The involvement of agonist monoclonal antibody against human CD28 in stimulating proliferative ability and the phenotypic traits of CIK

Objective To investigate the role of agonist monoclonal antibody against human CD28 involved in inducing proliferative ability and cell phenotype traits of CIK.Methods Ficoll separation method was first employed to obtain healthy human peripheral blood mononuclear cells(PBMC),which had an adjusted concentration of 2 × 106/mL,and then were inoculated in 24 culture plates in well format,at 1mL/ hole.Two groups were added an inducer: 1) anti-CD3+IL-2 + IFN-γ,2) anti-CD3 + anti-CD28 + IL-2 + IFN-γ.The final concentration of inducer anti-CD3 was 1μg/m L,anti-CD28 at 0.5μg/mL,IL-2 at 1 000 units/mL,and IFN-γ at 100 ng/mL.Liquid was changed every two days.On the seventh day,cells were cultured.The numbers of cells were calculated in each well.The expressions of surface molecule CD8 +,CD4 +and CD56 + of CIK were analyzed using immunofluorescence single marker analysis.Immunofluorescence double labeling analysis was used for deducing surface molecule expression of CD4+/ CD25+,CD8+/ CD25+,CD4+/ Fas L+,and CD8+/Fas L+of CIK.Results When cells were cultured for up to seven days,agonist monoclonal antibodies against human CD28 group were identified in the total CIK cells,at a concentration of(78.2±7.3) × 106/ hole,which was significantly higher than that of the control group at(27.8±2.7) × 106/ hole.The difference was statistically significant(P0.05).The results of single marker method showed that CD4+cells,CD8 + cells and CD56+cells of CIK were involved in the induction of agonist monoclonal antibody against human CD28 at(68.4±6.1) %,(34.6±7.2) % and(15.1±3.9) %,respectively,compared to the control group with(52.5±3.6) %,(26.9±2.7) % and(7.7±1.2) %,respectively.The differences showed statistical significance(P0.05).The results of double labeling analysis showed that the agonist monoclonal antibodies against human CD28 were involved in the induction of CIK cells in CD4+Fas L + T cells,CD8 + Fas L+T cells,CD4+CD25+T cells and CD8+CD25+T cells at(36.6±4.7) %,(40.7±3.2) %,(60.1±5.3) % and(58.5±4.1) %.Compared to the control group with(21.9±3.9) %,(24.3±5.1) %,(45.6±4.6) % and(44.6± 3.4) %,the differences were statistically significant(P0.05).Conclusion Agonist monoclonal antibodies against human CD28 could enhance the capability of CIK amplification and increase activation of membrane-type molecular expression,suggesting that agonist CD28 monoclonal antibody could improve CIK tumor killing ability.