Journal: Cancer immunology research

Article Title: Modified Hypoxia-Inducible Factor Expression in CD8 + T Cells Increases Antitumor Efficacy

doi: 10.1158/2326-6066.CIR-20-0561

Figure Lengend Snippet: (A) HIF transcription factors are post-translationally regulated. Oxygen-dependent hydroxylation at conserved proline (P) residues by PHD results in VHL)-mediated proteasomal degradation. Hydroxylation at a conserved asparagine (N) residue by FIH prevents recruitment of the coactivator p300/CBP resulting in inhibited transcriptional activity. Once released from VHL/PHD and FIH repression, HIF proteins heterodimerize with ARNT, translocate to the nucleus, bind hypoxia-responsive elements (HRE) and initiate transcription of target genes. (B) Mutation of key amino-acid residues modulates HIF regulation. Mutation of conserved prolines (P402, P577 in mouse HIF1α; P405, P530 in mouse HIF2α) and of conserved asparagine (N813 in mouse HIF1α and N851 in mouse HIF2α) into alanine (A) prevents hydroxylation by PHD and FIH, respectively. (C) Retroviral vector design for ectopic HIF expression. After genomic integration, the retroviral long terminal repeat (LTR) promoter drives expression of a polycistronic peptide containing Thy-1.1 (THY), HIF1α and HIF2α interspersed with furin cleavage sites and self-cleaving picornavirus 2A sites. Post-translational processing results in separation of the elements. Surface and nuclear localization sequences target Thy-1.1 to the cell surface and HIF isoforms to the nucleus, respectively. (D) Nuclear extracts from HEK cells transfected with vectors encoding HIF1α alone, HIF-2α alone or both probed for mouse HIF1α, HIF2α and Lamin B. Vector control (VC) encodes Thy-1.1 alone. (E) CD8+ T-cell transduction scheme. Primary CD8+ T cells were purified from mouse (C57BL/6J) splenocytes and activated by TCR triggering for 24 hours before transduction with retroviral particles. Transduced T cells were expanded in the presence of IL2 for further 3-5 days before subsequent analysis. (F) Example of CD8+ T-cell transduction. Representative flow cytometry plot showing retrovirally (RV)-transduced cells expressing Thy-1.1 on the cell surface (red box). (G) Nuclear extracts from Thy-1.1+CD8+ T cells transfected with vectors encoding HIF1α or HIF2α probed for mouse HIF1α, HIF2α and Histone 3. Vector control (VC) encodes Thy-1.1 alone.

Article Snippet: Western blotting Nuclear protein extracts (15-20 μg) from HEK293 cells transfected with HIF-encoding vectors or from Thy-1.1–purified CD8 + T cells transduced with HIF-encoding vectors were prepared with the NE-PER nuclear extraction kit (Thermo Fisher, #78833) probed with polyclonal antibodies against HIF1α (NB-100-449 or NB-100-105 Novus Biologicals), HIF2α (AF2997, R&D Systems), Lamin B (sc-6217, Santa Cruz), and Histone 3 (4499S, CST) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Residue, Activity Assay, Mutagenesis, Retroviral, Plasmid Preparation, Expressing, Transfection, Control, Transduction, Purification, Flow Cytometry

Journal: Cancer immunology research

Article Title: Modified Hypoxia-Inducible Factor Expression in CD8 + T Cells Increases Antitumor Efficacy

doi: 10.1158/2326-6066.CIR-20-0561

Figure Lengend Snippet: (A) OVA-specific OT-I splenocytes were activated with an H-2Kb–restricted OVA peptide (SIINFEKL) for 24 hours before transduction with HIF1α- or HIF2α-encoding retroviral vectors. After 5 days of expansion in the presence of IL2, live CD8+Thy-1.1+ cells were sorted by flow cytometry followed by RNA extraction and RNA-seq (n = 3 independent transductions per vector). (B) Violin plot representing transcript frequency in Log2 counts per million (CPM) of 12155 mapped transcripts. Solid vertical line: median. Dashed vertical line: quartiles. Red circles represent transcripts defining CD8+ T-cell identity. (C) Mean-difference plots showing Log2 fold change of transcripts in HIF1α- and HIF2α-transduced relative to vector control (VC)-transduced CD8+ T cells. Pink and green circles: differentially expressed transcripts as defined by a false discovery rate (FDR) < 0.01 and Log2 fold change >1 or <−1. Grey circles: non differentially expressed transcripts. (D) Bar chart summarizing total number of up- and down-regulated transcripts. Values over bars: total number of differentially expressed genes. (E) Scattered dot plot showing absolute Log2 fold change of differentially expressed transcripts in each transduction. Lines: median and interquartile range. Values over plots: median Log2 fold change. α, P < 0.01; Kruskal-Wallis with Dunn’s multiple comparison test. (F) Heatmap representing correlation in transcript frequency between HIF1α and HIF2α-transduced CD8+ T cells. Values in boxes: Spearman’s rank correlation coefficient.

Article Snippet: Western blotting Nuclear protein extracts (15-20 μg) from HEK293 cells transfected with HIF-encoding vectors or from Thy-1.1–purified CD8 + T cells transduced with HIF-encoding vectors were prepared with the NE-PER nuclear extraction kit (Thermo Fisher, #78833) probed with polyclonal antibodies against HIF1α (NB-100-449 or NB-100-105 Novus Biologicals), HIF2α (AF2997, R&D Systems), Lamin B (sc-6217, Santa Cruz), and Histone 3 (4499S, CST) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Transduction, Retroviral, Flow Cytometry, RNA Extraction, RNA Sequencing, Plasmid Preparation, Control, Comparison

Journal: Cancer immunology research

Article Title: Modified Hypoxia-Inducible Factor Expression in CD8 + T Cells Increases Antitumor Efficacy

doi: 10.1158/2326-6066.CIR-20-0561

Figure Lengend Snippet: (A) Fitness of HIF-transduced CD8+ T cells over time. CD8+ T cells were transduced with HIF1α and HIF2α-coding vectors and cultured for 21 days in the presence of IL2. Cells were restimulated with CD3/CD28 beads on days 7 and 14. Fitness was calculated as the difference in % of Thy-1.1+ cells in culture relative to day 7 (Δ% Thy-1.1+). VC: vector control. (B) Proliferation of HIF2α-transduced CD8+ T cells. Cells were loaded with CellTrace Violet (CTV) proliferation dye 6 days after transduction and were restimulated with αCD3/CD28 beads for 3 days. Proliferation was determined by CTV dilution in flow cytometry. Left: representative histograms pre-gated on live, singlet, CD8+. Thy-1.1+ events. Right: summary data showing CTV mean fluorescence intensity (MFI). n = 7 independent transductions. Lines: median and interquartile range. (C) Fitness of HIF2α-transduced cells after restimulation. Fitness was calculated as the difference in % of Thy-1.1+ cells in culture between restimulated and unstimulated cultures (Δ% Thy-1.1+). n = 3 independent transductions. Lines: median and interquartile range. (D) Mean-difference plots showing Log2 fold change of TCR chain-coding transcripts in HIF2α-transduced relative to VC-transduced CD8+ T cells. Green circles: differentially expressed transcripts as defined by a false discovery rate (FDR) < 0.01 and Log2 fold change >1 or <−1. Grey circles: non differentially expressed transcripts. Trbv12-1 codes the Vβ5 segment of the OT-I TCRβ chain. (E) Expression of TCR Vα2 and TCR Vβ chains, and CD3 determined by flow cytometry in OT-I CD8+ T cells transduced with vectors encoding HIF1α and HIF2α, HIF1α alone or HIF2α alone (day 3 to 5 post-transduction). Data expressed as Log2 fold change of MFI relative to VC-transduced cells. Each data point represents an independent transduction (n=4-24). Results are pooled from a minimum of two independent experiments. (F) Surface expression of OT-I TCR chains in HIF2α-transduced OT-I cells on day 4 post-transduction. Flow cytometry zebra plots pre-gated on live, singlet, CD8+ events showing surface expression of OT-I TCR Vα2 and TCR Vβ5 chains in transduced (Thy-1.1+; top row) and non-transduced (Thy-1.1−; bottom row). Values are the percentage of events within the double-negative quadrant. (G) Frequency of TCR-negative cells. n=5 independent transductions. Lines: median and interquartile range. (H) Surface expression of CD3 and the constant region of the TCRβ chain in HIF2α-transduced polyclonal and OT-I CD8+ T cells. Flow cytometry zebra plots pre-gated on live, singlet, CD8+, Thy-1.1+ events. Values are the percentage of events within the double-negative quadrant. (I) Frequency of TCR-negative cells in HIF2α-transduced CD8+ T cells cultured with DMSO or 10 μM PT2977 (HIF-2α inhibitor). n=5 independent transductions. Lines: median and interquartile range. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC.

Article Snippet: Western blotting Nuclear protein extracts (15-20 μg) from HEK293 cells transfected with HIF-encoding vectors or from Thy-1.1–purified CD8 + T cells transduced with HIF-encoding vectors were prepared with the NE-PER nuclear extraction kit (Thermo Fisher, #78833) probed with polyclonal antibodies against HIF1α (NB-100-449 or NB-100-105 Novus Biologicals), HIF2α (AF2997, R&D Systems), Lamin B (sc-6217, Santa Cruz), and Histone 3 (4499S, CST) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Transduction, Cell Culture, Plasmid Preparation, Control, Flow Cytometry, Fluorescence, Expressing, Comparison

Journal: Cancer immunology research

Article Title: Modified Hypoxia-Inducible Factor Expression in CD8 + T Cells Increases Antitumor Efficacy

doi: 10.1158/2326-6066.CIR-20-0561

Figure Lengend Snippet: (A) IFNγ secretion determined by intracellular cytokine flow cytometry in OT-I CD8+ T cells transduced with vectors encoding HIF1α and HIF2α, HIF1α alone or HIF2α alone and restimulated for 4 hours with 1 μM OVA (SIINFEKL) peptide. Values are the percentage within the IFNγ+ gate. Pre-gated on live, singlet, CD8+. Thy-1.1+ events (B) Summary data expressed as % IFNγ+ cells. Each data point represents an independent transduction (n=3-7). Results are pooled from a minimum of two independent experiments. (C) Real-time cytotoxicity assay. Upper row: B16F10-OVA, MC38-OVA and LLC-OVA cell density over time after addition of HIF2α-transduced OT-I CD8+ T cells. Tumor cells were seeded 5 hours prior. Bottom row: endpoint cytotoxicity. n=6-12 replicate wells. Grey horizontal area: interquartile range of no T-cell control. (D) Real-time cytotoxicity assay with DMSO- or PT2977-treated HIF2α-transduced OT-I CD8+ T cells. Left: B16F10-OVA cell density over time after addition of T cells. Right: endpoint cytotoxicity. n=6-12 replicate wells. Grey horizontal area: interquartile range of no T cell control. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC.

Article Snippet: Western blotting Nuclear protein extracts (15-20 μg) from HEK293 cells transfected with HIF-encoding vectors or from Thy-1.1–purified CD8 + T cells transduced with HIF-encoding vectors were prepared with the NE-PER nuclear extraction kit (Thermo Fisher, #78833) probed with polyclonal antibodies against HIF1α (NB-100-449 or NB-100-105 Novus Biologicals), HIF2α (AF2997, R&D Systems), Lamin B (sc-6217, Santa Cruz), and Histone 3 (4499S, CST) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Flow Cytometry, Transduction, Cytotoxicity Assay, Control, Comparison

Journal: Cancer immunology research

Article Title: Modified Hypoxia-Inducible Factor Expression in CD8 + T Cells Increases Antitumor Efficacy

doi: 10.1158/2326-6066.CIR-20-0561

Figure Lengend Snippet: (A) ACT model. C57BL/6J mice were injected subcutaneously with 5×105 OVA-expressing B16-F10 (B16-F10-OVA) and 4 days later were lymphodepleted with 300 mg/kg cyclophosphamide. On day 8, 0.5-1 × 106 HIF-transduced (Thy-1.1 enriched) OVA-specific OT-I CD8+ T cells were adoptively transferred into tumor-bearing mice. Peripheral blood was sampled at day 15 and analysed by flow cytometry. Tumor growth was monitored every 2-3 days until day 60. (B) Frequency of HIF-transduced OT-I cells per million PBMCs in peripheral blood. n = 8-13 animals pooled from two independent experiments. Grey horizontal line: median of VC group. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC. (C) B16-F10-OVA tumor growth after ACT. B16-F10-OVA tumor volume measured until day 60 after mice received VC-HIF1α- or HIF2α-transduced OT-I cells on day 8. Thin lines: tumor growth from individual animals. Thick line: centered sixth order polynomial curve. Shaded area: 99% confidence level interval. n = 9-25 animals per group pooled from two (HIF1α) or four (HIF2α) independent experiments. (D) Survival curves for tumor growth shown in (D). Threshold for survival was set at 200 mm3. Grey line: no ACT. Black line: ACT of VC-transduced OT-I. Pink or green lines: ACT of HIF-1α- or HIF-2α-transduced OT-I, respectively. α, P < 0.01; log-rank (Mantel-Cox) test relative to VC.

Article Snippet: Western blotting Nuclear protein extracts (15-20 μg) from HEK293 cells transfected with HIF-encoding vectors or from Thy-1.1–purified CD8 + T cells transduced with HIF-encoding vectors were prepared with the NE-PER nuclear extraction kit (Thermo Fisher, #78833) probed with polyclonal antibodies against HIF1α (NB-100-449 or NB-100-105 Novus Biologicals), HIF2α (AF2997, R&D Systems), Lamin B (sc-6217, Santa Cruz), and Histone 3 (4499S, CST) and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Injection, Expressing, Flow Cytometry, Comparison