Establishment Of Leukemia Cell Lines Expressing IL-21/15and Determination Of Their Anti-tumor Effects

Cancer biotherapy is the most inspiring area of cancer therapy. More and more innovative methods appeared and is clinically tried. Cytokine Induced Killer (CIK) cells are among them. Tumor cell vaccines are composed of tumor cells and adjuvant. Antigens on the surface of tumor cells, with the assistance of adjuvants, can stimulate the immune system and elicit the production of memory cell. The specificity of CIK cells and the the proliferation stimulation of tumor cell vaccines have much to be desired. GVAX, which consist of2prostatic tumor cell lines expressing GM-CSF, has proven a potential treatment for prostatic cancer, non-small lung cancer and metastatic melanoma.In this research, we tried the combination of newly defined cytokines and tumor cells for their tumor associated antigen. Leukemia cell lines and IL-21and IL-15were chosen and stable constitutively expression were achieved. The ability of their activation and proliferation on lymphocytes and their potential possibility as tumor vaccines were explored.At first, leukemia cell lines were infected with lentivirus carrying IL-21or IL-15and RFP genes. Then, Fluorescent microscope was used for observing the expression of RFP and flow cytometry was used for analyzing the percentage of RFP-positive cells. The concentration of Interleukins in the medium was detected by ELISA. After stimulation with the Interleukin stable-expressing leukemia cell lines, the amplification folds of PBMC were calculated by cell counter and the surface markers were analyzed by flow cytometry, while their cytotoxicity towards different tumor cells were assessed with CCK8test.As a result, four leukemia cell lines stable-expressing IL-21were established, namely, K562-IL-21, THP-1-IL-21, Jurkat E6-1-IL-21and RPMI8226-IL-21. Four leukemia cell lines stable-expressing IL-15were established, namely, K562-IL-15, THP-1-IL-15, Jurkat E6-1-IL-15and RPMI8226-IL-15.Stimulated by IL-21secreting leukemia cells, the lymphocytes amplification of3different samples varied from1.147±0.057to2.725±0.345times and showed no difference (P>0.01) between and there was no difference among the amplification folds of lymphocytes stimulated by different numbers of leukemia cells (P>0.01), which varied from1.127±0.152to2.213±0.200. The percentage of CD3+T cell and CD56+NK cell decreased (P<0.05) while CD19+B cells increased (P<0.05) after stimulation. The killing rates of activated lymphocytes are much higher than that of the control. The activated lymphocytes had powerful killing capacity on their stimulating leukemia cells and7other tumor cells and the killing rates ranged from (28.6819.31)% to (78.45±0.61)%.Stimulated by IL-15secreting leukemia cells, the lymphocytes amplification of3different samples varied from1.23±0.227～1.59±0.178times and showed no difference (P>0.01) and there was no difference among the amplification folds of lymphocytes stimulated by different numbers of leukemia cells (P>0.01), which varied from1.300.277～1.48±0.222. The percentage of CD3+T cell and CD19+B cells decreased (P<0.05) while CD56+NK cell increased (P<0.05) after stimulation. The killing rates of activated lymphocytes are much higher than the control. The activated lymphocytes had powerful killing capacity on their stimulating leukemia cells and7other tumor cells and the killing rates ranged from (34.84±3.98)%to (78.45±0.61)%.In conclusion, all six leukemia cell lines stable-expressing Interleukin could activate PBMC to kill different tumor cells and show a potential ability as tumor cell vaccines.