The Influence Of LSD1 On T Cell And NK Cell Phenotype

Objective: 1. To investigate the influence on the development of T cell and NK cell lineages after deletion of LSD1. 2. To explore the specificity and efficiency of YFP labeled natural killer cells through Vav-Cre induced YFP reporter system in mice.Methods: This project was divided into two part: Part one:Using thymus of C57BL/6 mice, made into single cell suspension, detecting the expression of LSD1 in T cells DP, SP stage by FACS, q RT-PCR; hybriding mice with LSD1(fl/fl) and Lck-Cre+ genotypes, then screened the LSD1(fl/fl) Lck-Cre+ conditional knockout mice from these hybridization mice. In our experiments, LSD1(wt/wt) Lck-Cre- mice(6 weeks) were used as the control group(WT) and LSD1(fl/fl) Lck-Cre+ conditional knockout mice(6 weeks) as the experimental group(KO). Thymus, spleen, lymph node tissue were made into single cell suspensions, after that, analyzed the percentage changes of T cells at various stages through CD4, CD8, CD44 and CD25 cell surface markers by flow cytometry analysis, The same method of analysis to be used in NK cells the percentage change through NKp46, NK1.1 cell surface markers. Part two:Hybrid ROSA26R-YFP and Vav-Cre+ transgenic mice, the double positive gene in mice offspring of ROSA26R-YFP and Vav-Cre were screened through the genotyping analysis, analyzed the expression efficiency of YFP of immune organs thymus, spleen, lymph nodes and bone marrow cells by flow cytometry, screened NK cells of lymph node, spleen and bone marrow by analyzing NK1.1 and CD122 cell markers, and analysis of NK cell groups in the percentage of YFP positive cells by flow cytometry.Results: Part one:(1) Detecting the expression of LSD1 in T cells DP, CD4(SP), CD8(SP) stage by q RT-PCR, the results showed that LSD1 was significantly expressed, and the expression in DP cells was the highest during DP, CD4(SP), CD8(SP) cells, while there was no significant difference in expression of LSD1 in CD4 and CD8 cells, and the expression of LSD1 was gradual reduction in T cell development from DP to SP phase.(2) Through generations of hybrid LSD1(fl/fl) and Lck-Cre+ mice, we obtained three LSD1(fl/fl) Lck-Cre+ conditional knockout mice, which are derived from different parents.(3) Cell counting results showed that, the total thymus T cells of the LSD1 knockout mice(KO) decreased about 10 times compared with the control group of mice(WT)(KO 2.5±0.8×107 vs WT 23.0±7.2×107, P<0.05), while the total number of lymph node T cells(KO 11.0±2.5×107 vs WT13.0±3.7×107, P>0.05) and spleen T cells(KO 12.1±2.8×107 vs WT15.3±1.3×107, P>0.05) had no significant difference.(4) The percentage analysis of DP cells by flow cytometry(KO 63.5±12.4 vs WT 86.2±1.2, P>0.05), CD4 cells(KO 15.3±4.8 vs WT 7.9±0.8, P>0.05) and CD8 cells(KO 10.7±6.1 vs WT 4.5±1.8, P>0.05) in thymus T cells had no significant difference. The percentage of CD8 in the lymph node T cells(KO 13.4±1.0 vs WT 28.3±0.6, P<0.05) compared with the control group decreased by a factor of 2.1, and there were no significant differences between CD4 cells and spleen T cells of CD4 and CD8( P>0.05).(5) Flow cytometry analysis of LSD1 knockout mice(KO),the percentage of thymic DN1 compared with the control group( KO 51.0±11.9 vs WT 16.7±1.3, P<0.05) has increased by about 3.1 times, while DN2, DN3, DN4 were reduced to varying degrees, but there was no significant difference(P>0.05).(6) Analysis of the percentage of NK cells in thymus by flow cytometry(KO 4.2±1.2vs WT 0.6±0.3) increased by about 6 times, the percentage of NK cells in the lymph node cells(KO 8.9±1.0vs WT 2.2±0.8) increased by about 4 times, the percentage of NK cells in spleen cells(KO 22.0±2.2 vs WT 8.7±1.3) increased approximately 2.5 times(P<0.05). Part two: There were 17 progeny of ROSA26R-YFP and Vav-Cre hyberd mice. Among them, there were 11 double positive(ROSA26R-YFP(+ /-) Vav-Cre) mice; the percentage analysis of YFP positive cells in lymph node, spleen, thymus, bone marrow cells by flow cytometry were 73.87±1.51 、 56.07±1.47 、 86.17±1.74 、53.60±3.56, while in the control group, the percentage in the corresponding organs were 0.27±0.01 、 1.33±0.91, 0.11±0.01, 0.29±0.03, these differences were statistically significant(P<0.01). In non-immune organs like kidney, two types mice had no significant expression of YFP(double positive mice were 0.72%±0.43%, 0.92% ±0.27% in control group mice, P>0.05). And YFP positive percentage of NK cells in lymph node, spleen and bone marrow(76.94±0.84、81.66±1.18、88.92±0.77) were significantly increased comparing with the negative control group(P<0.01).Conclusion: This study initiative proved the influence of LSD1 on the development of T cell and NK cell lineages. Deletion of LSD1 may hinder the development of T cell lineages, and promote the NK cell lineages. Labeled NK cells in the mice with specificity and efficiency by Vav-Cre mediated YFP reporter gene system, but the functional analysis of the differentiation of T cells to NK cells need to be further proved.