswere presented by LS-mDCs, and then full antigens of LS174-Tadenocarcinoma cells-specific DC-CIK-CTL effector cells (LS-effector) weregenerated. Using similar methods, imDCs pushed with purified EpCAMpolypeptide antigen to generated EpCAM antigen mature DCs that namedEp-mDCs. Ep-mDCs and autologous CIK cells were co-cultured in vitro, partof CIK cells turn to EpCAM antigen-specific cytotoxic CTL after EpCAMantigens information were presented by Ep-mDCs, and then EpCAM antigen-specific DC-CIK-CTL effector cells (Ep-effector) were generated.Detected the expressions of surface molecules CD80, CD86, CD83andHLA-DR in imDCs, LS-mDCs and Ep-mDCs by Flow cytometry (FlowCytoMetry, FCM) respectively, and analyzed the statistical differences. Set upCIK group, LS-effector group and Ep-effector group. Detected the changes ofpercentages of CD3+CD8+T cells and CD3+CD56+cells in CIK group and ofCD3+CD8+T cells and CD3+CD4+T cells in LS-effector group andEp-effector group at day0, day7, day14(d0, d7, d14), respectively, andanalyzed the statistical differences. Detected the killing rates of each group ofeffector cells to EpCAMhighLS174-T and EpCAMlowPaCa-2at different E:Tby Cytotoxicity assay (CCK-8method), and analyzed the statisticaldifferences.Results:1The expression of EpCAM molecules were99.95%and6.70%inLS174-T and Paca-2cells, respectively.2The expressions of surface molecules CD80, CD86, CD83andHLA-DR in LS-mDCs and Ep-mDCs were all higher than imDCs(P <0.05),but no significant difference between LS-mDCs and Ep-mDCs(P>0.05).3The phenotypic changes of each effector cells group cultured in vitroafter d0, d7, d14. In CIK group, the percentage of CD3+CD8+T cells was80.47±1.38%at d14which higher than25.10±0.53%at d0(P <0.05),CD3+CD56+cells was13.37±0.61%at d14which higher than5.73±0.68%atd0(P <0.05). In LS-effector group and Ep-effector group, the percentage ofCD3+CD8+T cells respectively were76.60±2.96%and81.80±2.63%at d14which both higher than25.10±0.53%at d0(P <0.05, P <0.05), CD3+CD4+Tcells respectively were23.33±1.80%and25.77±1.50%at d14which bothlower than30.07±0.85%at d0(P <0.05, P <0.05). In LS-effector group andEp-effector group, the values of CD3+CD4+T/CD3+CD8+T respectively were0.30±0.03and0.30±0.01which both lower than1.20±0.02at d0(P <0.05, P<0.05).4With the target ratio (E: T) increased, the killing rates of effector cells in each group were all raised. In CIK group, the killing rates of CIK toLS174-T were39.59±9.37%,49.48±11.71%and69.39±6.65%at E:T=5:1,E:T=10:1and E:T=20:1respectively, the killing rates at E:T=20:1was higherthan that at E:T=5:1(P <0.05). The killing rates of CIK to PaCa-2were37.68±6.09%,47.10±7.61%and66.33±10.04%at E:T=5:1, E:T=10:1andE:T=20:1respectively, the killing rates at E:T=20:1was higher than that atE:T=5:1(P <0.05). In LS-effector group, the killing rates of LS-effector toLS174-T were53.01±6.91%,66.26±8.64%and74.71±8.36%at E:T=5:1,E:T=10:1and E:T=20:1respectively, the killing rates at E:T=20:1was higherthan that at E:T=5:1(P <0.05). The killing rates of LS-effector to PaCa-2were41.99±6.87%,52.48±8.58%and65.56±8.62%at E:T=5:1, E:T=10:1and E:T=20:1respectively, the killing rates at E:T=20:1was higher than thatat E:T=5:1(P <0.05). In Ep-effector group, the killing rates of Ep-effector toLS174-T were63.34±1.85%,71.07±8.48%and93.57±5.68%at E:T=5:1,E:T=10:1and E:T=20:1respectively, the killing rates at E:T=20:1was higherthan that at E:T=5:1(P <0.05). The killing rates of Ep-effector to PaCa-2were43.40±5.65%,59.42±5.67%and78.37±9.63%at E:T=5:1, E:T=10:1andE:T=20:1respectively, the killing rates at E:T=20:1was higher than that atE:T=5:1(P <0.05).5Compaired the differences of killing rates of effector cells in eachgroup to LS174-T cells and PaCa-2cells at E:T=20:1, respectively. In CIKgroup, the killing rates of CIK to LS174-T and PaCa-2were69.39±6.65%and66.33±10.04%at E:T=20:1, and there was no significant difference betweenthem (P>0.05). In LS-effector group, the killing rates of LS-effector toLS174-T and PaCa-2were74.71±8.36%and65.56±8.62%at E:T=20:1, andthere was no significant difference between them (P>0.05). In Ep-effectorgroup, the killing rates of Ep-effector to LS174-T and PaCa-2were93.57±5.68%and78.37±9.63%, the former was higher than the latter (P<0.05)6Compaired the differences of killing rates among different effector cellsgroup to LS174-T cells and PaCa-2cells at E:T=20:1. To kill LS174-T cells, the killing rates of CIK group, LS-effector group and Ep-effector group were69.39±6.65%,74.71±8.36%and93.57±5.68%, respectively. There weresignificant differences among the three groups (P <0.05), the killing rates ofEp-effector group was higher than LS-effector group (P <0.05) and CIK group(P <0.05), there was no significant difference between LS-effector group andCIK group (P>0.05). To kill PaCa-2cells, the killing rates of CIK group,LS-effector group and Ep-effector group were66.33±10.04%,65.56±8.62%and78.37±9.63%, respectively. There was no significant difference betweeneach two groups (P>0.05).Conclusion:1EpCAM is differentially expressed on many adenocarcinoma cells. Theexpression of EpCAM molecules were99.95%and6.70%in LS174-T andPaca-2cells that has made this a viable target for immunotherapy of LS174-Tcolon adenocarcinoma cells.2ImDCs pushing with LS174-T cells lysate or EpCAM antigen wereboth able to promote their maturation and to generate LS-mDCs and Ep-mDCs.LS-mDCs and Ep-mDCs with antigen-presenting ability to present fullantigens information of LS174-T cells and EpCAM antigen information toautologous CIK to promote them turn to EpCAM antigen-specific cytotoxicCTL and full antigens of LS174-T adenocarcinoma cells-specific cytotoxicCTL.3CIK with immune killing activity can be induced from T lymphocytesderived from PBMCs in vitro. LS-mDCs and Ep-mDCs were both able toactivate autologous CIK cells then to generate LS-effector and Ep-effectorwhich were with the function of an efficient immune destruction.4The efficient killing activity of CIK, LS-effector and Ep-effector toLS174-T cells were highly and raised with the increase of E:T. The killingrates of Ep-effector group was higher than LS-effector group and CIK group.The killing rates of CIK to LS174-T and PaCa-2were approximate, but thekilling rates of Ep-effector to LS174-T was higher than that of PaCa-2. Theresult implied that Ep-effector has a higher specific cytotoxicity for EpCAM. |
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