Journal: Frontiers in Immunology

Article Title: EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells

doi: 10.3389/fimmu.2023.1191866

Figure Lengend Snippet: Characteristics of cell lines employed in this study.

Article Snippet: Polyclonal antibody (pAb) Goat anti-human Ig-PE (cat# 2040-09, Southern Biotech, Birmingham, AL, USA), monoclonal antibodies (mAb): anti-CD27-APC (cat# 302810, clone O323, BioLegend, San Diego, CA, USA) (also used as CD27 mAb), anti-EGFR-FITC (cat# sc-120 FITC, clone 528, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD25-APC (cat# 302610, clone BC96, BioLegend), anti-CD4-FITC (cat# 300506, clone RPA-T4, BioLegend), anti-CD8-Brilliant Violet 421 (cat# 344748, clone SK1, BioLegend), mouse (IgG2A) (mAb 425) (Cat# EWI020, Kerafast, Boston, MA, USA), anti-Myc mAb Alexa Fluor 647 (cat# 2233, clone 9B11, Cell Signaling, Danvers, MA, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells

doi: 10.3389/fimmu.2023.1191866

Figure Lengend Snippet: CD27 is a target for re-activation of tumor infiltrating cytotoxic and exhausted lymphocytes. (A) Normalized TCGA PAN CANCER epidermal growth factor receptor (EGFR) expression levels from epithelial cancers (black): BLCA (Bladder urothelial carcinoma), ACC (Adrenocortical carcinoma), BRCA (Breast invasive carcinoma), CESC (Cervical squamous cell carcinoma and endocervical adenocarcinoma), CHOL (Cholangiocarcinoma), COAD (Colon adenocarcinoma), ESCA (Esophageal carcinoma), HNSC (Head and Neck squamous cell carcinoma), KIRC (Kidney renal clear cell carcinoma), KIRP (Kidney renal papillary cell carcinoma), LIHC (Liver hepatocellular carcinoma), LUAD (Lung adenocarcinoma), LUSC (Lung squamous cell carcinoma), OV (Ovarian serous cystadenocarcinoma), PAAD (Pancreatic adenocarcinoma), READ (Rectum adenocarcinoma), PRAD (Prostate adenocarcinoma), STAD (Stomach adenocarcinoma), THCA (Thyroid carcinoma), UCEC (Uterine Corpus Endometrial Carcinoma), and non- epithelial cancers (gray): DLBC (Diffuse large B-cell lymphoma) and UVM (Uveal melanoma) were plotted in violin plots to visualize their relative EGFR expression. (B) Normalized CD27 expression levels from all 20 epithelial cancer types described in (A) were matched with lymphocytic infiltration signature scores via TCGA participant barcodes and plotted against each other. A linear regression was performed to visualize the correlation between CD27 expression and the lymphocytic infiltration signature score (R-squared = 0.6895, p < 0.0001). Statistical significance was determined using an F-test. (C) Single-cell tumor immune atlas RNA sequencing dataset based on 526,261 cells from 217 patients and 13 cancer types, revealing CD27 expression within different immune cell subtypes. (D) Proportion of CD27 + cells in each T cell type described in (C) , statistical comparisons are shown in Supplementary Table 2 . (E) Volcano plots of the differential gene expression analysis in CD27 + vs CD27 - terminally exhausted and (F) cytotoxic CD8 + T cells calculated using the FindMarkers function from Seurat with MAST as the method of choice.

Article Snippet: Polyclonal antibody (pAb) Goat anti-human Ig-PE (cat# 2040-09, Southern Biotech, Birmingham, AL, USA), monoclonal antibodies (mAb): anti-CD27-APC (cat# 302810, clone O323, BioLegend, San Diego, CA, USA) (also used as CD27 mAb), anti-EGFR-FITC (cat# sc-120 FITC, clone 528, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD25-APC (cat# 302610, clone BC96, BioLegend), anti-CD4-FITC (cat# 300506, clone RPA-T4, BioLegend), anti-CD8-Brilliant Violet 421 (cat# 344748, clone SK1, BioLegend), mouse (IgG2A) (mAb 425) (Cat# EWI020, Kerafast, Boston, MA, USA), anti-Myc mAb Alexa Fluor 647 (cat# 2233, clone 9B11, Cell Signaling, Danvers, MA, USA).

Techniques: Activation Assay, Expressing, RNA Sequencing, Gene Expression

Journal: Frontiers in Immunology

Article Title: EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells

doi: 10.3389/fimmu.2023.1191866

Figure Lengend Snippet: CD27xEGFR selectively binds EGFR and CD27 on tumor cells and T cells. (A) CD27xEGFR is designed in a scFv-scFv-IgG1 format with binding domains targeting CD27 (scFv1F5) and EGFR (scFv425) connected to an IgG1 tail containing LALAPG Fc mutations L234A, L235A, and P329G. (B) Association and dissociation of His-CD27 (500 nM) and/or His-epidermal-growth-factor-receptor (EGFR) (125 nM) against surface bound CD27xEGFR (8 µg/mL) as measured by biolayer interferometry (n = 3). (C) Flow cytometry plot displaying EGFR expression of stained (DiD) A431 cells (left). Dose-dependent binding (represented as normalized mean fluorescent intensity (MFI) to the highest MFI value) of CD27xEGFR on A431 tumor cells (n = 3) (right). (D) Flow cytometry plot displaying CD3 and CD27 expression on primary human T cells (left) Dose-dependent binding (represented as normalized MFI to the highest MFI value) of CD27xEGFR on primary human T cells (n = 5) (right). (E) Binding (represented as normalized MFI to the highest MFI value) of CD27xEGFR to HT1080 tumor cells ectopically expressing CD27 and its (partial) binding abrogation by pre-incubation of excess amounts of mAb 425 (EGFR block), an anti-CD27 mAb (CD27 block) or both (double block) (n = 3). Statistical significance was determined using one-way ANOVA test with Dunnett’s correction (F) Representative doublet formation between EGFR + A431 tumor cells and CD27 + primary human T cells upon incubation with CD27xEGFR with the corresponding bar graph on the right (n = 3). Statistical analyses were done using a paired t-test. Data are presented as mean with shaded areas and error bars denoting standard deviation. “**” indicates (p < 0.01), “*” indicates (p < 0.05).

Article Snippet: Polyclonal antibody (pAb) Goat anti-human Ig-PE (cat# 2040-09, Southern Biotech, Birmingham, AL, USA), monoclonal antibodies (mAb): anti-CD27-APC (cat# 302810, clone O323, BioLegend, San Diego, CA, USA) (also used as CD27 mAb), anti-EGFR-FITC (cat# sc-120 FITC, clone 528, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD25-APC (cat# 302610, clone BC96, BioLegend), anti-CD4-FITC (cat# 300506, clone RPA-T4, BioLegend), anti-CD8-Brilliant Violet 421 (cat# 344748, clone SK1, BioLegend), mouse (IgG2A) (mAb 425) (Cat# EWI020, Kerafast, Boston, MA, USA), anti-Myc mAb Alexa Fluor 647 (cat# 2233, clone 9B11, Cell Signaling, Danvers, MA, USA).

Techniques: Binding Assay, Flow Cytometry, Expressing, Staining, Incubation, Blocking Assay, Standard Deviation

Journal: Cells

Article Title: Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder

doi: 10.3390/cells11121939

Figure Lengend Snippet: ( A ) A focal mosaic of PCDH19 expression was created by injecting an AAV expressing EGFP-Cre recombinase into the right visual cortex (V1) of the PCDH19flox/flox mouse. The opposite, non-injected hemisphere was used as the internal control. ( B ) PCDH19 mosaic mice experienced a transient period of unexpected mortality. Data collected from 70 mice injected at P1 (blue) differs significantly from that of control littermates (N = 48 mice, black; Mantel-Cox test p < 0.01). Yellow area indicates the period of adolescence in mice. ( C ) PCDH19 mice have a normal diurnal alternation of resting phases during the day (light on) and of high locomotor activity during the night (light off, shaded area; darkness from 7 PM till 7 AM). Bars represent the speed averaged in 5 min bins measured in three control and three PCDH19 mice in a 48-h period. Recordings start at 12 AM. ( D ) Density maps showing the arena occupation during a 2-h period of the active phase. ( E ) PCDH19 mice are hyperactive during the night phase. ( F ) PCDH19 mice spend less time than controls in resting states that can be ascribed to sleep (N = 8 for both control and PCDH19 mice; data pooled from 36 and 38 days of video recording). Significant differences are indicated with asterisks (** p < 0.02, Mann–Whitney test).

Article Snippet: Mosaic PCDH19 expression was induced in the occipital cortex of PCDH19 conditional knockout mice by focal injection of an AAV expressing EGFP-Cre recombinase (Addgene #105545-AAV1; pAAV.CMV.HI.eGFP-Cre.WPRE.SV40).

Techniques: Expressing, Injection, Control, Activity Assay, MANN-WHITNEY

Journal: Cells

Article Title: Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder

doi: 10.3390/cells11121939

Figure Lengend Snippet: In vivo 2-photon calcium imaging in PCDH19 mosaic brain. ( A ) Example field of view with Cre-negative red neurons (top grey square; expressing jRGECO1a only) and Cre positive (PCDH19 knockout) green neurons (bottom square) that express jRGECO1a in the cytoplasm and EGFP-Cre recombinase in the nucleus. ( B ) Calcium transients (green trace) measured over the entire field of view. The vertical axis displays the number of pixels affected by calcium activity over time (see Supplemental Information). The black trace shows the LFP recorded from the same hemisphere with an electrode positioned just outside of the imaged field at a depth of 250 µm. The inset shows magnified traces in correspondence of the black bar. Notice how Ca 2+ transients are phase locked to the USs. ( C ) Frequency of calcium transients in red and green cells of mosaic mice and controls. Each dot represents the average value for each active neuron. Significant differences are indicated with asterisks (*** p < 0.0005, ** p < 0.005; Mann–Whitney test). Transient frequency is significantly increased in mosaics compared to control mice both for Cre+ (PCDH19 KO) neurons and for Cre- neurons. ( D ) Box plots of the percentage of Ca 2+ transients occurring within USs, demonstrating a significant reduction in synchronization with the SWA oscillation in mosaic mice compared to control mice (* p < 0.02; Mann–Whitney test, whiskers indicate data range). Each dot represents the value obtained by all neurons in the imaging field of PCDH19 mosaic mice. The filled magenta dots represent the values obtained from mosaic animals with a hyperexcitable (>5 β oscillations per hour) phenotype, the blue dots from all other mosaics.

Article Snippet: Mosaic PCDH19 expression was induced in the occipital cortex of PCDH19 conditional knockout mice by focal injection of an AAV expressing EGFP-Cre recombinase (Addgene #105545-AAV1; pAAV.CMV.HI.eGFP-Cre.WPRE.SV40).

Techniques: In Vivo, Imaging, Expressing, Knock-Out, Activity Assay, MANN-WHITNEY, Control

Journal: Neuro-oncology Advances

Article Title: Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities

doi: 10.1093/noajnl/vdab180

Figure Lengend Snippet: EGFRvIII-/+ model system. (A) Detection of EGFR and EGFRvIII by Western blot analysis. β-Actin served as loading control. (B) EGFRvIII expression was detected by flow cytometry using an EGFRvIII-specific antibody (L8A4). (C) Colony formation, representative images. (D) Plating efficiency (average number of colonies per well divided by the number of seeded cells. (E) Proliferation. (F–H) Cell cycle distribution measured by flow cytometry. (F) Representative DNA content profiles, (G) cell cycle distribution and (H) quantification of S-phase cells ( n = 3; mean with SEM; P values are obtained by two-tailed Student’s t -test. * P < .05).

Article Snippet: Primary antibodies: EGFR (1:1000, rabbit, Cell Signaling Technology, #2232); pEGFR (1:1000, rabbit, Cell Signaling Technology, #4407); β-Actin (1:40000, mouse, Sigma-Aldrich, #A-2228); ATM (1:1000, rabbit, Cell Signaling Technology, #2873); pATM (1:1000, rabbit, GeneTex, GTX61739); ATR (1:1000, mouse, Santa Cruz, #SC-515173); pATR (1:1000, rabbit, Cell Signaling Technology, #58014); Chk1 (1:1000, mouse, Cell Signaling Technology, #2360); pChk1 (1:1000, rabbit, US Biological, #C4200-05); Chk2 (1:1000, mouse, BD Transduction Laboratories, #2360); pChk2 (1:1000, rabbit, Cell Signaling Technology, #2661); RPA (1:1000, mouse, Santa Cruz, SC-56770); pRPA (1:1000, rabbit, Boster, #02067); RNaseH (1:1000, rabbit, Abcam, #ab229078).

Techniques: Western Blot, Control, Expressing, Flow Cytometry, Two Tailed Test

Journal: Cell

Article Title: The intrinsic and extrinsic effects of TET proteins during gastrulation

doi: 10.1016/j.cell.2022.06.049

Figure Lengend Snippet: Generation of fluorescently labeled Tet -TKO mESCs clones and embryos, related to Figure 1 (A) Targeting strategy using CRISPR/Cas9-mediated genome editing and 3 gRNAs against Tet genes for generation of Tet KO mESC clone. Exons and conserved domains were shown in black and green/blue boxes, introns marked in lines. (B) Screening for clones with reduced 5hmC intensity after gene targeting using dot blot. Tet -TKO3 and Ctrl3 mESCs lines were marked in rectangles. (C) DNA sequencing validation for Tet -TKO3 mESCs clone. Protospacer adjacent motif (PAM) sequences are marked in red. Intron sequences are shown in lower-case letters. (D) Western blot validation for Tet -TKO3 mESCs clone. Following confirmation for lack of expression of TET2 protein, TKO3 clone was further validated for the lack of TET1 and TET3 protein expression. (E) Generation of Tet -TKO4 mESCs clone. Tet triple floxed mESCs were derived from blastocysts followed by Cre recombinase treatment . Validation of the deleted alleles was performed by PCR. f / f , homozygous floxed allele, Δ / Δ , homozygous deleted allele. Sizes of each allele were indicated on the right. (F) Flow cytometric analysis of reporter activity for day 5 (Ctrl3 and Tet -TKO3) and day 8 (Ctrl1, Ctrl2, Tet -TKO1, and Tet -TKO2) embryoid bodies. (G) Embryo size (extraembryonic mesodermal part excluded, log 2 of μm 2 ) comparisons between E7.5 control and Tet -TKO whole-embryo chimeras. Wilcoxon-Mann-Whitney test, two-tailed. Data are represented as mean ± SD. (H) Representative images of E7.5 control whole-embryo chimeras. Scale bars, 100 μm. (I) Representative images of E8.5 Tet -TKO3 and Tet -TKO4 whole-embryo chimeras. Scale bars, 100 μm.

Article Snippet: AAV encoding GFP/Cre recombinase fusion protein: AAV-GFP/Cre , , Addgene plasmid: #49056.

Techniques: Labeling, Clone Assay, CRISPR, Dot Blot, DNA Sequencing, Biomarker Discovery, Western Blot, Expressing, Derivative Assay, Activity Assay, Control, MANN-WHITNEY, Two Tailed Test

Journal: Cell

Article Title: The intrinsic and extrinsic effects of TET proteins during gastrulation

doi: 10.1016/j.cell.2022.06.049

Figure Lengend Snippet: scRNA-seq profiling of Tet -TKO whole-embryo chimeras, related to Figure 1 (A) Distribution of unique reads per cell in control and Tet -TKO whole-embryo chimeras. Cells with number of UMIs < 2,000 or > 12,000 were excluded in subsequent analysis. (B) Number of cells for individual control and Tet -TKO whole-embryo chimeras alongside their inferred E t . (C) Gene expression in Tet -TKO and control whole-embryo chimeras compared with WT for a selection of cell types. The comparison was performed between query cells from whole-embryo chimeras and their projected WT cells . The dashed line indicates a 2-fold change in expression. Genes with over 50% fold change were labeled in black. (D) Frequency distribution of cell types in Tet -TKO, control whole-embryo chimeras and WT embryos spanning E t 7.75–E t 8.1 as calculated in Figure 2 F. Wilcoxon-Mann-Whitney rank sum test, two-tailed. ns, not significant; ∗ , q value < 0.05 (Benjamini-Hochberg procedure). (E) Whole-mount in situ hybridization analysis of Noto , Cdx1 , and Twist1 expression in E8.5 Tet -TKO whole-embryo mutant and time-matched WT embryos. The number of embryos analyzed for each probe in WT and Tet -TKO embryos, respectively, is Noto (8, 2), Cdx1 (4, 3), and Twist1 (4, 3). Scale bars, 100 μm. (F) Time distribution of sampled cells from Tet -TKO whole-embryo chimeras compared with those of WT embryos with matched transcriptional time. (G) Genotyping of Tet triple floxed embryos following AAV-mediated Cre treatment. For each Tet gene, the absence of floxed allele and the presence of excised allele were tested. Note that the WT band is likely due to maternal blood contamination from the DNA sampling (see ). +/+, WT allele. Sizes of each allele were indicated on the right. (H) Phase-contrast images of E7.5 Tet- TKO embryos recovered post Cre recombinase treatment. Scale bars, 100 μm. (I) Cell-type composition as calculated in Figure 1 D for each Tet -TKO embryo recovered post Cre recombinase treatment. E t is indicated in parentheses. (J) Fraction of major lineages per embryo.

Article Snippet: AAV encoding GFP/Cre recombinase fusion protein: AAV-GFP/Cre , , Addgene plasmid: #49056.

Techniques: Control, Gene Expression, Selection, Comparison, Expressing, Labeling, MANN-WHITNEY, Two Tailed Test, In Situ Hybridization, Mutagenesis, Sampling

Journal: Cell

Article Title: The intrinsic and extrinsic effects of TET proteins during gastrulation

doi: 10.1016/j.cell.2022.06.049

Figure Lengend Snippet:

Article Snippet: AAV encoding GFP/Cre recombinase fusion protein: AAV-GFP/Cre , , Addgene plasmid: #49056.

Techniques: Recombinant, Virus, Transfection, Bicinchoninic Acid Protein Assay, Methylation, Western Blot, In Situ, Plasmid Preparation, Expressing, Labeling, Software