Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: CD70 expression is a relevant marker of rGBM. (A) Manhattan plot of the top upregulated RNAseq genes in the recurrent patient-derived GBM cancer stem cells (rGBM CSCs) BT241, compared with the publicly available the Cancer Genome Atlas database, identified CD70 (circled) as a target candidate upregulated in the CSC subpopulation, compared with tumor bulk. (B) Analysis of CD70 mRNA levels in the five TGCA primary/recurrent patient matched GBM pairs depicts an CD70 increase in three pairs on recurrence, alongside a switch towards the Mesenchymal subtype (GBM subtype classification: C, classical; P, proneural; M, mesenchymal). (C) Among GBM CSCs from , the two in-house matched p/rGBM CSCs pairs display an increase of CD70 cell surface expression on tumor recurrence. (D) Dot plot representation of CD70 cell surface expression in rGBM CSCs compared with primary (p-) GBM CSCs and normal brain cells (astrocytes, neural stem cells), assessed by flow cytometry (Singh lab brain tumor database). (E) Volcano plot of the top up- and down-regulated cell surface proteins of pair two from , as assessed by glycocapture proteomics. CSC, cancer stem cell; GBM, glioblastoma; rGBM, recurrent GBM.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: CD70 is a dedicated player in GBM maintenance and tumor formation. (A) GBM CSCs were sorted into positive and negative populations and proliferation was assessed by PrestoBlue assay (B) Limiting dilution analyses of CD70 positive and negative cells in pGBM (GBM8) and rGBM (BT241). (C) Cell surface CD70 expression after shRNA knockdown in three CD70 HIGH GBM lines, as assessed by flow cytometry. (D) Silencing of CD70 expression by shRNA (shCD70) knockdown and sphere formation ability was assessed compared with shGFP (control shRNA). (E–I) Immunocompromised mice (NSG, a minimum of six mice per condition) were intracranially injected with shGFP or shCD70 CSCs. (E, F) Tumor area of CD70-silenced CSCs compared with control knockdown CSCs was measured using formalin-fixed, H&E-stained mouse brain slices (right, representative image). (G, H) Kaplan-Meier survival curves comparing mice engrafted with shCD70 CSCs compared with shGFP CSCs. The two remaining BT241 shCD70 mice at the end of experiment showed an absence of tumor by H&E staining at experimental endpoint (data not shown). (I) MRI images representative of xenografts from shGFP and shCD70 transduced GBM CSC line BT241. Images on the right are control images of normal mouse brain. (*P<0.05; **p<0.01; ***p<0.001; ****p<0.0001). CSCs, cancer stem cells; GBM, glioblastoma; pGBM, primary GBM; NSG, NOD/SCID gamma; shRNA, short hairpin RNA.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: Prestoblue Assay, Expressing, shRNA, Knockdown, Flow Cytometry, Control, Injection, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: Generation and in vitro characterization of CD70-Specific CAR-T Cells. (A) Binding curve comparing CD70-specific Fabs to commercial standard antibody. (B) Anti-CD70 Fab’2 is specific against CD70, assessed by cytotoxicity assay under combination treatment with 2ºADC, against GBM cells expressing high (GBMCSC BT241) or no (HEK293) CD70. (C) Schematic representation of CAR structure. (D) Successful transduction of CAR-T vectors as observed by NGFR+ cells in ConCAR-T cells and NGFR+Myc+ cells in CD70 CAR-T cells, displayed as a representative flow plot. (E) Testing of CAR-T cell activation; IFN-γ and TNF-α cytokine release during coculture of GBM CSC BT241 with CD70 CAR-T, compared with ConCAR-T cells, as analyzed by ELISA (n=3). (F) Cytotoxicity assay to assess CD70CAR killing capacity compared with ConCAR after coculturing for 24 hours, tested at various effector to target (E:T) ratios (n=3). (***p<0.001; ****p<0.0001). CAR, chimeric antigen receptor; CSCs, cancer stem cells; GBM, glioblastoma; MFI, mean fluorescence intensity.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: In Vitro, Binding Assay, Cytotoxicity Assay, Expressing, Transduction, Activation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: CD70 CAR-Ts are efficacious against recurrent GBM tumors in vivo . NSG mice (at least n=6 per group) were intracranially implanted with 100,000 human BT241 ffLuc GBM cells. Upon successful engraftment, mice were treated with 1×10 6 CD70CAR-T or ConCAR-T cells, delivered intracranially once a week for 2 weeks. (A) CD70 CAR-T treated mice showed decreased tumor signal, as assessed by bioluminescence measurement (right, representative image of radiance measurement in the region of interest). A lower tumor burden was observed in the CD70 CAR-T group compared with the control group, as measured on (B) formalin-fixed, H&E-stained mouse brain slices (representative image on the right), and (C) an extended survival (Kaplan-Meier curve) (***p<0.001). CAR, chimeric antigen receptor; GBM, glioblastoma; NSG, NOD/SCID Gamma.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: In Vivo, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: Modeling of CD70s influence on GBM TIME. (A) CD70 expression kinetics on in-house, activated T cells and (B) levels of CD69 and cMyc displayed by CD70CAR or ConCAR-T cells 9 days post-transduction, evaluated by flow cytometry. (C) CD70-enriched or -silenced Jurkat cells were transduced with either ConCAR or CD70CAR. After 8 days, CD69 and CD70 levels were assessed by flow cytometry. (D) TIME cells extracted from patient tumor samples were analyzed by flow cytometry, evaluating the pattern of expression of CD27 in non-lymphoid (CD45+CD3-) and M1 populations (CD45+CD3-CD68+HLADR+). (E) Average expression of CD27 on CD4/CD8 lymphoid population, and CD70 expression on the lymphoid population (CD3+). CAR, chimeric antigen receptor; GBM, glioblastoma; TIME, tumor immune microenvironment.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: Expressing, Transduction, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

doi: 10.1136/jitc-2021-003289

Figure Lengend Snippet: CD70 and CD27 expression in GBM and immune cells. (A, B) ScRNAseq profile of human GBM and immune infiltrate (Neftel 2019). Cell-type annotated UMAP (A) and expression of CD70 (top) and CD27 (bottom) visualized on UMAP and stratified by cell type (all cells included), Neftel GBM subtype (GBM only), and Richards GBM subtype (GBM only) (B). (C, D) ScRNAseq profile of human glioblastoma stem cells (Richards 2021). Cell-type annotated UMAP (C) and expression of CD70 (top) and CD27 (bottom) visualized on UMAP and stratified by Neftel GBM subtype and Richards GBM subtype (D). (E, F) ScRNAseq profile of immune cell infiltrates in murine GL261 tumors (Ochocka 2021). Cell-type annotated UMAP (E) and expression of CD70 (top) and CD27 (bottom) visualized on UMAP and stratified by cell type (F). Astrocyte-like, BAM, border-associated macrophage; DC, dendritic cell; Develop., Developmental; Expr, expression; GBM, glioblastoma; Injury Resp, injury response; MES1, mesenchymal type 1; MES2, mesenchymal type 2; NK, natural killer; NPC1, neural progenitor cell type 1; NPC2, neural progenitor cell type 2; OPC, oligodendrocyte progenitor cell.

Article Snippet: Guide RNAs (gRNAs) targeting AAVS1 (5’-GGGGCCACTAGGGACAGGAT-3’) and CD70 (A: 5’- GCTGAGCCTGTGCGAAGCGC-3’; B: 5’-ATGGGACCAAAGCAGCCCGC-3’ were obtained from TKOv3 and cloned into a single-gRNA lentiCRISPRv2 construct (Addgene 52961).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Gain-of-function human UNC93B1 variants cause systemic lupus erythematosus and chilblain lupus

doi: 10.1084/jem.20232066

Figure Lengend Snippet: C haracterization of TLR8 signaling in THP-1 cells. (A) ISG15 expression, assessed by intracellular staining and flow cytometry, in THP-1 cells stimulated for 16 h with CL264 (10 µg/ml), CL307 (TLR7 agonist) (1 µg/ml), R848 (10 µg/ml), TL8-506 (1 µg/ml), CpG-A (5 µM), and CpG-B (5 µM). Relative MFI: mean fluorescence intensity of stimulated condition over NS condition. Mean ± SEM of n = 3 experiments. (B) Relative mRNA levels of indicated genes in THP-1 cells stimulated for 16 h with CL307 (1 µg/ml) or TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over NS condition. Mean ± SEM of n = 4 experiments. (C) V5-tagged UNC93B1 expression, assessed by intracellular staining of V5 and flow cytometry, in THP-1 cells stably expressing EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct), or non-transduced (NT). Mean ± SEM of n = 3 experiments. (D) V5-tagged UNC93B1 expression, assessed by intracellular staining of V5 (PE) and flow cytometry, in THP-1 cells stably expressing WT, L330R, or R525P UNC93B1, or an EV (pTrip-CMV-Puro-2A construct). Overlayed histogram of PE intensity is shown. Representative experiment of n = 2. (E) Western blot of non-transduced (NT), EV- and UNC93B1-V5-transduced THP-1 protein lysates (pTrip-SFFV-GFP-2A construct), using a conformational anti-UNC93B1 antibodies. Endogenous UNC93B1 migrates at 65 kDa, while the overexpressed, V5-tagged UNC93B1 migrates at a higher molecular weight. (F) Relative mRNA levels of indicated genes in THP-1 cells transduced with EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct) and stimulated for 16 h with R848 (1 µg/ml), assessed by qPCR, normalized to HPRT mRNA, and expressed as fold induction over WT NS condition. Mean ± SEM of n = 3 experiments. Two-way ANOVA with Dunnett’s post-hoc test, except for IFNB1 : one-way ANOVA with Holm–Sidak post-hoc test. (G) Syntenin-1 expression level in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying two single sgRNA targeting syntenin-1/ SDCBP gene (sgSyn-1a and sgSyn-1b), harvested 7 days after transduction and selection, and assessed by western blot. Vinculin is a loading control. Representative experiment of n = 3. (H) Baseline ISG score (median of the relative mRNA levels of the ISGs IFI27 , IFI44L , OAS1 , and IFIT1 ), ISGs, IFNB1 , and inflammatory cytokine ( IL6 , IL8 , and TNF ) expression in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying two single sgRNA targeting syntenin-1/ SDCBP gene (sgSyn-1a and sgSyn-1b), at baseline, harvested 7–10 days after transduction and selection. mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over EV1. Mean ± SEM of n = 6–10 experiments. (I) ISGs and inflammatory cytokine ( IL6 , IL8 , and TNF ) expression in THP-1 cells stably transduced with EVs (EV1, EV2) or vectors carrying sgRNA targeting syntenin-1/ SDCBP gene, stimulated for 24 h with TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a percentage of the averaged EV1-EV2 fold stimulation over the NS condition. Mean ± SEM of n = 6–10 experiments. (H and I) Kruskal–Wallis test with Dunnett’s post-hoc analysis (for ISG score and IFNB1 ); mixed-effects analysis (REML; restricted maximum likelihood) with uncorrected Fisher’s test (for ISGs and inflammatory cytokines). (J and K) NF-κB SEAP reporter assay in control (sgNTgt) or syntenin-1 KO (sgSyn-1b) THP-1 Dual cell pools stably transduced with EV, WT, and variant UNC93B1 (pTrip-SFFV-GFP-2A construct) stimulated for 16 h with (J) TL8-506 (0.1 µg/ml) and (K) CL307 (5 µg/ml). Data are expressed as fold induction over WT NS. Mean ± SEM of n = 7–8 experiments. (L) ISG score (median of the relative mRNA levels of the ISGs IFI27 , IFI44L , OAS1 , and IFIT1 ) in control (sgNTgt) or TLR8 KO (sgTLR8) THP-1 cell pools stimulated for 24 h with TL8-506 (1 µg/ml). mRNA was assessed by qPCR, normalized to HPRT mRNA, and expressed as a fold induction over control (sgNtgt) in the NS condition. Mean ± SEM of n = 2 experiments. (M) PLA assessing UNC93B1-TLR8 association (yellow dots) with anti-V5 (for UNC93B1) and anti-TLR8 specific antibodies, as in , in unstimulated TLR8 KO THP-1 cell pools stably transduced with WT UNC93B1-V5. No PLA signal is detected. Nuclei (blue) were stained with DAPI, scale bar: 5 μm. Two-way ANOVA with Dunnett’s post-hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Article Snippet: Single-guide RNAs (sgRNA) targeting syntenin-1/SDCBP , TLR8 , or non-targeting controls (sgNtgt) ( ) were either designed using the CRISPOR tool ( http://crispor.tefor.net ) (sgSyn-1a), taken from (sgSyn-1b) or taken from , and cloned into lentiviral construct lentiCRISPRv2-hygro, a gift from Brett Stringer (Flinders University, Adelaide, Australia) (plasmid #98291; Addgene ) , following the protocol provided on the plasmid website.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, Stable Transfection, Variant Assay, Construct, Western Blot, Molecular Weight, Transduction, Selection, Control, Reporter Assay