RNAseq of lymphocyte populations in Graft vs Host Disease. RNAseq of lymphocyte populations in Graft vs Host Disease

In this experiment we harvested donor T cells from recipient allogeneic mice and examined changes in gene expression in FLICA (caspase-1 activation) positive and negative T cell populations Overall design: Splenocytes were harvested from the indicated donor mouse strain and T cells were isolated by negative selection using a negative isolation kit (EasySep Mouse T Cell Isolation kit; STEMCELL Technologies) or a cocktail of biotinylated antibodies (anti-B220 [RA3-6B2], anti-CD11b [M1/70], anti-CD11c [N418]), followed by incubation with magnetic nanoparticles conjugated to streptavidin (BD IMag Streptavidin Particles Plus; BD Biosciences) and magnetic isolation. Purity (>90%) was confirmed by flow cytometry staining for T cells (anti-CD3) (Fig. S1). 0.75-1x106 T cells + 1x107 BM cells were resuspended in PBS with heparin and intravenously injected into BALB/c recipient mice via tail vein injection. Human HLA-A2 negative PBMCs were isolated from adult healthy donors. 10 million PBMCs were resuspended in PBS with heparin and injected into NSG recipient mice. On the day of transplantation, BABL/c recipients received a total body lethal dose of irradiation (8 Gy) and NSG recipients received (2.5 Gy) from an X-ray source (RS 2000 Biological Research Irradiator). Body weights were recorded and clinical signs observed throughout the experiment. On day 7 (BALB/c) or day 10 (NSG), mice were sacrificed for ex vivo analyses. For survival experiments, experiments were carried out for 80 days. When mice reached weight loss >25% of their initial body weight or hit end of life criteria, mice were considered clinically dead.

本实验中，我们从同种异体供体小鼠中获取供体T细胞，并检测了FLICA（caspase-1激活）阳性与阴性T细胞群体的基因表达变化。实验整体设计如下：从指定供体小鼠品系中分离脾细胞，采用阴性分选试剂盒（EasySep小鼠T细胞分离试剂盒；STEMCELL Technologies）或生物素化抗体混合物（抗B220 [RA3-6B2]、抗CD11b [M1/70]、抗CD11c [N418]），与链霉亲和素偶联磁纳米颗粒（BD IMag链霉亲和素磁珠加强型；BD Biosciences）孵育后进行磁分选以纯化T细胞。通过流式细胞术检测T细胞表面标志物CD3（抗CD3抗体）验证细胞纯度（>90%），详见补充图S1。将0.75×10^6~1×10^6个T细胞与1×10^7个骨髓（BM）细胞重悬于含肝素的磷酸盐缓冲液（PBS）中，经尾静脉注射至BALB/c受体小鼠体内。从健康成人供体中分离人HLA-A2阴性外周血单个核细胞（PBMC），取1×10^7个PBMC重悬于含肝素的PBS中，注射至NSG受体小鼠体内。移植当天，采用X射线辐照仪（RS 2000生物研究辐照仪）对BALB/c受体小鼠给予致死剂量全身辐照（8 Gy），对NSG受体小鼠给予2.5 Gy辐照。实验全程记录小鼠体重并观察临床症状。分别于移植后第7天（BALB/c小鼠组）或第10天（NSG小鼠组）处死小鼠进行离体分析。生存实验的观测周期为80天，当小鼠体重较初始体重下降超过25%或达到濒死标准时，判定为临床死亡。