Fig 1: Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin−/− mice followed by induction of PIFS. C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 107 GFP+ CD8+ splenocytes (n = 10) or 107 Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.
Fig 2: IL-2-positive DC coculture promotes PA T cells’ survivalin vivo. (A) PA T cells were prepared in vitro, shown as the flow chart, then the cells were i.v. injected into 6–8 week old C57BL/6 wild type mice, 1 × 106 cells per mouse (5 per group). Experimental group mice were injected with resting PA T cells, PA T co-cultured with WT or Il2-/- splenic CD11c+ DCs. Mice injected with PBS were the negative control. (B) After one month, the cells from spleen and LNs were collected, and in 24-well-plate 1 × 106 or 2 × 106 cells added to 2 × 106 feeder population of irradiated B6 splenocytes which were prior-treated with 3,000 rad ? irradiation. The clonal expansion experiment was carried out in vitro by adding soluble OVA. IFN-? and IL-2 in the supernatant were measured by ELISA. Every dot stands for one mouse. Results are representative of two independent experiments. (C) PA T cells from CD45.1+ OT-II mice were prepared in vitro, then the cells were i.v. injected into 6-8 weeks CD45.2+C57BL/6 wild type mice, 1 × 106 cells per mouse. Experimental group mice were injected with resting PA T cells, PA T co-cultured with B6 or Il2-/- splenic CD11c+ DCs. After three months, the host mice were re-challenged by injected 50 µg OVA and 10 ug LPS per mice, 72 h later, the cells from spleen and LNs were collected, then analyzed for the total cell number of CD4+CD45.1+ cells in host mice by FCAS. Every symbol stands one mouse. Results are representative of two independent experiments. **P < 0.01, ***P < 0.001 (Unpaired Student’s t test)
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