Research Of Anti-tumor Ability Of Human Peripheral Blood Lymphocyte Subsets

ObjectiveTry to compare anti-tumor ability among different human peripheral bloodlymphocyte, so as to find the most efficient tumoricidal lymphocyte subset. Then it isnecessary to explore a method to amplify the subset in vitro, with the maintenance ofanti-tumor capacity of this subset. Finally, the tumoricidal ability of amplifiedsubsets should be evaluated in vitro and in vivo.MethodsPeripheral blood was collected from healthy volunteers, labeled with humanlymphocyte lineage marks including CD3, CD56, CD8and CD4, for analysis andsorting by flow cytometry. Considering important roles of NK cells and CTLs intumor immunity,subsets of CD3~-CD56~+CD8~+cells, CD3~-CD56~+CD8-cells,CD3~+CD56~+cells, CD3~+CD56-CD8~+cells and CD3~+CD56-CD8bricells were sortedand compared by their kill rates on eight kinds of diversely originated human tumorcell lines in vitro. When the most efficient tumor killer subsets were found,phenotype and cytokine secretion of the subset was detected by flow cytometry. Thenwe try to amplify the subset in vitro, by adding interlukin-2and allogeneic dendriticcells in vitro. To make sure that the amplified subset was originated only from theselected subset in fresh human blood, different lymphocyte subsets in human bloodwere separated and cultured in vitro. Then anti-tumor capability was evaluated on theamplified subset both in vitro and in vivo.ResultsCD8~+CD56~+cells had the highest kill rates on60%of all tumor cell linesalmost in each blood sample. It was shown that activated CD8~+CD56~+cellsexpressed CD25and CD69, secreted IL-2, TNF-α and IFN-γ, but did not expressCD107a. When sorted from fresh human blood, only the subset of CD8~+CD56~+cells could be amplified to CD8~+CD56~+cells. Then it was established that CD8~+CD56~+cells could be amplified in vitro by adding IL-2and allogeneic dendritic cells. Theamplification of the subsets did not cause any damage to their killing capacity ontumor cell lines. Amplified CD8~+CD56~+cells showed high tumoricidal efficiency onHela cells in vitro and on HepG2cells in vivo.ConclusionCD8~+CD56~+cells had the highest kill rates against tumor cells and the mostwidely anti-tumor capacity. These cells expressed markers expressed on activated Tcells and secreted cytokines such as IL-2, TNF-α and IFN-γ. However, CD107a wasnot detected on these cells, indicating another mechanism to kill tumor cells exceptthe way by granzyme. CD8~+CD56~+cells could be amplified when stimulation of IL-2and allogeneic dendritic cells, and did not alter their phenotype during theirproliferation. The amplified CD8~+CD56~+cells also maintain their high tumoricidalcapacity both in vitro and in vivo.