Functional Characterization Of Foxp3 Gene Transduction Into The Human Natural Killer Cell Lines NKL/NK-92

ObjectNatural killer cells are a key component of the innate immune system and are responsible for initial defences in the surveillance against malignant cells and virally infected cells in the early stage of immune response. Recent research highlights the fact that NK cells are also regulatory cells that could thus limit or exacerbate immune responses by engaging in reciprocal interactions with dendritic cells, macrophages and T cells. NK cells could modulate the regulation of the adaptive and innate immune system to sustain the immune homeostasis. But until now, no evidence about the regulatory NK subsets which exhibited phenotypes and functions similar to regulatory T cells was reported.Foxp3, an X chromosome-encoded forkhead transcription factor family member, is exclusively expressed by regulatory T cells and indispensable for the development and function of Treg cells. In addition, the expression of Foxp3 is crucial for the immunosuppressive activity of regulatory T cells. Foxp3 mutation could lead to lacking of regulatory T cells and autoimmune diseases development. Many reports showed that Naive Foxp3-CD4+T cells could be converted to Foxp3+ T cells by induction with TGF-βas well as gene-transfection, and the induced Treg cells share most phenotypic and functional features of natural Treg cells. More interesting, a recently study found that DC cells shown immunosuppressive activity by ectipic expression of Foxp3. These reports indicated that Foxp3 take functions not only in regulatory T cells but also in other immune cells, ectipic expression of Foxp3 in immune cells might induce immunosuppressive activity.Foxp3 was seen as the'master regulator'of the Treg cell lineage, and no expression of Foxp3 was found in NK cells. According to this, we supposed that ectipic expression of Foxp3 in NK cells may help to investigate the function of Foxp3 and discovery of regulatory NK subsets.Allogeneic hematopoietic stem cell transplantation, a medical procedure involving transplantation of bone marrow or other blood-forming cells, is an effective tool in the treatment of acute leukemia, but often accompanied with graft-versus-host disease (GVHD). NK cells could suppress GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects, and causing no GVHD themselves. Meanwhile, CD4+CD25+Foxp3+ regulatory T cells also contributed to the reduction of GVHD. So, ectipic expression of Foxp3 in NK cells may induce immunesuppressive function which could enhance the suppressive function on GVHD and improve the effectiveness of transplantation.Methods1. Electroporation was used to transfer hFoxp3 gene into NKL/NK-92 cells;2. RT-PCR and Western blot were used to analysis the expression of hFoxp3 in NKL/NK-92 cells;3. MTT was used to determine the proliferation of hFoxp3 transfected NKL/NK-92 cells;4. MTT was used to determine the cytotoxity of hFoxp3 transfected NKL/NK-92 cells against hepotocarcinomar cells;5. Real-time PCR and FACS were used to analysis the expression of cytotoxicity-associated genes in hFoxp3 transfected NKL/NK-92 cells;6. ELISA was performed to detect the concentration of IL-10 in the culture supematants of hFoxp3 transfected NKL/NK-92 cells;7. Luciferase assay was performed to analysis the activity of IL-10 promotor in hFoxp3 transfected HEK293T cells;8. FACS was used to analysis the proliferation and the expression of activity marker CD69 in PBMCs and MTT was used to determine the cytotoxity of PBMCs; IL-10 Neutralization antibody was used to investigate the role of IL-10 in this process.Results1. Ectopic expression of hFoxp3 in NKL/NK-92 cells; 2. The proliferation of NKL/NK-92 cells was reduced by ectopic expression of hFoxp3 gene;3. The cytotoxicity of NKL/NK-92 cells against hepatocarcinoma cells was decreased by hFoxp3 gene in vitro;4. The expression of inhibitory receptor NKG2A in NKL/NK-92 cells was up-regulated by hFoxp3 gene (in mRNA level);5. Eptic expression of hFoxp3 induced IL-10 expression and secretion in NKL/NK-92 cells;6. The activation and proliferation of hPBMC were inhibited by hFoxp3 gene-modified NKL cells by a IL-10 dependent mechanism.ConclusionFoxp3 is a key marker of CD4+CD25+ regulatory T cells and appears highly specific for Tregs. However, induction of Foxp3 in naive CD4+ T cell or DC cells could create Treg-like phenotype. Whether NK cells could be endowed with regulatory properties by transduction of Foxp3 gene is unknown. In this study, a recombinant vector (pRV.GFP Foxp3) or control vector (pRV.GFP WWRR) was transferred into NKL/NK-92 cells by an electroporation method. We found that ectopic expression of hFoxp3 in human NK cells resulted in the decrease of cytotoxicity of NK cells, which concomitant with the up-regulation of inhibitory receptor NKG2A. Interestingly, the immunosuppressive cytokine, interleukin-10, was induced in NK cells by Foxp3 over-expression. Further investigation showed the expression of IL-10 is directly regulated by Foxp3 to some degree. Meanwhile, NKL.Foxp3 cells suppressed the activation and cytotoxic activity of human PBMCs stimulated with PMA and Ionomycin. This immunosuppressive process could be reversed by adding anti-IL-10 neutralizing antibody in the culture medium. These findings indicated that IL-10-secreting NKL.Foxp3 cells display suppressive properties and obviously attenuate the activation of CD3+T cells. For the first time, we observed that ectopic expression of Foxp3 could induce high production of IL-10 and negatively regulatory activity in NK cells. These findings suggested that NK cells acquired IL-10+ phenotype by Foxp3 gene, and NK cells could be endowed with regulatory properties by transduction of Foxp3 gene. In addition, since both NK cells and IL-10 play important roles in suppressing graft versus-host disease (GVHD) in allogeneic bone marrow transplantation, Foxp3 gene-modified NK cells may be potential useful in GVHD or other autoimmune diseases.