[In vitro anti-tumor effect of cytotoxic T lymphocyte activated by antigen- loaded dendritic cells from peripheral blood mononuclear cells treated with calcium ionophore A23187 and GM-CSF].
OBJECTIVE
To evaluate the effect of calcium ionophore (CI) A23187 and human recombinant granulocyte/macrophage colony stimulating factor (rhGM-CSF) on the cultivation of dendritic cell (DC) from healthy human peripheral blood mononuclear cell (PBMC) and to evaluate the in vitro effect of DC stimulated by K562 cell lysate on inducing specific cytotoxic T lymphocyte (CTL) against K562 cell.


METHODS
Human PBMCs isolated from healthy subjects were separated into two groups. In Group A, the cells were cultured with additional rhGM-CSF, recombinant human interleukin 4 and recombinant human tumor necrosis factor-α only as control group. In Group B, the cells were cultured in the presence of rhGM-CSF and CI A23187. The cells in both groups were pre-incubated with K562 cell lysate at 37°C for 30 min. The cells were harvested after a 4-day cultivation. Morphology of DC was continuously observed under inverted microscope. The surface antigens of induced cells were analyzed by flow cytometry (FCM). Then the proliferation of allogeneic T cell and the specific cytotoxicity of T cell primed with DC were examined by colorimetry. Also, the nonspecific inhibition of DC loaded K562 cell lysate against K562 cell was detected.


RESULTS
Typical morphological features of DC could be observed in both groups. The expressions of CD83, CD1a, CD86 and CD40 were stronger in Group B than those in control group (45.2% ± 1.8%, 31.5% ± 3.9%, 40.1% ± 7.8%, 36.4% ± 6.3% vs 16.9% ± 1.3%, 20.4% ± 3.4%, 26.5% ± 2.2%, 22.3% ± 3.0%) (all P < 0.05). The expression of CD14 was weaker in Group B than that in control group (5.7% ± 0.8% vs 19.0% ± 1.6%) (P < 0.05). As compared with the control group, DC in Group B loaded with K562 lysate could evidently stimulate the proliferation of allogeneic T cell (P < 0.05, exclusion of effector-to-target ratio of 1:40) and inhibit the growth of K562 cell (P < 0.05). In addition, both groups of DC-stimulated CTL had specific cytotoxicity against K562 cell. At the effector-to-target ratios of 10:1 and 40:1, the DC-stimulated CTL of Group B had stronger cytotoxicity against K562 cell (both P < 0.05).


CONCLUSIONS
In combination with rhGM-CSF, CI A23187 induces PBMC into DC in a more effective way. DC loaded with K562 lysate can stimulate CTL and maintain high immunocompetence with specific cytotoxicity against K562 cell.
Peng
Wei-bin
WB
Department of Gastroenterology, Guangzhou First People's Hospital, Affiliated Hospital of Guangzhou Medical College, Guangzhou 510180, China.
Sha
Wei-hong
WH
Li
Yu-yuan
YY
Nie
Yu-qiang
YQ
D000941:Antigens / Q000276:immunology*; D000001:Calcimycin / Q000276:immunology / Q000494:pharmacology*; D003713:Dendritic Cells / Q000276:immunology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism; D007476:Ionophores / Q000276:immunology / Q000494:pharmacology; D020014:K562 Cells; D011994:Recombinant Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000187:drug effects* / Q000276:immunology*
D000941:Antigens; D007476:Ionophores; D011994:Recombinant Proteins; D015847:Interleukin-4; D000001:Calcimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor