| Objective: Leukemia is one of the common cancers in hematological system with an increasing incidence. At present ,though some leukemia patients could avhieve complete remission through chemotherapeutics and bone marrow transplantation, finally drug resistance and relapse would lead to treatment failure in majority of patients. Therefore, it is urgently needed to explore more effective treatments to improve the patient's prognosis and quality of life. There is growing body of research evidence that the immune factors in the control of hematopoietic malignancies play an important role. Immune therapy is becoming hot spots in the treatment of hematologic malignancies.IL-23(interleukin-23) found in the year 2000 is a cytokine excreted by activated dendritic cell (DC) and macrophage cells, it belongs to IL-12 family. It can promote proliferation of memory T cell and induce T cells and natural killer cell (NK cells) to produce interferon-γ(IFN-γ). Therefore IL-23 takes a very important part in immune defence, tumor immunity and some autoimmunity diseases. IL-2 is a self-secreted growth factor produced by T cells, it can stimulate the proliferation and differentiation of cytotoxic Tlymphocyte (CTL) and enhance the cytotoxicity of NK cell. It has now been confirmed that IL-2 has significant anti-tumor effect in vivo, but the effect depends on continuous infusion of exogenous high-dose IL-2 and associated with serious side effects. To reduce the toxic side effects, in recent years, there is a lot of reports about the low-dose IL-2 and other cytokines on proliferation and function of T cells in vitro, and most show synergistic effect.IL-23 can promote T cells to produce less IFN-γ, and will not have serious side effects in the clinical application, so it is possible to become a safer choice of cancer therapy. Now there are no reports on the antileukemia effect of IL-23 combined with IL-2 on leukemia cells at home and abroad. In this study, We investigated these two kinds of cytokines in different concentrations either alone or in combination, to study their impacts on proliferation and anti-leukemia activity of human peripheral blood mononuclear cell. We want to provide a new treatment for leukemia, and improve the clinical outcome of hematological malignancies.Materials and method: We separated human peripheral blood mononuclear cells (PBMC) by ficoll density gradient centrifugation, and cultured them with the culture medium Containing different concentrations of IL-23 alone or in combination with IL-2 . Cell proliferation and PBMC's killing activity for leukemia cell lines K562 was investigated by MTT assay. Changes in cell phenotype of PBMC before or after induction were detected by Flow cytometry (FCM). The results were analyzed by SPSS16.0, and we established the standard of statistic significance asα=0.05.Results: 1. Proliferation of human PBMC activated by IL-23 alone or combined with IL-2: Absorbance (A) of PBMC after treated with various concentrations of IL-23 ng/mL ( 2,10,50) and IL-23 ng/mL +IL-2 IU/mL (2 +10,10+10,50+10,2+100,10+100,50+100) for 1 day were 0.536±0.047,0.628±0.018,0.660±0.036 and 0.643±0.030,0.710±0.031,0.768±0.022,0.732±0.020,0.798±0.047,0.864±0.053, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 3 days were 0.580±0.014,0.654±0.037,0.706±0.014 and 0.690±0.014,0.741±0.014,0.808±0.021,0.791±0.008,0.831±0.023,0.886±0.030, respectively; Absorbance (A) of PBMC after treated with various concentrations of cytokines for 5 days were 0.642±0.009,0.727±0.025,0.796±0.008 and 0.788±0.013,0.841±0.009,0.893±0.023,0.897±0.023,0.958±0.012,1.077±0.151, respectively. Cell proliferation in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the proliferation of PBMC, IL-23 combined with IL-2 can further enhance the proliferation. Within in a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the proliferation of PBMC in a time-and concentration-dependant manner.2. Anti-leukemia cell line K562 activity of human PBMC activated by IL-23 alone or combine with IL-2: The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 ng/mL ( 2,10,50) and IL-23 ng/mL +IL-2 IU/mL (2 +10,10+10,50+10,2+100,10+100,50+100) for 1 day were(8.6±0.42) %,(11.8±0.59) %,(15.2±0.57)% and (10.9±1.50) %,(13.0±0.91) %,(17.9±0.65) %,(16.4±1.07) %,(20.2±1.19)%,(23.5±0.77) %, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 3 days were:(18.7±1.40) %,(23.1±0.25) %,(25.4±0.56) % and (21.4±1.06) %,(24.3±0.47) %,(30.7±0.51) %,(26.4±0.59) %,(32.0±0.98) %,(39.9±1.59)%, respectively; The rate of killing K562 cells of PBMC after treated with various concentrations of IL-23 and IL-23 +IL-2 for 5 days were:(26.2±0.37) %,(30.1±0.68) %,(32.9±0.54)% and (29.9±0.73)%,(34.5±0.64)%,(38.4±1.83) %,(35.9±1.13) %,(43.4±3.92) %,(52.2±3.38) %, respectively. The rates of killing K562 cells of PBMC in experiment groups after treated for 1d, 3d, 5d were significantly different (P<0.05) , at the same time, there was a significant difference between concentration groups and time groups (P<0.05). IL-23 can promote the rate of killing K562 cells of PBMC, IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells by PBMC in a time-and concentration-dependant manner.3. Flow cytometric analysis of PBMC surface markers activated by IL-23 alone or combined with IL-2 : Surface markers of PBMC after treated with IL-23 50ng/mL and IL-23 50ng/mL +IL-2 100IU/mL for 5 days were: CD3+ cells were (80.7±4.37)% and (83.2±4.04)%,CD16+CD56+ cells were (8.4±0.28)% and (12.7±0.9)%,CD4+ cells were (45.2±1.4)% and (47.0±1.72)%,CD8+ cells were (34.5±2.53)% and (35.4±2.11)%. CD4/CD8 ratio were 1.31and 1.33,while the control was 1.27. In both sets of pairs of PBMC as compared with the control group, the increase in cell phenotype were significantly different (P<0.05). The expression of CD16+CD56+ cells between the two groups have significant difference (P<0.05), but there was no significant difference between the two groups of the expression of CD3+ cells,CD4+ cells and CD8+ cells (P>0.05).Conclusions: 1. IL-23 can promote the proliferation of PBMC, and IL-23 combined with IL-2 can further enhance the proliferation with a concentration and time dependent fashion.2. IL-23 can promote the rate of killing K562 cells by PBMC, and IL-23 combined with IL-2 can further enhance the rate. Within a certain concentration range, with increasing concentration and time, IL-23 combined with IL-2 can significantly promote the rate of killing K562 cells of PBMC.3. Surface markers of PBMC after treated with IL-23 alone or combined with IL-2 were increased, and IL-23 combined with IL-2 can further enhance the antigen expression of CD16/56, while CD4/CD8 ratio remains unchanged. We speculate that there was a relationship between Anti-leukemia activity and the proliferation and differentiation of T cells and NK cells.
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