Effect Of A Immunomodulator Composed Of Muramyl Dipeptide And Anti-CD10 Monoclonal Antibody On The Activation Of Memory T Cells In Vitro

Objective:Memory T lymphocytes have the capacity of long-term self-renewal and survival in comparison with effector T lymphocytes.While interacted with the same antigen again,they can proliferate rapidly and cause more intense cellular immune responsein the body,establishinga long-lasting anti-tumor immunity.In this paper,we aim to explore the impact of the new immunomodulator that combine muramyl dipeptide with anti-CD10 monoclonal antibody(MDP-Ab),which is intended for the treatment of leukemia by activating the specific memory T lymphocytes in peripheral blood of healthy children.Methods:1.Peripheral blood mononuclear cells(PBMC)of healthy children were isolated by Ficoll-Hypaque method and cultured in RPMI-1640 medium containing 10%fetal bovine serum.The PBMCs(1×106/m L)were divided into three groups: unstimulated group(anti-CD28 Mab,0.5?g/m L),MDP-Ab group(anti-CD28 Mab+MDP-Ab,20?g/m L),BCG group(anti-CD28 Mab+BCG,20?g/m L).The supernatant of cells was collected at the time points of 0,12,24,48 and 72 hours respectively,and the IFN-?levels were measured by ELISA.2.The whole blood was diluted with PBS 1: 1,and were divided into four groups:control,MDP-Ab,BCG and PHA(anti-CD28 MAb+PHA 20?g/m L).After 48-hour incubation,the cells were collected.The flow cytometry was applied to analyze the expression of surface molecules of CD3?CD4?CD45RA?CD69 and cytokines of IFN-?.Results:1.Morphology of cells: Compared with the control group,the cells in the MDP-Ab group and the BCG group showed cell aggregation after 24 hours of culture,and the cell morphology in the cell mass was discernible.As the time went on,the number of cell clusters increased,which was the most obvious in BCG group.2.The levels of IFN-? in the supernatant of the control group at different time point(0,12,24,48 and 72 hours after treatment)were(401.76±17.10)pg/ml,(374.64±24.30)pg/ml,(376.53±28.96)pg/ml,(380.02±29.15)pg/ml,and(390.17±33.64)pg/ml.In parallel,the levels in MDP-Ab group were(402.04±23.22)pg/ml,(421.49±24.09)pg/ml,(456.76±41.99)pg/ml,(479.81±21.43)pg/ml,(449.10±13.66)pg/ml.The levels in BCG group were(405.34±30.48)pg/ml,(404.98.70±37.08)pg/ml,(444.33±22.94)pg/ml,(502.20±15.52)pg/ml and(517.84±21.54)pg/ml.The levels of IFN-?in the MDP-Ab group and BCG group were significantly higher than those of the control group at anytime point(F=13.19,F=5.87,p<0.05;analyzed by repeated measurement design).The overall effect of prolonged culture time in BCG group was significant(F=26.60,p <0.01),but in MDP-Ab group was not significant(F=1.65,p =0.21).The levels in BCG group were(405.34±30.48)pg/ml,(404.98.70±37.08)pg/ml,(444.33±22.94)pg/ml,(502.20±15.52)pg/ml and(517.84±21.54)pg/ml.The interaction between BCG stimulation and culture time was significant(F=5.49,p < 0.01).Besides,there were significant differences between the two treatment groups and the control groupat 24 and48 hours after stimulation(p<0.05).3.The expression of intracellular cytokine of IFN-?: ? CD3+CD4+CD45RAIFN-?+%: After 48-hour incubation,the percentage of CD3+CD4+ CD45RA-IFN-?+cells in PBMC in MDP-Ab group,BCG group and PHA group were 0.61±0.20(p=0.034),1.05±0.45(p=0.057)and 0.27±0.19(p=0.137)respectively,compared to the control group(CD3+CD4+CD45RA-IFN-?+%= 0.010±0.017).The proportion of CD3+CD4-CD45RA-IFN-?+cells was undetermined.4.The expression of cell surface molecules of CD69: ? CD3+CD4+ CD45RACD69+%: The CD3+CD4+CD45RA-CD69+cells% in MDP-Ab(8.99±2.75,p=0.01),BCG group(13.69±2.79,p<0.01)and PHA group(8.44±3.25,P>0.05)was significantly higher than that in control group(4.70±1.16).? CD3+CD4-CD45RA-CD69+%: The CD3+ CD4+ CD45RA-CD69+ cells% in MDP-Ab(3.91±1.73,p<0.05),BCG group(6.76±3.28,p<0.01)and PHA group(5.15±1.79,p>0.05)was significantly higher than that in control group(1.41±0.31).? The CD3+CD4+CD45RA-CD69+ cells% in MDP-Ab(8.99±2.75)was significantly higher than CD3+CD4-CD45RA-CD69+%(3.91±1.73),(p<0.05).The CD3+CD4+CD45RA-CD69+cells% in BCG(13.69±2.79)was significantly higher than CD3+CD4-CD45RA-CD69+%(6.76±3.2 8),(p<0.05).Conclusion: MDP-Ab can stimulate the secretion of IFN-? in memory T lymphocytes of healthy children,and induce CD69(early activation marker)expression on CD4+CD45RA-T lymphocyte.The immune response led by MDP-Ab is similar with that of BCG stimulation,suggesting that MDP-Ab can induce BCG antigen-specific CD4+ T lymphocyte activation in vitro.The study provides further theoretical support for application of the MDP-Ab in leukemia immunotherapy.