Co-stimulation of multiple activating factors on proliferation and phenotype of T lymphocytes in peripheral blood in vitro

AIM: To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro.METHODS: Peripheral blood mononuclear cells(PBMCs) were separated by fractionation on Ficoll-Hypacue gradient.According to adding different cytokines(CD3 mAb,CD28 mAb,IFN-γ,IL-1α,IL-2 and IL-15),the experiments were divided into seven groups.Effects of different cytokines on the proliferation of PBMC were counted by automated hematology analyzer five categories.The phenotypes(CD3,CD4,CD8,CD28,CD16,CD56+CD16,CD3+CD8+,CD3+CD4+,CD3+ CD56+,CD45RO) expressing on the surface of costimulatory cells were detected by flow cytometry,and the cytotoxicity of costimulatory cells on SGC-7901,SW-1990 and SW-116 cell lines was examined by lactate dehydrogenase release method.RESULTS: The proliferation has significant difference when adding different cytokines into PBMCs culture system,the highestest proliferation multiples group is the one contains cytokines CD3,CD28,IFN-γ,IL-2,IL-1α,IL-15 and IL-21,which proliferation multiple is 255.3±6.3 at the tenth day of cell culture,obviously higher than the other culture systems which only contains CD3,IFN-γ and IL-2(166.6±5.5)(P0.05).Part of cells'phenotype changed when adding different activating factors.Without IL-15,the proportion of CD16+CD56+(NK) cells and CD3+CD56+ cells was higher than the other groups;CD45RO+ memory cells is most evident when delayed adding IL-15 and IL-21 for three days.The cytotoxicity of PBMCs cultured for ten days with different activating factors had significant difference,the highest was the one which delayed adding IL-15and IL-21 for three days(76.2%,60.3% and 70.6%,respectively.),higher than the cell culture groups containing CD3,IFN-γand IL-2(54.9%,44.6% and 50.4%,respectively)(P0.05).The cultured cells had the strongest cytotoxicity on SGC-7901 gastric adenocarcinoma cells.CONCLUSION: The PBMCs' proliferation,phenotype and cytotoxicity had significant difference after being activated by different stimulating factors,adding matching stimulating factors into the culture system have great value on cell-directed culture.