IL-35Promotes Immune Escape Of Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults, but the pathogenesis of it is very complicated and until now it has not been completely clarified. Recent studies demonstrated that gene mutation, chromosomal translocations and immune dysfunction have been important factors in the tumorigenesis of AML. CD4+CD25+regulatory T cells (Tregs) is a special population of CD4+T cells, which can not only directly target effector immune cells in a cell-cell contact manner, but also can suppress the immune response via secreting some inhibitory cytokines, such as IL-10and TGF-β. Thus, Tregs is considered to control immunologic tolerance to self-antigens and play a role in suppressing antitumor immune responses. In our previous studies, we and many other groups have found that AML progression resulted in a progressive Tregs accumulation both in the peripheral blood and bone marrow. Those Tregs does not only inhibit the effector T cells (Teffs) proliferation and secreting cytokines,but also inhibits the adoptive cytotoxic T cells (CTLs) proliferation and spreading in AML. In addition, the classical immunosuppressive cytokines IL-10and TGF-P secreted by Tregs have also been considered to be involved in the pathophysiological process of AML. However, recent studies have reported that Tregs depletion by a brief course of anti-CD25monoclone antibody or IL-2diphtheria toxin (IL-2DT) transiently reduced AML disease burden but does not permit long-term survival, meanwhile, blockade of IL-10and TGF-β does not absolutely abrogate the suppressive activity of Tregs, suggesting other mechanisms may be responsible for Tregs-mediating suppression. IL-35, as a novel cytokine identified in2007, belongs to IL-12cytokines family and plays an important role in the development of immune tolerance. In mice, IL-35may be specifically produced by Tregs and required for their maximal suppressive activity in vitro and in vivo. In contrast to mice counterpart, its expression in human Tregs is still of controversial discussion. The data have demonstrated that IL-35significantly suppresses the Teffs proliferation and responses, as well as promotes myeloid-derived suppressor cells (MDSCs) accumulation and angiogenesis. Moreover, the predominant mechanism of IL-35suppression is related to its ability to improve Tregs differentiation, proliferation and function. Especially, IL-35mediates a novel Tregs subpopulation named iTr35, which cascades the immunomodulatory effects of Tregs and constitutes a key mediator of infectious tolerance and tumor immune escape. However, to our knowledge, the exact role and mechanism of IL-35on AML is yet to be elucidated.In this study, we collected28newly diagnosed adult AML patients, and followed by27complete remission ones and7relapsed ones, meanwhile,30age-matched healthy adults were selected as controls. Then, we explored the following four issues:Firstly, the expression of IL-35in AML; Secondly, the source of IL-35in AML; Thirdly, the impact and mechanism of IL-35on AML cells immune escape; Lastly, the methods to improve the adoptive immune cells killing ability against AML cells via targeting IL-35. The purpose of this study is to understand deeply the mechanisms of AML cells immune escape and provide a potential target or new approach for the immunotherapy of AML.Finally, we found that:(1) To investigate the peripheral immune micro-environment of AML patients, we conducted a systematic analysis of immune cell subsets and their related-cytokines. Compared with healthy control group and complete remission AML group, we found NKT, B, CD4+T, CD8+T, Thl, Th17cells and IL-15, sCD25, IL-2, IFN-y, IL-17were significantly decreased but NK, Th2, Tregs cells and IL-4, IL-10, TGF-β were significantly increased in newly diagnosed AML group and relapsed AML group. These data suggested that AML patients have been in a significant immune suppression status, favoring the survival of malignant hematopoietic cells.(2) Either on mRNA level or on protein level, we both found IL-35were increased in newly diagnosed AML and relapsed AML groups compared with healthy control and complete remission AML groups. So, we believed that IL-35is positively correlated to AML occurrence and development and may play a suppressive role in antitumor immune response.(3) To further investigate the source of IL-35in AML, Tregs and Teffs were both isolated from AML patients and cultured in vitro with anti-CD3and anti-CD28monoclonal antibody stimulation. Then, we found that the high production of IL-35either on mRNA or protein level was only presented in stimulated Tregs, but not for Teffs and un-stimulated Tregs. Therefore, we believed Tregs-derived from AML patients expresses IL-35in a stimulation-dependent manner. Similarly, AML cells were also isolated from AML patients and assessed the ability to produce IL-35. Then we found the AML cells-derived from most patients expressed neither the p35subunit nor the EBI3subunit, or only expressed the p35subunit. But a part of AML cells-derived from few patients both expressed the p35subunit and the EBB subunit, which indicated that those AML cells were another source of IL-35in some AML patients. In addition, we tested and compared the expression of iTr35between AML patients and healthy adults, and found that iTr35was significantly increased in AML patients and highly produced IL-35either in resting or activated status.(4) In the functional studies, we firstly investigated that IL-35inhibited the AML patients-derived Teffs proliferation and the cytotoxicity against AML cells. Then, Tregs-isolated from AML patients were cultured with plate-bound anti-CD3and anti-CD28monoclonal antibody for72hours, and IL-35was added at24hours after the start of culture. We found IL-35significantly induced the proliferation of Tregs with elevation of IL-35both on mRNA and protein levels. Next, we co-cultured these IL-35-expanded Tregs with Teffs, and found them remained highly suppressive against Teffs. Similarly, we analyzed iTr35and found IL-35also significantly promoted its proliferation and cytotoxicity against Teffs.(5) We found AML cells expressed constitutively the IL-35R subunits IL-12RP2and gp130, and the stimulation of IL-35significantly up-regulated the expression of them, suggesting IL-35might directly interact with AML cells. Then, we found IL-35significantly induced the proliferation of AML cells as compared with PBS control group. Moreover, we confirmed that VP-16increased the apoptosis of AML cells, but IL-35pre-stimulation significantly down-regulated the apoptosis of AML cells induced by VP-16.(6) Cytokine-Induced Killer Cells (CIK) cells are cytotoxic immune effector cells that are readily expandable and express the T-cell marker CD3, as well as the NK-cell marker CD56, and are now considered as the primary candidate for adoptive cellular immunotherapy for AML. However, in this study, we found Tregs and Tregs-secreted IL-35concomitantly expanded by a time-dependent way during the generation of CIK cells and significantly decreased CIK cells cytotoxicity against AML cells. We known CIK cells are usually generated by the in vitro culture of mononuclear cells with IL-2because of it is a critical lymphocyte stimulator, but other common yc-signaling cytokines, such as IL-15, may be alternative choices for the optimal culture of CIK cells. In this study, we found that the application of IL-15instead of IL-2during the generation of CIK cells in vitro downregulated the production of Tregs and Tregs-related IL-35, but resulted in a significant enhancement of CIK cells-mediated cytotoxicity against AML cells, suggesting that IL-15may improve the cytotoxicity of CIK cells by inhibiting the production of Tregs and IL-35.(7) Dendritic cells (DCs) are the most powfull antigen-presenting cells, recent developments and advances are made in the generation of CIK cells combining with DCs. In this study, compared with conventional CIK cells, our data reified that Tregs and Tregs-related IL-35were significantly down-regulated in DC-CIK cells, but DC-CIK cells exhibited stronger proliferation ability and were more effective on killing AML cells. We believed that DC-CIK cells may be used for the induction of a specific immune response against AML cells by blocking the properties of Tregs and Tregs-related IL-35.In conclusion, our study demonstrated that AML patients have been in a significant immune suppression status, favoring the survival and proliferation of AML cells. The novel cytokine IL-35, which might be derived from Tregs and iTr35, even some AML cells, was highly expressed in AML patients and significantly correlated with the clinical development of malignancy. IL-35promoted AML cells immune escape by inhibiting Teffs as well as promoting Tregs and iTr35, and also directly promoted the proliferation and inhibited the apoptosis of AML cells. What’s more, Tregs and Tregs-secreted IL-35concomitantly expanded during the generation of CIK cells and significantly decreased their cytotoxicity against AML cells, whereas co-culture with DCs or the application of IL-15instead of IL-2during the generation of CIK cells improved the cytotoxicity of them by inhibiting the production of IL-35. Thus, our findings suggest that IL-35promotes immune escape of AML cells, and may represent a new potential therapeutic target for AML immunotherapy.