Journal: bioRxiv
Article Title: Modified Hypoxia Inducible Factor expression in CD8+ T cells increases anti-tumor efficacy
doi: 10.1101/2020.06.18.159137
Figure Lengend Snippet: (A) CD8 + OT-I cells transduced with HIF-1ɑ- and HIF-2ɑ-coding vectors prior to adoptive cell transfer (ACT). 6 days after transduction cells were Thy-1.1 enriched using magnetic bead sorting and adoptively transferred shortly after into tumor-bearing mice. (B) Frequency of adoptively transferred HIF-transduced OT-I cells in peripheral blood of tumor-bearing mice at day 15. Representative flow cytometry dot plots (pre-gated on CD45+ live singlets) showing TdTomato (adoptive T-cell marker) and Thy-1.1 (transduction marker). Values are events per million PBMCs within the gate. VC: vector control. No ACT: no adoptive cell therapy. (C) Complete response rate (% of tumor-free animals at day 60) and median survival (extrapolated from survival curves shown in Figure 8E) for groups of animals receiving ACT of VC-, HIF-1ɑ- or HIF-2ɑ-transduced OT-I cells. Dashed lines: VC reference. (D) Adoptive cell therapy (ACT) model. C57BL/6j mice were injected subcutaneously with 5×10 5 OVA-expressing B16-F10 (B16-F10-OVA) and 11 days later were lymphodepleted with 300 mg/kg cyclophosphamide. On day 14, 0.5×10 6 HIF-2ɑ-transduced (Thy-1.1 enriched) OVA-specific OT-I CD8+ T cells were adoptively transferred. Tumors were harvested 5 days later, dissociated into a single-cell suspension and analysed by flow cytometry. (E) Frequency of adoptively transferred HIF-2ɑ-transduced OT-I cells within B16-F10-OVA tumors, 5 days after ACT. Representative flow cytometry dot plots (pre-gated on CD45+ live singlets) showing CD45.1 (adoptive T-cell marker) and CD45.2 (endogenous leukocyte marker). Values are the percentage of total CD45.2 events within the gate. (F) Frequency of tumor-infiltrating HIF-2ɑ-transduced OT-I cells as a percentage of CD45+ cells (top) or per gram of tumor (bottom). n=3-5 animals. Lines: median and interquartile range. Grey horizontal line: median for VC group. (G) As (F) but for tumor-infiltrating endogenous CD8+ T cells. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC.
Article Snippet: Transduced CD8+ T cells were expanded in the presence of 10 U/ml recombinant human IL-2 (Sigma) Human CD8+ T cells were purified from donor PBMCs (Cambridge Bioscience or NHSBT) by negative selection magnetic bead sorting (Miltenyi) and activated in complete RPMI supplemented with 30 U/ml IL-2 with anti-human CD3/CD28 dynabeads (Thermo Fisher) at a 1:1 cell-to-bead ratio.
Techniques: Transduction, Flow Cytometry, Marker, Plasmid Preparation, Injection, Expressing, Suspension, Comparison
Miltenyi Biotec is a verified supplier 
Miltenyi Biotec manufactures this product 
