OBJECTIVE: A lot of studies have suggested that a certain amount of T cells may be involved among cytokine-induced killer (CIK) cells. The aim of the present study was to prove whether an antigen-specific killing effect on tumor cells is involved during the CIKs-induced killing process. METHODS: Bone marrow mononuclear cells (BMMNCs) derived from healthy subjects were separately cultured to generate dendritic cells (DC) and CIKs. A human mammary cancer cell line MCF-7/ADR, expressing P-gp antigen, was frozen-thawed and the lysate including P-gp antigen was obtained. The DC pulsed with or without tumor antigen lysate was co-cultured with CIK (pulsed-DC + CIK and DC + CIK), and CIK cultured alone was used as control. The cell phenotype of DC and CIK was analyzed by flow cytometry. The secretion of IL-12 and IFN-gamma was assayed by ELSA. The antitumor effect of the three CIK groups targeted at MCF-7/ADR cells expressing P-gp antigen and MCF-7 cells was detected by MTT. RESULTS: Pulsed-DC + CIK group and DC + CIK group showed a higher expression level of DC mature phenotypes than those before co-culture with CIK, with a significant difference (P = 0.003, P = 0.001, respectively). The phenotypes (CD3, CD8, CD56) of CIK in pulsed-DC + CIK group and DC + CIK group was higher than those in CIK group (P = 0.003, P = 0.011, respectively). Among the three CIK groups, pulsed-DC + CIK group had the highest phenotypes on CD3+ CD56 (pulsed-DC + CIK vs. DC + CIK, P = 0.001; pulsed-DC + CIK vs. CIK, P < 0.001) and CD3 CD8 (P = 0.002, P = 0.002, respectively). Among the three groups, the pulsed-DC + CIK group showed the lowest CD45RA phenotype (pulsed-DC + CIK vs. DC + CIK, P < 0.001; pulsed-DC + CIK vs. CIK, P = 0.004). Among the three groups the secretion of IL-12 and IFN-gamma had the highest level in pulsed-DC + CIK group, with a value of 254 +/- 14.5 pg/ml and 3100 +/- 286 pg/ml, respectively. The antitumor killing effect on MCF-7/ADR cells had a significant difference between any two groups (pulsed-DC + CIK VS. DC + CIK, P = 0.039; pulsed-DC + CIK VS. CIK, P = 0.002; DC + CIK vs. CIK, P = 0.049). The highest was in pulsed-DC + CIK group and the lowest was in CIK group. The CIK group showed a significantly lower antitumor effect on MCF-7 cells than the other two groups (pulsed-DC + CIK vs. CIK, P = 0.007; DC + CIK vs. CIK, P = 0.048), but no significant difference between the pulsed-DC + CIK and DC + CIK groups. CONCLUSION: In the present study, DC and CIK cells have been successfully obtained and cultured from bone marrow mononuclear cells. After their co-culture, not only both their specific phenotypes were increased, but also the associated cytokines were secreted. An improved antitumor killing effect and some possible specific immunocytotoxicity were observed. Our findings provided a basis for experimental and clinical research on bio-immunotherapy targeted at multi-drug resistant tumor cells.
OBJECTIVE: A lot of studies have suggested that a certain amount of T cells may be involved among cytokine-induced killer (CIK) cells. The aim of the present study was to prove whether an antigen-specific killing effect on tumor cells is involved during the CIKs-induced killing process. METHODS: Bone marrow mononuclear cells (BMMNCs) derived from healthy subjects were separately cultured to generate dendritic cells (DC) and CIKs. A human mammary cancer cell line MCF-7/ADR, expressing P-gp antigen, was frozen-thawed and the lysate including P-gp antigen was obtained. The DC pulsed with or without tumor antigen lysate was co-cultured with CIK (pulsed-DC + CIK and DC + CIK), and CIK cultured alone was used as control. The cell phenotype of DC and CIK was analyzed by flow cytometry. The secretion of IL-12 and IFN-gamma was assayed by ELSA. The antitumor effect of the three CIK groups targeted at MCF-7/ADR cells expressing P-gp antigen and MCF-7 cells was detected by MTT. RESULTS: Pulsed-DC + CIK group and DC + CIK group showed a higher expression level of DC mature phenotypes than those before co-culture with CIK, with a significant difference (P = 0.003, P = 0.001, respectively). The phenotypes (CD3, CD8, CD56) of CIK in pulsed-DC + CIK group and DC + CIK group was higher than those in CIK group (P = 0.003, P = 0.011, respectively). Among the three CIK groups, pulsed-DC + CIK group had the highest phenotypes on CD3+ CD56 (pulsed-DC + CIK vs. DC + CIK, P = 0.001; pulsed-DC + CIK vs. CIK, P < 0.001) and CD3 CD8 (P = 0.002, P = 0.002, respectively). Among the three groups, the pulsed-DC + CIK group showed the lowest CD45RA phenotype (pulsed-DC + CIK vs. DC + CIK, P < 0.001; pulsed-DC + CIK vs. CIK, P = 0.004). Among the three groups the secretion of IL-12 and IFN-gamma had the highest level in pulsed-DC + CIK group, with a value of 254 +/- 14.5 pg/ml and 3100 +/- 286 pg/ml, respectively. The antitumor killing effect on MCF-7/ADR cells had a significant difference between any two groups (pulsed-DC + CIK VS. DC + CIK, P = 0.039; pulsed-DC + CIK VS. CIK, P = 0.002; DC + CIK vs. CIK, P = 0.049). The highest was in pulsed-DC + CIK group and the lowest was in CIK group. The CIK group showed a significantly lower antitumor effect on MCF-7 cells than the other two groups (pulsed-DC + CIK vs. CIK, P = 0.007; DC + CIK vs. CIK, P = 0.048), but no significant difference between the pulsed-DC + CIK and DC + CIK groups. CONCLUSION: In the present study, DC and CIK cells have been successfully obtained and cultured from bone marrow mononuclear cells. After their co-culture, not only both their specific phenotypes were increased, but also the associated cytokines were secreted. An improved antitumor killing effect and some possible specific immunocytotoxicity were observed. Our findings provided a basis for experimental and clinical research on bio-immunotherapy targeted at multi-drug resistant tumor cells.