Interactions Between Bm-mesenchymal Stem Cell From Acute Leukemic Children And Natural Killer Cell From Cord Blood: Evidence In Vitro And Vivo

Mesenchymal stem cells (MSCs) constitute a rare population of adult stem cells that can be isolated from a simple bone marrow except hematopioetic stem cell. MSCs are known for their characteristic of being multipotent stem cells and capable of forming bone, cartilage, and other mesenchymal tissues[1]. Moreover, MSCs are a component of the bone marrow stroma that have been shown to support hemopoiesis by providing suitable cytokines and growth factors. Besides their regeneration capability, MSCs possess immunomodulatory functions[2], being able to suppress immune reactions both in vitro and in vivo in a non-major histocompatibility complex (MHC) restricted manner. So the therapeutic potential of bone marrow-derived MSCs has recently been brought into the spotlight of many fields of research, not only in blood cancer but also in regenerative medicine and tissue engineering. Currently, both allogeneic bone marrow MSCs and autologous bone marrow MSCs are used in clinic or clinical trials. However, research has showed that the homing of allogeneic bone marrow MSCs are uncertain[3], but autologous MSCs can homing effectively[4]. Moreover, the utility of autologous MSCs should be more safe clinically because fewer infection would occur. So much attention has been put into the autologous bone marrow MSCs. For children with leukemia, the clinical application of autologous MSCs might be unsafe because some leukemia cells would mess into the MSCs during the collection. Research by Li-Ping Wu et al showed that the biological characteristics of bone marrow MSCs from children with leukemia have no significant difference compared with that from normal children[5]. However, it is worthy of further study to evaluate whether there are changes at the molecular level and in the functional representation for this kind of MSCs, especially in the functional studies. Keys for the clinically safe application of autologous bone marrow MSCs from leukemia children may lies on the answers to questions such as whether bone marrow MSCs from leukemia children would be a vicious choice or whethere it would weaken the GVL effects and the ability for eradication of MRD of NK cells, T cells or other immune effector cells and then increasing relapse rate ultimately.Natural killer (NK) cells are major effector cells of the innate immunity and are generally thought to play a fundamental role in antiviral and antitumor responses. As the developing comprehension to their biological and the mechanisms of activation, transfusion of NK cells is becoming a important means to clear residual tumor cells after chemotherapy and HSCT, therfore, more and more efforts are been focusing on its effects as adoptive immunotherapy. Because, the level of NK cells from original source is low (5%-7% in lymphocytes of peripheral blood, and higher in umbilical cord blood), and currently few effective expansion systems ex vivo can be used, its clinical application are restricted. Therefore, it is of great significance to explore efficient expansion system for NK cells in vitro.One of the functions of MSCs is its immunomodulatary effect. Data from researches have demonstrated that MSCs can inhibit T-cell responses induced by mitogens or allo-antigens[6-8]. But the mechanisms underlying such immunosuppressive activity are only in part understood. Acting as major effector cells of innateimmunity, NK cells, like T cells are known to have strong cytolytic activity against tumor or virus-infected cells. As mentioned above, MSCs can inhibit T-cell responses induced by mitogens or alloantigens. However, whether MSCs have a similar impact on the NK cells is still unclear. If the answer is negative, then what is the corelationship between the 2 kinds of cells? In fact, little information is available regarding the cellular interactions between NK cells and MSCs, especially about BM-MSCs from leukemia children.Therefore, with these questions, on the basis of our precursory study about the biological characteristics of MSCs from leukemia children, in this research we attempt firstly to optimize the expansion system for the purified NK cells from cord blood, then delineate the effect of MSCs on NK cells in vitro, with respect to their proliferation, cytotoxic potential and cytokine secretion during activation and then analyze the underlying mechanisms, thereby extending our comprehension on the immunomodulatory properties of MSCs. Furthermore, NOD/SCID mouse are used to establish leukemia model, we exploit this model to investigate the interaction between MSCs and NK cells and their impact on the mouse leukemia model. We hope this research can act as an outpost of exploration for follow-up study of application of immunotherapy relating to CB-NK and leukemia childrens autologous MSCs.Objectives: To investigate optimizing expansion of purified NK cells from cord blood, then delineating the effect of MSCs on NK cells in vitro, with respect to their proliferation, cytotoxic potential and cytokine secretion during activation and then analyzing the underlying mechanisms, thereby extending our knowledge on the immunomodulatory properties of MSCs. Furthermore, NOD/SCID mouse are used to establish leukemia model, to exploit this model to study the interaction between MSCs and NK cells and their impact on the model.Methods: NK cells were isolated from cord blood with miniMACS (magnetic cell-selection) and NK Cell Isolation Kit II. The isolated cells were cultured for 15 days in RPMI-1640 supplemented with 10% FBS and different combinations of IL-2 and IL-12, IL-15. Cultures were fed with flesh media and cytokines every 3 days, and were evaluated for cell expansion, cytotoxicity at the end of the culture period. BM-MSCs from AL children were isolated, cultured and purified in vitro with combination of density gradient centrifugation and adhere segregating culture methods and identified by their morphology characterization, differentiation potential into adipocytes and osteoblasts, phenotype analysis with flow cytometry. MSCs with or without Interleukin-2 (IL-2) were cultured for 5 days with NK cells in 48-well plates or with physically separated NK cells in transwell plates. In above culture system different ratio of NK cells were used. Then the proliferation and cytotoxicty of NK cells were measured with CCK8-kit, the level of IFN-γand Perforin in cultures were dermined by enzyme linked immunosorbant assay (ELISA). Finally, K562 leukemia cells with or without MSCs, were injected into the NOD/SCID mice via lateral tail vein within 24h after the injection of cyclophosphamide in 2mg. Two weeks later, the injected mice begin to showed symptoms of bearing tumor, then NK cells with or without MSCs are injected to the suffering mouse via lateral tail vein to determine whether the tumor was eliminated according to the general situation, survival analysis and pathology tests.Results: The results showed that in group IL-2+IL-l5 and IL2+IL-l5+IL-l2, cells were expanded 50.46±4.31 and 52.35±6.72 fold respectively, much higher than others (p<0.01), but no significant difference between themselves (p>0.05). And the purity of CD3~-CD56~++16~+ NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines was significantly higher than those that were not expanded at different E︰T ratio (p<0.01), although the cytotoxicity of IL-2+IL-l5+IL-l2 group was slightly higherthan that of IL-2+IL-l5 group, but no significant difference between themselves (p>0.05). BM-MSCs of acute leukemia children could be sucessfully cultured in vitro, grow in colonys. The phenotypes analysis showed that the MSCs were positive for CD105, CD29 and CD13 but negative for CD34 and CD45. It also could be induced into adipocytes and osteocytes in appropriate conditions. In MSCs-NK co-cultures, compared with those in the control group, at a MSCs︰NK cell ratio of 1︰5, the proliferation of NK cells was strongly inhibited, but in the groups that MSCs stimulated 24h with IL-2 before, such effect is weakened unexpectedly, and if the MSCs︰NK cell ratio was lower than 1︰100, the the effect of inhibition was lossed. In transwell culture system, in which MSCs could not contact with NK cells directly, no inhibitive effect on NK cells'proliferation was showed. Also, it is showed that cytotoxicity of NK cells against K562 are suppressed and the level of IFN-γand Perforin secreted by NK cells are reduced. All of these effects seemed to be in a dose-dependent manner. There was little differce between transwell groups and directed contiguous coculture groups. But in the group of MSCs stimulated 24h with IL-2 before the co-culture, the suppressions were not obsvious. It indicated that the inhibitory effect of MSCs on the immune cells may be reversible and regulatable. Furthermore, In vivo part, the mouse inoculated K562 cells only being compared with the mouse co-infusion of MSCs and K562 cells, no obvious changes in body weight, symptom such as sluggish, survival time and pathological biomarks were observed. According to above observations, compared with the mouse in which non-NK cells were infused, the biomarks in mouse infused with NK cells or NK cells together with MSCs showed significant differences statistically. No differences were showed between mouse infused with NK cells and those infused with NK cells together with MSCs.Conclusions: It is concluded that NK cells can be eficiently expanded in culture with IL-2+IL-l5. And there are differences in the functions of NK cells cultured with different cytokines. IL-2 and IL-15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. We demonstrate that at different NK cells to MSCs ratios, MSCs suppress proliferation, cytokine secretion, and cytotoxicity of NK cells against target K562 cell. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors. Our research data suggest that different machanisms lie for different MSCs-mediated NK cell suppressions. And MSCs stimulated by IL-2 are susceptible to lysis by activated NK cells. On the other hand, it indicates that the immunoloregulation of MSCs is reversible and ajustable. Finaly, data of our researches demonstrate that MSCs from children with leukemia exert no effect on engraftment of K562 cell, and does not affect the antitumor effect of NK cells from cord blood in NOD/SCID. Overall, these data improve our knowledge about interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graftrejection, and cancer immunotherapy.