The study of special killing effect of CD3AK on anti-CEA-positive tumor enhanced by DC loaded with CEA-rV

Objective： To survey the special killing activity of CD3AK on anti-CEA-positive tumor enhanced by umbilical cord blood dendritic cell （DC） loaded with CEA recombinant vaccinia virus （CEA-rV）. Methods： Freshly isolated umbilical blood mononuclear calls （UBMC） were cultivated for 3 h. Suspension cells and attached calls were used to induce CD3AK calls and DC separately. DC was loaded with CEA-rV on the 3rd day to prepare CEA-rV＋DC. CD3AK cells were co-cultured with CEA- rV＋DC on the 8th day, to prepare CEA-rV＋DC＋CD3AK. The killing activity of each effector＇s cell, which included UBMC, CD3AK, DC＋CD3AK and CEA-rV＋DC＋CD3AK, was measured respectively by MTT reduction assay. Results： （1） 4 target cells were con- firmed by CEA monoclonal antibody of rabbit anti-human. Lovo and A549 were really CEA positive cell lines, while Bel-7402 and K562 were CEA negative cell lines. （2） It was showed by flow-cytometry that the mature DC cultured at 10th day expressed MHC I, II molecules such as CD86, CDS0, CD83 and CD40 highly, but CD123 lowly. The expression rates of CD86, CDS0, CD83 and CD40 was 82.7%, 51.1%, 57.5% and 69.4%, respectively. The appearances and intra-cellular structures of DC were observed through light and electron microscope. The diameter of mature DC was 15-20 μm presented the irregular morphologic appearanca, much prominences and pseudopodium. There were abundant mitochondria and endoplasmic reticulum in DC endochylema. （3） The rates of CD3, CD4, CD8 and CD28 in CD3AK cells group were 2 folds higher than that in UBMC group by FACS. It was said that the numbers of the mature T lymphocyte in CD3AK cells group were much greater than that in UBMC group. （4） The killing activities to 4 target cells of 3 effector＇s cells, which included CEA-rV＋DC＋CD3AK, DC＋CD3AK and CD3AK, were much greater than that of UBMC （P〈0.01）. Moreover, comparing with the killing activities of 4 effector＇s： CEA-rV＋DC＋CD3AK group 〉 DC＋CD3AK group 〉 CD3AK group 〉 UBMC group. It showed that, cytokine, DC and CEA-rV could efficiently elevate the killing activity of UBMC on broad-spectrum tumor cells. （5） Comparing with the killing activities of CEA-rV＋DC＋CD3AK and CD3AK cells to CEA positive and negative cells, the killing activities of CEA-rV＋DC＋CD3AK to CEA positive tumor calls, Lovo and A549 calls （P〈0.01） were remarkably better than that to CEA negative tumor cells BEL-7402 and K562 cells （P〈0.05）. It was said that the CEA-rV＋DC could obviously enhance the killing activity of CD3AK on CEA positive tumor cells. Comparing with the killing activities of CEA-rV＋DC＋CD3AK and DC＋CD3AK cells, the killing activity of CD3AK on CEA negative tumor cells was no statistical difference （P〉0.05）. However, the killing activity to CEA positive cells of CEA-rV＋DC＋CD3AK group was notably higher than that of DC＋CD3AK group. Namely, CEA-rV could distinctly promote the special killing activity to CEA positive tumor cells of CD3AK, but could not do it to CEA negative tumor cells. Conclusion： CEA-rV＋DC could obviously enhance the special killing activity of CD3AK on CEA positive tumor cell lines, while the DC only couldn＇t. The results indicated that the CEA-rV played an important role during the special killing activity of CD3AK cells to CEA positive tumor cells.

Study of Multi-directional Derivation of Cord Blood Mononuclear Cells and Observation of Killing Activity to MGC-803 Gastric Cancer Cell Strain In Vitro

Objective： To study multi-directional derivation of cord blood mononuclear cells to CD3AK, LAK and CIK cells as well as changes of killing activity to gastric cancer cell strain in vitro. Methods： CD3mAb and IL-2 were used to induce CD3AK cells, and IL-2 was used to induce LAK cells; IFN-γ was used in the beginning, then IL-1, CD3mAb and IL-2 were used to induce CIK cells after 24 h for observing amplification and analyzing their relationship. The phenotypes of the cultured CIK cells were analyzed by flow cytometry. Subsequently, by using MGC-803 gastric cancer cell strain as target cells, the killing activity of CD3AK, LAK and CIK cells was evaluated by using MTT method. Results： The amplification activity of CD3AK and CIK cells was all far higher than LAK cells （P〈0.05）. The amplification activity had no obvious difference between CIK cells and CD3AK cells at prophase, but that was far higher in CIK cells than CD3AK cells at about 20^th day （P〈0.05）. The flow cytometry revealed that the amount of CD3^＋ CD56^＋ cells, major effector cells after CIK cells being cultured was significantly increased （P〈0.05）, moreover, the amount of CD8^＋ cells was significantly increased as well （P〈0.05）. The killing activities of CD3AK and CIK cells to the MGC-803 gastric cancer cell strain were all significantly higher than LAK cells, while the killing activity of CIK cells was stronger than CD3AK cells （P〈0.05）. Conclusion： CIK cells have stronger amplification activity and killing activity, and can be taken as more effective killing cells applied to the tumor adoptive immunotherapy.