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.