Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), Alt (10μg) or PBS (25μl) over 1 week and culled 24h after the final dose. The frequency of ILC2 (GFP+ CD45+ Linneg CD3-NKp46-CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung and (C) lung draining lymph nodes (mediastinal). Live viable precision cut lung slices of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow). Images of 1024μm x 1024μm field of view (FOV) were taken under a 20x objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4-) CD4+ T cells (CD4+GFP-) location in PBS, rIL-33 and Alt treated mice, scale bar, 150 μm. (E) Number of ILC2 (GFP+CD4-) in lung sections per FOV taken under a 10x objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where precision cut lung slices were prepared. Representative images show two regions of the lung slice from a rIL-33 treated mouse showing distribution of ILC2 and CD4+ T cells, scale bar 150 μm. n = 4 mice per group (Mock(PBS)), n= 6 mice per group (Alt or rIL-33 treatment). Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p <0.001 and, **** p < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Staining, Microscopy

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose), over 1 week and culled 24h after the final dose. Live viable precision cut lung slices (PCLS) of 200μm thickness were obtained and stained for CD31 (Magenta, the lung structure and blood vessels), CD4 (cyan, T cells, orange arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), and time-lapse video taken (1024μm x 1024μm field of view (FOV), 45 min duration under a 20x objective using an inverted confocal microscope) (A) Static image depicting the location of ILC2 and CD4+ T cells, scale bar 100 μm. (B) Zoomed in section of the blood vessel in figure 2A, scale bar 20 μm. (C) High power images of boxed cells in figure 2B showing differences in pattern of cell movement (oscillatory vs amoeboid movement). ILC2 and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length and (H) track displacement were quantified. Representative images shown in (A-C) are from rIL-33 treated mice, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F-H) in box and whiskers plots, each dot represents an individual cell. Data are representative from 4 experiments where n = 6 mice per treatment. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Staining, Microscopy

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or Alt (10μg) over 1 week. Live precision cut lung slices were obtained and ILC2 dynamics were compared between the two and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13eGFP mice after rIL-33 treatment (one 512μm x 512μm field of view (FOV) in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2 at separate time-points, scale bar 20 μm. n ≥ 4 mice per group. Data representative of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and, **** p < 0.0001. Quantifications from (A-E) are representative of 4 experiments, where n = 6 mice per treatment (3 slices per mouse were imaged). For (F) IVM images are representative of 6 individual IL-33 treated mice. *p < 0.05, **p < 0.01, ***p < 0.001, and, **** p < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Intravital Microscopy

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose. (A) The percentage of murine ILC2 (CD45+LinnegNKp46-CD3-) expressing CCR1, CCR4 and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2 and (E) quantified CCL8 deposits in PCLS stained for CD31 (Magenta, the lung structure and blood vessels), CCL8 (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow), images of 1024μm x 1024μm FOV, scale bar 150 μm. Human ILC2 lines were generated and migration to varying concentrations of (F) PGD2 and (G) CCL8 were determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8 and PGD2. IL13-eGFP mice treated with rIL-33 were also treated with 5μg purified anti-mouse CCR8 antibody i.p., rCCL8 i.n. or an isotype control and PCLS obtained and stained. (I) Localisation of ILC2 in live PCLS. (J) Number of ILC2 per FOV under 10x objective. Time-lapse imaging of 45 min duration was performed and ILC2 (K) track from centroid, (L) track length and (M) track speed and (N) track displacement were quantified. In box and whiskers graphs each data point represents an individual cell. Balb/c mice treated with rIL-33 were further treated with rCCL8, αCCR8 or Isotype control antibody. (O) Percentage of IL-13+IL-5+ILC2 (CD45+lin-NKp46-CD3-GATA-3+). (P) Representation Histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 3 individual donors. Data representative of 3 experiments. For panels I-R, n = 5 mice per group. Data representative of 2 experiments *p < 0.05, **p < 0.01, ***p < 0.001 and,**** p < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Expressing, Staining, Generated, Migration, Purification, Control, Imaging

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) for 24h. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots and (C) track speed spider plot. IL13-eGFP mice were treated with 3 doses of rIL-33 (1μg per dose) or PBS (25μl), over 1 week and culled 24h after the final dose PCLS obtained. (D) SHG imaging of PCLS revealing collagen fibres, representative maximum intensity projections, scale bar 50μm. (E) GLCM analysis of SHG imaging. (F) Images of Fibronectin expression and localisation. PCLS stained for CD31 (Magenta, the lung structure and blood vessels), Fibronectin (cyan, yellow arrow), EpCAM (Red, to visualise bronchial epithelium) and GFP (ILC2, white arrow) and images of 1024μm x 1024μm field of view (FOV) were taken, scale bar 150μm. For panels A-C, n = 3 donors (in triplicate). Data representative of 3 experiments. For panels D-F n = 6 (in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001 and,****, ****P < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Control, Imaging, Expressing, Staining

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV or serum free coating (control) and imaged after 12 hours. (A) Bright field images depicting change in shape. (B) Actin remodelling following staining with Phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections), scale bar 5μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data representative of 2 experiments. *** P < 0.001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques: Control, Staining, Imaging

Journal: Science immunology

Article Title: Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

doi: 10.1126/sciimmunol.aav7638

Figure Lengend Snippet: IL13-eGFP mice treated with rIL-33 were further treated with (BAPN) along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLS were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length and (E) track displacement. Total ILC2 () in (F) Lungs, (G) BAL and (H) Blood were enumerated. For panels A-E n ≥ 4 mice per group. Data representative of 4 experiments. For panels F-H n = 6 mice per group. Data representative of 2 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: ILC2 were enriched from whole blood using RosetteSepTM Human ILC2 Enrichment Kit (STEMCELL Technologies) and further sorted by FACs using CD45 + Lineage neg (CD1a, CD3, CD4, CD5, CD8, CD11c, CD14, CD16, CD19, CD20, CD34, FcγRI and CD123) (Biolegend), CD161 + , CD127 + , CRTH2 + and C-Kit var (Biolegend).

Techniques:

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: nPKC-θ signatures are enriched in CTCs and metastatic tissues and are associated with poor patient survival in immunotherapy-resistant disease. ( A ) Immunohistofluorescence analysis of PKC-θ expression in CD4 + and CD8 + T cells isolated from healthy donor liquid biopsies. Bar/dot plots show the Fn/c (nuclear to cytoplasmic ratio) of PKC-θ phosphorylated at threonine 568 (PKC-θ-Thr568p). A score below 1 indicates cytoplasmic bias. Data are from three separate patients, n ≥ 20 cells per patient. Representative images are shown for each dataset. PKC-θ-Thr568p (red); CD8/CD4 (purple), and DAPI (cyan) were used to visualize expression and nuclei; scale bar represents 10 µM. ( B ) Immunohistofluorescence analysis of PKC-θ expression in human MCF-7 inducible model (MCF-7-IM) and MDA-MB-231 breast cancer cells. Human MCF-7 epithelial cells (MCF-7epi) were activated with PMA to induce EMT and generate MCF-7 mesenchymal-like (MCF-7mes) breast cancer cells. Bar graphs show the mean nuclear fluorescence intensity (NFI), cytoplasmic fluorescence intensity (CFI), and Fn/c for PKC-θ-Thr568p, n ≥ 20 cells per group. Representative images are shown for each dataset. PKC-θ-Thr568p (green) and DAPI (blue) were used to visualize nuclei. Scale bar represents 10 µM. ( C ) Contrast-enhanced CT scans of tumors in CR and PD metastatic melanoma patients at baseline and 12 weeks after treatment with immunotherapy (nivolumab). Red circles indicate tumor lesions. Tumor lesions are reduced in CR compared with baseline, while PD shows increased tumor burden. ( D ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence intensity in circulating tumor cells (CTCs) isolated from immunotherapy-responsive (CR, partial response (PR)) or resistant (stable disease (SD), PD) melanoma patients defined using RECIST 1.1 criteria. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified using ASI digital pathology. Representative images are shown for each cohort (six patients were profiled per cohort, n ≥ 20 cells per group); scale bar represents 10 µM. ( E ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence in FFPE sections of primary melanomas from patients ( n = 18 patients) with CR, SD, or PD. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified by ASI digital pathology ( n ≥ 40 cells per patient sample, four samples per patient). Representative images for each dataset are shown, scale bar represents 30 µM. ( F ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence intensity in FFPE sections from breast cancer brain metastases ( n = 30 patients) and primary breast cancer biopsies ( n = 15 patients). The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified by ASI digital pathology ( n > 40 cells per patient sample). Representative images for each dataset are shown (top); scale bar represents 30 µM. ( G ) Percent change in tumor lesion from baseline for a single patient with metastatic melanoma (Patient D) who was resistant to first-line treatment with pembrolizumab and displayed PD (baseline, 12 weeks) as defined by RECIST 1.1 criteria but subsequently responded to second-line nivolumab and ipilimumab to show a CR (24 weeks, 36 weeks). ( H ) CT scan showing overall tumor burden in Patient D at baseline, SD, and PR (partial response). ( I ) Mesenchymal protein expression (PKC-θ-Thr568p, CSV, and ABCB5) was profiled in CTCs isolated from Patient D at 0 (baseline) and 12-, 24-, and 36-weeks post-immunotherapy. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were determined by ASI Digital Pathology. Representative images for each dataset are shown ( n ≥ 20 cells per group); scale bar represents 10 µM. ( J ) Metastatic melanoma patients ( n = 18 patients) were scored for the Fn/c of PKC-θ from four liquid biopsies over 12 months, with Fn/c categorized as <3 or ≥3 (Fn/c >1 indicates nuclear bias, whereas <1 indicates cytoplasmic bias). These patients were tracked for an additional two years (total 36 months), and their survival data are plotted as Fn/c <3 or ≥3. Statistical significance is denoted by ns (not significant), * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.0001.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Immunohistofluorescence, Expressing, Isolation, Fluorescence, Computed Tomography

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: PKC-θ is enriched in the nuclei of dysfunctional CD8+ T cells isolated from stage IV metastatic cancers. ( A ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from immunotherapy-responsive (complete response, CR; partial response PR) or resistant (PD, progressive disease) melanoma patients defined using RECIST 1.1 criteria. The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 20 cells per group). ( B ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from healthy donors (HD), stage IV metastatic breast cancer patients (Mets), or stage IV breast cancer patients with brain metastases (Brain Mets). The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 40 cells per group). ( C ) The amino acid sequence of ZEB1 indicating the top eight peptides for peptide phosphorylation by PKC-θ. The top peptide sequences are displayed in a table with the mean signal intensity (2 SD above the mean was considered a positive phosphorylation event). ( D ) Top peptides positive for phosphorylation and their overlap with the ZEB1 amino acid sequence as well as the structure of ZEB1, adapted from . ( E ) Duolink ® proximity ligation assay (PLA) for PKC-θ and ZEB1 in CD8+PD1+ T cells isolated from immunotherapy-resistant or responder melanoma patients. Representative images are shown for PKC-θ/ZEB1, scale bar represents 10 µM. Graphs represent the PLA signal intensity of the Duolink ® assay; data represent n ≥ 100 cells/sample. Graphs plot the percentage of PLA signal positive cells out of total cells for (A). Data represent n ≥ 100 cells/sample. ( F ) FFPE sections from primary breast cancers ( n = 6 patients, >500 cells counted per patient) or breast cancer brain metastases ( n = 20 patients, >500 cells counted per patient) were processed for high-resolution microscopy using the BondRX platform. FFPE sections were fixed and immunofluorescence microscopy performed probing with primary antibodies targeting CD8, PKC-θ (T53p), and ZEB1 with DAPI. Plots represent the % population of CD8+ T cells positive for PKC-θ and ZEB1 out of total CD8+ T cells. Example images are shown with 20 µM scale bar. ( G ) CD8+ cells were isolated from melanoma patient liquid biopsies (responder = complete response (CR) or resistant, where primary = primary resistance, secondary = secondary resistance, PD = progression of disease) and stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CI) and pre-clinically screened with either vehicle control or nPKC-i2. Samples were then fixed and immunofluorescence microscopy performed with primary antibodies targeting ZEB1, PKC-θ, and CD8. Representative images for each dataset are shown in . Graph represents the mean TNFI for PKC-θ and ZEB1 measured using ImageJ to select the nucleus minus background ( n > 20 cells/sample). ( H ) Plot profiles for each cohort for ZEB1 and PKC-θ are also depicted (red = ZEB1, green = PKC-θ) with the Pearson correlation coefficient (PCC) used to quantify colocalization between fluorophore-tagged proteins indicated and plotted. −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. Statistical significance is denoted by ns (not significant), ** p ≤ 0.005 and **** p ≤ 0.0001.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Isolation, Sequencing, Phospho-proteomics, Proximity Ligation Assay, Microscopy, Immunofluorescence, Control

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: nPKC-θi2 disrupts the nuclear ZEB1/PKC-θ complex and induces cytokine production in CD8+ T cells. ( A ) Graphs depicting the % inhibition or induction based on protein expression were also plotted for each protein target relative to untreated sample. ( B ) Percent of PKC-θ+/ZEB1+/CD8+ T cells in samples isolated from melanoma patients responsive (PR/CR) or primary/secondary resistant (PD) to immunotherapy. CD8+ T cells were treated with nPKC-θi2 before activation ex vivo with PMA/ionomycin. ( C ) Gene expression of key effector cytokines IL2, IFNG, and TNFA in PBMCs isolated from resistant and responder patients either treated with vehicle control or nPKC-θi2 before activation ex vivo with PMA/ionomycin. ( D ) Protein expression of TNF-α and IFN-γ in CD8+ T cells isolated from primary or secondary resistant or responder patient liquid biopsies and treated with nPKC-θi2. Graphs show the % CD8+ increase in expression of TNF-α or IFN-γ in CD8+ T cells stimulated with PMA/ionomycin in addition to treatment with nPKC-θi2. One-way ANOVA was used to compare groups, where ns (not significant), **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Inhibition, Expressing, Isolation, Activation Assay, Ex Vivo, Gene Expression, Control