positive selection magnetic bead sorting  (Miltenyi Biotec)

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: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

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: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

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) Heatmaps showing Log2 fold change of transcripts involved in functional aspects of CD8+ T cells. (B) Expression of differentiation markers determined by flow cytometry in CD8+ T cells transduced with vectors encoding HIF-1α and HIF-2α, HIF-1α alone or HIF-2α alone (day 3 to 5 post-transduction). Data expressed as Log2 fold change of median fluorescence intensity (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. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC. Histograms are representative flow cytometry plots for each parameter and are pre-gated on live, singlet, CD8+Thy-1.1+ events.

Article Snippet: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

Techniques: Functional Assay, Expressing, Flow Cytometry, Transduction, Fluorescence, 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: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

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: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

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: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

Techniques: Injection, Expressing, Flow Cytometry, 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) Retroviral vector design for co-expression of an anti-CD19 CAR and FIH-insensitive (N847A; PPA) human HIF2α. LTR: long terminal repeats. Fu-2A: furin and picornavirus 2A self-cleaving sequence. (B) Expression of GZMB, CD25 and ICOS on GFP+ human CD8+ T cells 6 days after transduction with vector control (VC), CD19CAR, or CD19CAR+HIF2. Cells were cultured with DMSO or 10 μM PT2977 (HIF2α inhibitor) from day 1 post-transduction. n = 6 donors. Mean Fluorescence Intensity (MFI) is normalized to DMSO-treated VC-transduced cells of respective donors. Lines: median and interquartile range. (C) Cytotoxicity of VC- or CAR-transduced human CD8+ T cells against GFP+CD19+ RAJI lymphoma cells after 20 hours of co-culture at a 1:1 effector-to-target ratio. Representative flow cytometry showing transduced (GFP+, CD45RO+) CD8+ T cells and RAJI targets (GFP+, CD19+) after gating on live, singlet GFP+ events. Summary data of specific cytotoxicity of 11 donors. Lines: median and interquartile range. (D) Experimental scheme for ACT of human CD19CAR-transduced CD8+ T cells. Immunocompromised NSG mice were injected with 1×106 luciferase-expressing CD19+ Raji cells followed by (ACT) of 2.5×105 VC-, CD19CAR- or CD19CAR+HIF2-transduced CD8+ T cells 4 days later. Tumor burden assessed by whole-body bioluminescence imaging up to day 45. Adoptive cell engraftment monitored on day 10 by sampling peripheral blood. (E) Frequency of human CD8+ T cells in peripheral blood on day 10 after ACT. n = 5-10 mice. Lines: median and interquartile range. α, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test relative to VC. β, P < 0.01; one-way ANOVA with Dunnett’s multiple comparison between PT2977- and DMSO-treated counterparts. (F) Top: Tumour burden after ACT shown as bioluminescent signal (photons/second; thick line: group median; thin lines: individual animals). Horizontal shaded area and line: range ad and median tumor burden at baseline. Bottom: Log10 fold change in tumor burden relative to baseline (median and interquartile range) at different time points. α,β P < 0.01; one-way ANOVA with Tukey’s multiple comparison test relative to CD19CAR or VC, respectively. Right: Representative images of bioluminescent whole-body imaging. Scale ranges from 1×106 to 1×108 photons/sec/cm2 on a logarithmic scale.

Article Snippet: CD8 + T-cell sourcing, activation and restimulation CD8 + T cells from female and male mice were purified from spleens by CD8α positive selection magnetic bead sorting (Miltenyi, #130-117-044) and activated in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol (Thermo Fisher, #21985023), with 2 μg/ml Concanavalin A (Sigma, #C5275) and 10 ng/ml recombinant human IL7 (R&D Systems, #207-IL-005) or anti-mouse CD3/CD28 dynabeads (Thermo Fisher, #11453D) at a 1:1 cell-to-bead ratio for 24 hours before transduction.

Techniques: Retroviral, Plasmid Preparation, Expressing, Sequencing, Transduction, Control, Cell Culture, Fluorescence, Co-Culture Assay, Flow Cytometry, Injection, Luciferase, Imaging, Sampling, Comparison

Journal: Immunology

Article Title: Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation

doi: 10.1111/imm.12245

Figure Lengend Snippet: Highly skewed frequency of peripheral blood cell subsets early after autologous stem cell transplantation (ASCT). Expression of cell surface markers examined before and after ASCT by flow cytometry. (a) Representative flow cytometry plots, showing CD4 ( y -axis) versus CD8 ( x -axis) expression on cells gated on live CD3 + T cells 1 month before and 1, 12 and 31 months after ASCT in patient 1. Percentages of events in each gate are shown. (b) Total T (CD3 + , left) and B (CD19 + , middle) cell and monocyte (CD14 + , right) proportions before ASCT and at 1 month, 3–12 months and 2–3 years after ASCT. All gated on live cells according to forward and side scatter, some using live–dead discrimination. (c) CD4 + (left) and CD8 + (middle) T-cell proportions, gated on live CD3 + cells, and the CD4/CD8 ratio (right). (d) Naive (CD45RA + , left) and memory (CD45RO + , middle) T-cell proportions, gated on live CD3 + CD8 + T cells. Throughout, horizontal bars in summary plots represent the mean, n = 2–5 (where samples were available).

Article Snippet: CD4 + and CD8 + T-cell populations were separated using CD4 + -positive selection magnetic bead sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and the CD4 − fraction was used as the source of CD8 + T cells.

Techniques: Transplantation Assay, Expressing, Flow Cytometry

Journal: Immunology

Article Title: Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation

doi: 10.1111/imm.12245

Figure Lengend Snippet: Highly oligoclonal CD4 + T-cell receptor β variable region (TCRVb) repertoire early in immune reconstitution in patient 1, who obtained remission after autologous stem cell transplantation (ASCT). TCRVb CDR3 length spectratyping for Vb1, Vb8 Vb4, Vb15 and Vb21; samples tested from before and 1 and 12 months after ASCT (top to bottom as shown). Representative TCRVb CDR3 spectratypes are shown from patient 1.

Article Snippet: CD4 + and CD8 + T-cell populations were separated using CD4 + -positive selection magnetic bead sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and the CD4 − fraction was used as the source of CD8 + T cells.

Techniques: Transplantation Assay

Journal: Immunology

Article Title: Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation

doi: 10.1111/imm.12245

Figure Lengend Snippet: Two patterns of T-cell receptor (TCR) β repertoire: re-emergence of old and establishment of new T-cell clones after autologous stem cell transplantation (ASCT). Sub-cloning of the CDR3 of Vb15 (a), Vb4 (b) and Vb21 (c) in CD4 − T-cell subset (representing CD8 + T cells). (a) Vb15 with N region protein sequence: GVGG: white; DYEN: light grey; RGNS: dark grey and DLGS: black stripes. (b) Vb4 with N region protein sequence: RHIP: white and GTGE: light grey. (c) Vb21 with N region protein sequence: AAGA: white and HGTG: light grey. Throughout, unique N region sequences in black. 16–24 sequences per Vb analysed.

Article Snippet: CD4 + and CD8 + T-cell populations were separated using CD4 + -positive selection magnetic bead sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) and the CD4 − fraction was used as the source of CD8 + T cells.

Techniques: Clone Assay, Transplantation Assay, Subcloning, Sequencing