Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic pan DCs were isolated from naïve BALB/c mice and analyzed by flow cytometry to characterize pre-cDC1s and CD8α+ cDC1s. (A) Representative flow plot demonstrating gating strategy. Gated on CD11c+B220- (conventional DCs); pre-cDC1s are gated as CD24 high CD8α- and CD8α+ cDC1s are gated as CD8α+. (B) Mean absolute cell number of respective DC subsets shown with SEM. (C-F) Mean percent and MFI with representative histograms shown with SEM for expression of (C) PD-L1, (D) PIR-B, (E) CD70, and (F) ICOSL on CD8α+ cDC1s (black) and pre-cDC1s (cyan). Data is representative of two experiments. (n = 5 per group). Paired t tests were used to determine significance between DC subsets. **P<0.01, ***P<0.001.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and analyzed by flow cytometry. Data is representative of three independent experiments. (n = 4 per group) (A) Representative flow plots depicting relative cDC1 proportions in naïve DCs (left) and 2.43 mAb DCs (right). Gated on CD11c+B220- (conventional DCs). (B) Mean percent of CD8α+ cDC1s (black) and pre-cDC1s (cyan) in untreated and 2.43 mAb-treated DCs shown with SEM. Two-way ANOVA and Dunnett’s multiple comparisons used to determine significance. **P<0.01, ****P<0.0001. (C) Mean absolute cell number of pre-cDC1s (CD24 high CD8α-) in untreated (black) and 2.43 mAb-treated (magenta) groups shown with SEM. Unpaired t test used to determine significance. *P<0.05. (D) Schematic depicting experimental design for proposed allogeneic mixed leukocyte reaction (MLR) using 2.43 mAb treatment.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and plated in an allogeneic MLR. Allogeneic T-cell proliferation was assessed on day 3 and day 4 of co-culture. Data is representative of two independent experiments. (n = 4 per group) (A-B) ModFit Software analysis of CellTrace dye dilutions. (A) Representative histograms depicting proliferation of H2K b + T-cells following stimulation with naïve DCs (top) or 2.43 mAb DCs (bottom) on days 3 and 4. Proliferation Index (PI) for representative histograms is boxed. (B) Mean PI for naïve or 2.43 mAb DCs shown with SEM. (C-G) Flow analysis of the proliferative fraction of T-cells was determined by first gating on H2K b +CellTrace low T-cells. (C) Mean percent of proliferated T-cells that are CD4+ (left) and CD8+ (right) on day 3 shown with SEM. (D) Mean percent of proliferated CD4+ (left) and CD8+ (right) T-cells expressing PD-1 on day 3 shown with SEM. (E) Mean percent of proliferated T-cells that are CD4+ (left) and CD8+ (right) on day 4 shown with SEM. (F) Mean percent of proliferated CD4+ (left) and CD8+ (right) T-cells expressing PD-1 on day 4 shown with SEM. (G) Mean percent of proliferated T-cells (left) and CD4+ T-cells (right) that are Propidium Iodide+ shown with SEM. Unpaired t tests were used to determine significance between groups. *P<0.05.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Co-Culture Assay, Software, Expressing

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and plated in an allogeneic MLR. DC composition was determined by flow cytometry on day 2 of co-culture. Data is representative of two independent experiments. (n = 4 per group) (A) Representative flow plots depicting percent of H2K d +CD11c+ DCs in naïve DCs (left) and 2.43 mAb DCs (right) on day 2 of co-culture. (B) Mean percent of H2K d +CD11c+ DCs for naïve (black) or 2.43 mAb DCs (magenta) shown with SEM. Unpaired t test was used to determine significance between groups. **P<0.01. (C) Representative flow plots depicting percent CD8α+ cDC1s and pre-cDC1s in naïve (left) and 2.43 mAb DCs (right). (D) Mean percent CD8α+ cDC1s (black bars) and pre-cDC1s (teal bars) in naïve and 2.43 mAb DCs shown with SEM. 2way ANOVA and Šidák’s multiple comparison used to determine statistical significance. **P<0.01.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry, Co-Culture Assay, Comparison

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic DCs were isolated from naïve BALB/c mice and pre-cDC1s and CD8α+ cDC1s were cell sorted for RNA sequencing. (A) Enhanced volcano plot identifying genes that are up or down-regulated in pre-cDC1s compared to CD8α+ cDC1s. (B-C) Pathway results using Qiagen’s IPA software. P = 0.05 corresponds to a 1.3 -log p-value, and anything above this value is considered statistically significant. (B) Pathways predicted to be significantly inhibited (z-score < -2) in pre-cDC1s compared to CD8α+ cDC1s. (C) Pathways predicted to be significantly activated (z-score < 2) in pre-cDC1s compared to CD8α+ cDC1s. (D) Heat map showing relative gene expression levels for the PD-1/PD-L1 cancer immunotherapy pathway. Full documentation, methodology, and raw data for this data set is available online at: https://doi.org/10.25422/azu.data.14241902 .

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, RNA Sequencing, Software, Gene Expression

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: Multiplex fluorescent immunostaining and 3D image reconstruction of whole core needle biopsies using T3. a Diagram shows slicing and imaging of cross-section of cores to assess antibody penetration after multiplex immunostaining. b Cross section images from BALB-NeuT tumor core after immunostaining for Her2 (green), CD3 (yellow), CD8 (red), CD31 (cyan), F4/80 (magenta), and ER-TR7 (gray). Left column shows 10X objective scan of 0.84 mm diameter core. Right column shows 40X objective region-of-interest imaging of the center of the cross section (white dotted square) demonstrating staining of tumor cells, T cells, microvasculature, macrophages, and fibroblasts. Scale bars: 100 μm (left) and 20 μm (right). c Six-plex immunostaining, clearing, scanning and image analysis of a whole BALB-NeuT tumor core (top image) reveals 3D distributions of Her2 tumor cell marker (green), CD3 T cell marker (yellow), CD8 cytotoxic T cell marker (red), CD31 endothelial marker (cyan), F4/80 macrophage marker (magenta), and ER-TR7 fibroblast marker (gray), displaying each channel in pseudocolor individually and in a merged image. Scale bar: 500 μm. d High resolution 2D images of the cell markers in the core. Scale bar: 10 μm.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Multiplex Assay, Immunostaining, Imaging, Staining, Marker

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: 3D visualization and mapping of CTLs in whole head and neck cancer cores. a Cores obtained with an 18 gauge automated device from head and neck cancer samples from patients 1 and 2 (left images) were immunostained for EGFR (green), CD3 (yellow), CD8 (red), and CD31 (cyan), cleared and scanned. 3D rendering of whole cores are shown for each channel and a merged image. Scale bar: 500 μm. b High resolution 3D view (left) and X-Y tomographic visualization (right) of a volume within the rendered core models, showing distributions of CD3 + CD8 + CTLs (orange) and CD31 + endothelium (cyan) within the EGFR + (green) tumor tissue and stroma. Scale bar: 30 μm. c Serial tomographic sections of X-Y planes at different Z-stack depths showing CTLs (orange) and endothelium (cyan) within the EGFR + (green) tumor tissue.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: 3D mapping and analysis of cytotoxic T lymphocytes in whole mouse core needle biopsy. a 3D spatial mapping of CD3 + CD8 + cytotoxic T lymphocytes (CTLs) in cores from six NeuT tumors displays local clustering and inhomogeneity. Scale bar: 500 μm. b Enumeration of CTLs in virtual optical sections along the long axis of each core in a demonstrates heterogeneous distribution of CTLs. c Conventional IHC after T3 confirms non-uniform distribution of CD8 + cells. Core #1 in a was formalin fixed and paraffin embedded, sectioned, immunostained, counterstained, and scanned. Inset displays high-magnification image of CD8 + cells. Scale bar: 1 mm (top) and 10 μm (inset). d Manual counting of CD8 + cells after IHC. Every seventh 5 μm serial section was stained and counted.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: Spatial correlation between CTLs and microvasculature in tumor parenchyma in head and neck cancer. a Relative quantification of EGFR + parenchyma (%) in sets of three cores each from six excised head and neck cancer patient samples. Data are expressed as mean ± SEM. b Quantification of relative volumes (%, mean ± SEM, n = 3) of CD3 + CD8 + CTLs within the EGFR + parenchyma (orange) and the whole cores (green) for each patient sample. c Quantification of relative volumes (%, mean ± SEM, n = 3) of CD31 + microvasculature within the EGFR + parenchyma (orange) and the whole cores (green) for each patient. d Plot comparing normalized densities of CD3 + CD8 + CTLs and CD31 + microvasculature in EGFR + parenchyma for each patient (% CD3 + CD8 + ( b ) or % CD31 + ( c ) within EGFR + volume/% EGFR + volume ( a ), mean ± SEM, n = 3). Three groups (blue, red, and green) were defined using K-means cluster analysis ( P <0.05 for both X and Y axes), distinguishing one patient, 1, with a “hot”, inflamed tumor and another patient, 2, with a “cold”, non-inflamed tumor. e High resolution 2D images of CD3 + CD8 + CTLs and CD31 + endothelium in EGFR + parenchyma and stroma in Patients 1 (“hot”) and 2 (“cold”) cores. Scale bar: 10 μm. f 3D distance profiles of CD3 + CD8 + CTLs away from CD31 + blood vessels in Patient 1 (“hot”) core. Inset shows a color distance map between blood vessel and CTL surfaces.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Quantitative Proteomics