Cytokine Regulation Of NK Cell Development And Differentiation

NK cells are important lymphocytes in innate immune system for anti-tumor and anti-virus effects.In the early stage of viral infection,NK cells rapidly respond to kill the infected cells.The development and function of NK cells are regulated by many cytokines.Mouse NK cells can develop in vitro from BM precursor populations in a cytokine cocktail consisting of stem cell factor,IL-7,FLT31 and IL-15.Because cytokines network in vivo is more complex,it is important to know the exact effect of certain cytokine on NK cells.Cytokines or their receptors gene knockout mice are usually used as tools for their functional study,and we can learn the necessity of the cytokines for some cell types.Sometimes due to the compensatory effects,the deficiency of the cytokines signaling will not affect the phenotypes.Administration of cytokines to the mice is another method for studying the effect of cytokines during the cell development.For the half-life of cytokines is usually very short,and the injection of cytokines should be continuously,which will be complicated and expensive.For this reason,we have constructed a human immune system mice and two plasmids for continuously overexpressing hIL-21 and nIL-28B in vivo by hydrodynamics gene delivery.And on this basis,we study the effects of the two cytokines on the development of human and mice NK cells respectively.Ⅰ.Overexpression of hIL-21 improved reconstruction of NK cells in humanized miceHumanized mice is immunodeficient mice transplanted with human hematopoietic stem cells or human lymphocytes or organs,and they will gain of human immune system.For more than three decades of development,humanized mice model is becoming more complete.Human NK cells are still difficult to be reconstructed,and one reason of it is due to the recipients’ remaining immune cells.When severe immunodeficient mice were introduced to construct humanized mice,it got to only a slightly higher level of human NK cells.While administration of hIL-15 led to super-physiological levels of NK cells.To construct a humanized mice model with physiological levels of NK cells,we got the progresses as described below:We transplanted human peripheral blood mononuclear cells(PBMCs)to NOD/SCID mice,whose T cells and B cells are both deficient.The results showed that high levels of human immune system were reconstructed,but the cells were just coming from the engrafted mature lymphocytes without stem cells differentiation.With low percentage of stem cells,PBMCs are difficult to construct human immune system.Then we chose cord blood stem cells as donor cells,and transplanted them into NOD/SCID mice.High levels of human immune cells were reconstructed in the organs,but the levels of human T cells and NK cells were pretty low.Maybe the remaining NK cells in the recipient mice limited their reconstruction.In order to reduce the restriction of NK cells of recipient mice,we chose NOG mice as recipient mice,whose T cells,B cells and NK cells are all deficient.NOG mice transplanted with cord blood stem cells supported human immune system reconstruction with a higher level than that of NOD/SCID mice.To establish a microenvironment for NK cells development as in the humans,we overexpressed hIL-21 in the NOG mice by hydrodynamics gene delivery.We found that hIL-21 promoted human NK cells development in the mice,finally reaching the physiological levels.By imnunohistochemistry,we found the distribution of human immune cells were reconstituted extensively.In the peripheral organs,we detected CD4 and CD8 single positive T cells,and in the thymus,we also detected CD4 and CD8 double positive T cells,meaning human T cells may experience the positive selection.In conclusion,we reconstructed human immune system in the NOG mice,and overexpression of hIL-21 in vivo led to higher levels of reconstruction,especially for NK cells.What’s more,human T cells might experience positive selection in the thymus of the mice,and then they got to peripheral organs after maturation.This humanized mice model will be a powerful animal model for human NK cells research.Ⅱ.IL-28B improves NK cells proliferation and maturation in the miceType Ⅲ interferons are newly discovered cytokines that belong to interferon family,there are four members termed IFN-λ1(IL-29),IFN-λ2(IL-28A),IFN-λ3(IL-28B)and IFN-λ4.The downstream signaling events are similar between type Ⅰinterferons and type Ⅲ interferons,but during viruses infections,type Ⅲ interferons show their unique properties.NK cells play a critical role in anti-virus,but we know little about the role of type Ⅲ interferons in regulating NK cells.We constructed a plasmid to overexpress mIL-28B in vivo by hydrodynamics gene delivery,and confirmed the critical role of IL-28B in development of NK cells.We injected pLIVE-IL-28b or pLIVE-vector to wild type mice by hydrodynamics gene delivery,and NK cells from spleen,liver and lung of the mice were detected by flow cytometry.The results showed that IL-28B increased the percentages and absolute numbers of NK cells systemically.Then we found that the NK cells from IL-28B overexpressing mice showed a significant capacity of proliferation.After that,we detected the different development stages of NK cells by CD11b,CD27 and KLRG1.The results showed overexpression of IL-28B increased the percentages and absolute numbers of mature NK cells.In conclusion,overexpression of IL-28B in vivo promoted NK cells proliferation and maturation.We wondered if overexpression of IL-28B would affect the functions of NK cells,and then isolated NK cells from the IL-28B overexpressing mice,and detected the surface receptors NKG2A/C/E,NKG2D,Ly49CIFH,the secretory molecules IFN-y,perforin and active marker CD69.It was noted that there is no significant difference compared with control mice,suggesting IL-28B does not lead to overactivity of NK cells.Because IL-28Rα is only expressed ojn limited cell types,and there are still controversial opinions about whether IL-28B act on NK cells directly.We found that in IL-28B overexpressing mice,NK cells most significantly increased in the lung,and IL-28Ra was expressed higher on AMs than that on NK cells.To clarify the important role of IL-28B in promoting NK cells proliferation and maturation in vivo,IL-28B overexpressing mice were treated with CL2MDP-lip to deplete macrophages,and then we detected NK cells of the spleen,liver and lung.The results showed the percentages of NK cells in the macrophages depleted mice reduced,and they exhibited more inmmature phenotypes.These data suggested macrophages participated in the process of IL-28B mediated NK cells proliferation and maturation.We stimulated AMs with different concentrations of IL-28B in vitro,and co-cultured with CFSE-labeled NK cells.The results showed 1L-28B stimulated AMs promoted NK cells proliferation in vitro in a dose dependent manner and this process didn’t need cell-cell contact directly.We built a mouse model of influenza virus PR8 infection,and detected the mRNA and protein expression of IL-28.The results showed that influenza virus infection led to increased expression of IL-28 in the lung.After infected with influenza virus PR8,IL-28B expressing mice exhibited reduced bodyweight loss,a higher survival rate and less pathological injury.After IL-28B overexpressed in the ANfil3-/-mice,the protective effect on influenza virus infection was not as good as that in wild type mice,which suggested that the deficiency of NK cells impaired the protection role of IL-28B during influenza virus infection.Taken together,overexpression of IL-28B promoted NK cells proliferation and maturation in vivo in a macrophages dependent manner.IL-28B played a protective role in influenza virus PR8 infected mice model.On one hand,IL-28B limited the replication of virus,and on the other hand,more mature NK cells defended against the influenza virus.To get a better understanding of the biological characteristics of IL-28B will be significant in clinical applications.