| AML is the most common adult hematopoietic malignancy, but its pathogenesis is very complicated, it is widely accepted that both genetic and environmental factors play significant roles in the development of the disease, and the specific pathogenesis process involving multiple pathogenic factors in the interaction of multiple steps. There are numbers theory related to the pathogenesis of AML, and the theory of "Two-hit" is representative, which consider that the happening of AML include at least two stages in general: the decisive mutations of genes within a single cell which is caused by various reasons activate certain signaling pathways, and led to the cloned abnormal hematopoietic cells and strong proliferation and blocked apoptosis, further genetic changes and involved in some transcription factors, lead to the cloned abnormal hematopoietic cell differentiation blocking or disorder, eventually caused AML.It is believed that the dynamic balance of the expression and function of various immune cells and their related immune molecules is an important guarantee for the maintenance of the immune function of the body. Therefore, most of the diseases occur in addition to their own special pathogenesis, almost all related to the body’s functional defects caused by immune balance disorder. Our preliminary study suggests that immune dysfunction is also an important reason for the occurrence and development of AML. CD4+CD25+ regulatory T cell(Tregs) is a group of immune negative control cells, have immune suppressive function with down regulated immune response, through cell to cell contact or inhibit the secretion of cytokines to inhibit the activity of immune effector cells, plays an important role in immune regulation, maintenance of peripheral immune tolerance and prevention of autoimmune diseases. Recent studies have demonstrated that tumor immune escape mechanism governed by CD4+CD25+ regulatory T cells(Tregs) which is a key factor in the tumorigenesis of AML. IL-35, composed of an IL-12 subunit p35 and an IL-27 subunit Epstein-Barr virus induced gene 3(EBI3), is a newly identified heterodimeric cytokine. In mice, IL-35 is produced by Tregs specifically. But in the human body, the source and expression of IL-35 are not completely clarified. Now, it is generally accepted that IL-35 is a kind of new inhibitory cytokines, it can significantly induce the Tregs proliferation, suppresses the Teffs proliferation and responses. In addition, IL-35 can induced naive T cells into a novel Tregs subtype, which be named as “i Tr35”, to cascade amplify the immunoregulatory effects of Tregs. In view of the key role of IL-35 in the differentiation and functional activity of Tregs, we speculate that IL-35 could promote accumulation and function of Tregs in AML patients, eventually lead the balance between Teffs and Tregs to inhibit bias, which is conducive to the immune escape of AML cells. In addition, senescence gene SENEX may protect Tregs by inducing Stress induced premature senescence(SIPS), and participate in the occurrence and development of AML.In the early study, we collected the peripheral blood samples of AML patients, and made a preliminary study on the expression of IL-35 in AML and the effect and mechanism of inducing AML cell immune escape. The results showed that the expression of IL-35 was increased in AML patients, and it was closely related to the occurrence and development of the disease. IL-35 can promote the expression and function of Tregs and i Tr35, inhibit the function of Teffs, so as to mediate the immune escape of AML cells. This paper further studies the inhibitory cytokine IL-35 expression in bone marrow of patients with AML and its direct effect on AML cells. Combined with previous related research and this study, we explore the effects of IL-35 on the immune function of AML and its mechanisms.20 adult ND AML patients were enrolled into this study, and followed for two years. Among all the ND AML patients, 17 patients achieved CR, but relapse occurred in 5 patients. For controls, 7 age- and sex- matched iron-deficiency anemia adults were collected. And then we analyzed IL-35 expression and its effect on AML cells in bone marrow of patients with AML. The specific experimental methods and results are as follows:(1) we examined IL-35 by PCR, ELISA, FCM and IHC, and found that IL-35 was expressed highly in bone marrow of adult ND AML patients compared to controls. The results suggest that IL-35 is associated with the incidence of AML.(2) We examined the concentration of IL-35 protein in different stages of AML by using a unique human IL-35 ELISA kit. The concentrations of IL-35 in bone marrow were found to be significantly higher in ND group and relapse group compared to CR group and control group. The results show that IL-35 is closely related to the development of AML.(3) We examined Tregs in different stages of AML by FCM. The proportion of Tregs in bone marrow were found to be significantly higher in ND group and relapse group compared to CR group and control group. The Person correlation analysis showed that the levels of IL-35 were positively correlated to their respective levels of Tregs and Foxp3 in bone marrow of ND AML patients, preliminary suggested that Tregs may be one of the sources of IL-35, or other sources of IL-35 can significantly promote the expression of Tregs in patients with AML.(4) We cultured and intervened NB4 cells. Through analyzing the proliferation and apoptosis of NB4 cells which are stimulated by IL-35, we found that the IL-35 R subunits IL-12Rβ2 and gp130 are expressed in NB4 cells constitutively and the expression of them increased after IL-35 stimulation. Meanwhile, compared to PBS group, IL-35 also significantly promoted the proliferation of NB4 cells. Further experiments found that IL-35 pre-stimulated NB4 cells can significantly resist the apoptosis induced by Ara-C.(5) We cultured AML blasts which isolated from AML patients by FCM. By analyzing the proliferation of AML blasts which are stimulated by IL-35, we also found that IL- 35 significantly induced the expression of the IL-35 R subunits IL-12Rβ2 and gp130. Similarly, IL-35 significantly induced the proliferation of AML blasts. Apoptosis experiment also found that AML blasts which are pre-stimulated by IL-35 can significantly resist the apoptosis induced by Ara-C. The results indicated that IL-35 can binding to its receptor, and significantly promote the proliferation of AML cells and inhibit the apoptosis of AML cells.(6) We examined SENEX m RNA in different stages of AML by Real-time PCR, and found that SENEX m RNA was expressed highly in bone marrow of adult ND AML patients compared to controls. When the patients achieved complete remission, the expression of SENEX m RNA was down-regulated, but the levels of SENEX m RNA increased again when relapsed. The results suggest that SENEX gene is closely related to the development of AML.In conclusion, AML is a common hematological malignancy, whose pathogenesis is related to immune factors. It is generally believed that the accumulation of restrain cells and cytokines, making the balance of the immune system is breaking and the immune function is inhibited, so as to be in favor of AML cells immune escape, eventually lead to AML occurrence and development. In this study, we found that the inhibitory cytokine IL-35 was highly expressed in AML patients. IL-35 involved in AML by promoting Tregs and i Tr35 as well as inhibiting Teffs, and also directly promoted the proliferation and inhibited the apoptosis of AML cells. What’s more, SIPS by SENEX gene triggered have anti-apoptotic and immune-suppressing effect, so we speculate that the SENEX gene triggering senescence is the one of mechanisms for promoting immune escape in AML. |
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