Screen And Identification Of PCD86-RAE-1ε Transgenic Mice And Analysis Of Their Cellular Immune Function

Innate immunity and adaptive immunity are coordinated in the protective immune responses. The crosstalk between dendritic cells and NK cells not only has an impact on the innate immune response, but also affects the initiation, development and outcome of the acquired immune response. DCs activate resting NK cells by direct cell-to-cell contact and secreting cytokines, and augment anti-tumor immunity by triggering NK cell functions. Meanwhile, NK cells can promote DCs maturation and polarize ThO to Thl, thus enhancing adaptive immunity to mediate immune defense and immune surveillance.MICA (or other NKG2D ligands) mediates the crosstalk between DCs and NK cells directly, In addition, MICA antigens expressed on tumor cells prime NK cells and promote DC to uptake apoptotic debris and induce DCs to cross-present antigen, thus result in initiation of specific immune response. It remained unclear how activities of NK cells would be altered if there are sustained expression of NKG2D ligands on DCs in vivo.In previous study we had developed RAE-1ε transgenic mice which regulated RAE-1ε (a mouse NKG2DL) to express on cells under control of mice CD86gene promoter (pCD86-RAE-1ε mice). Preliminary screen of gene integration by PCR were completed. In this study, we identified RAE-1ε protein expression level and its tissue distribution. Meanwhile, cellular immune function in pCD86-RAE-1ε transgenic mice was observed. This study was divided into two parts as follows:Part I:Identification of RAE-1ε protein expression in pCD86-RAE-1ε miceObjective:To clarify whether and where RAE-1ε protein was expressed among ten founder mice which had been proved to be positive in genotype by PCR. Methods:Firstly the founder mice were sacrificed to collect lung, liver, thymus, intestines and other necessary organs. Whether RAE-1ε was expressed on liver cells or not was identified by Western-blot. RAE-1ε expression in various organizations which were mentioned above was detected by immunohistochemistry. Lastly, RAE-1ε expressions on spleen DCs, B cells and macrophage cells were detected by Flow cytometry (FCM). On this basis, offspring of positive transgenic mice were obtained by conventional mating method and screened by PCR.Results:1) Three positive transgenic mice which were confirmed to show RAE-1expression on APCs among10founder mice.2). pCD86-RAE-1ε homozygous transgenic mice will be expected to gain, s positive rate verified by PCR among the fourth generation offsprings from three positive mouse littermate is51.69%.Conclusions:Three positive transgenic founder mice were obtained through screen of gene integration level and identification of protein expression level, their offspring can be used in subsequent studies.Part Ⅱ:Analysis of cellular immune function in pCD86-RAE-1ε transgenic miceObjective:To observe how high and sustained expression of RAE-1ε on APCs affects relevant cellular immune function.Methods:At first, general vital signs of positive mice were observed. Then mononuclear cells of spleen were isolated both from positive mice and control mice. Frequency of NK, CD8+T, CD4+T cells, NKG2D expression, IFN-y secretion and cytotoxicity of NK and CTL cells were all detected by FCM. Next, spleen-derived DCs and B cells (high expression of RAE-1ε) from transgenic mice were co-cultured with spleen mononuclear lymphocytes from control mice ex vivo. NKG2D expression, IFN-γ secretion and cytotoxicity of NK and CTL cells from wide type mice were also detected by FCM. Finally, levels of serum sRAE-1ε and RAE-1ε antibody were measured by ELISA and compared between transgenic mice and wide type mice. Results:In pCD86-RAE-1ε transgenic mice, there were no other abnormalities of vital signs, except some mice showed a patch of hair loss. Although there were not significant changes of frequencies of NK, CD8+T, CD4+T cells, NKG2D expression, IFN-γ secretion and cytotoxicity of NK and CD8+T cells were all up-regulated. Meanwhile, NKG2D expression on CD4+T cells was enhanced, In addition, NKG2D expression and cytotoxic activity of NK cells from non-transgenic mice were significantly increased in stimulation with DCs/B cells from transgenic mice, whereas production of IFN-y was unchanged. However, NKG2D expression,cytotoxicity, IFN-y production of CTL cells from wide type mice didn’t show significant alterations with the similar stimulation. It suggested that NK cell activity may be enhanced directly stimulated with DCs/B cells of RAE-1ε high expression in short-term, but CD8+T cells couldn’t be influenced in this condition. Finally, there were no significant differences on levels of serum sRAE-1ε and RAE-1ε antibody between transgenic mice and wide type mice.Conclusions:Sustained high expression of RAE-1ε on antigen presentation cells may lead to spontaneous hair loss in mice. Activities of NK and CD8+T cells were up-regulated in the pCD86-RAE-1ε transgenic mice. However, whether there is an inevitable link between hair loss and enhanced function of NK and CD8+T cells need further study.