Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, Magnetic bead purified CD4+ T cells were stained with CFSE (2.5μM) and stimulated with plate-bound anti-CD3 antibody (2μg/ml) for three days together with Pam3CSK4 (4μg/ml), R-848 (1μg/ml), or ORNs (1μM) formulated with 12.5 μg/ml DOTAP. The proliferation of T cells was measured as the dilution of CFSE. One representative out of six experiments is shown. B, Isolated T cells were stimulated like in (A) and proliferation was calculated as the dilution of CFSE. Concentration of ORNs (R-0006, R-0002, R-1263) was 0.125μM, prepared with 1.55μg/ml DOTAP. Data shown represent mean ± SEM of five donors. C, Supernatants of the T cell cultures were harvested and IFN-γ production was measured in an ELISA. For stimulation without anti-CD3 antibody, 100 U/ml IL-2 were used. Data shown represent mean ± SEM of six donors. Significance of differences was determined by Student’s t-test.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Purification, Staining, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were isolated from different donors, stained with CFSE (2.5μM) and stimulated with anti-CD3 antibody (2μg/ml) and the indicated ligands on 96-well plates for three days. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). ODNs were used in the absence of DOTAP. For analysis PBMCs were stained with mAb to CD4, CD8 (B) and CD19 (C), and the proliferation of the different cell types was measured as dilution of CFSE fluorescence. Data shown represent mean ± SEM of eleven (A, B) and 3 donors (C).

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Fluorescence

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were stimulated with 2.5μg/ml TT for six days together with the indicated TLR agonists. ORN R-0006 and R-1263 were formulated with DOTAP (1μM ORN with 12.5μg/ml DOTAP). The recall antigen stimulation of CD4+ T cells within PBMCs was measured as the dilution of CFSE. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated with 2μg/ml SEB for three days and proliferation of CD4+ and CD8+ T cells was measured. Data shown represent mean ± SEM of six donors. C, T cells from one donor and CD3 depleted APCs from another donor were isolated and mixed in a 5:1 ratio (T cells:APCs in a MLR) for three days. For inhibition of IDO function, two different concentrations of 1-MT (100μM and 500μM) were added to the cultures. Proliferation of T cells in allogenic MLR was measured as the dilution of CFSE. One experiment out of three is shown. D, PBMCs were stimulated with the indicated ligands (1.3μM CpG C-Class ODN 2395 or ORN R-0006 with DOTAP 16.6μg/ml) or 20ng/ml IFN-γ for 24h. Plates were washed and RNA from plate bound monocytes was prepared. IDO mRNA levels relative to 1000 copies cyclophilin B are depicted. Data shown are mean ± SEM of three donors

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Isolation, Inhibition

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were stimulated with the different ORNs (4μM with 50μg/ml DOTAP) for 24h and stained with AnnexinV and PI together with anti-CD3 Ab. Percentage of the AnnexinV−, PI− population within the whole PBMCs or within the pre-gated CD3+ T cell population (B) is indicated. Data shown represent mean ± SEM of three donors.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Staining

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, Schematic overview of the transwell assay system. Flat bottom 96-well plates were coated with anti-CD3 Ab (2μg/ml) and T cells were cultured on these plates. In the transwell chamber T cell depleted PBMCs were stimulated. T cells and T cell depleted PBMCs from one donor were isolated and mixed either directly together (5×105 T cells with 1×105 T cell depleted PBMCs) for the control plate (C), or were stimulated in different chambers in the transwell system (B). Before mixing, T cells were stained with 2.5μM CFSE. Cells were stimulated for three days with the indicated ligands. ORNs were formulated with DOTAP (4μM with 50μg/ml DOTAP). As control T cells were stimulated with anti-CD3 Ab together with 100 U/ml IL-2. Data shown represent mean ± SEM of 4 donors.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Transwell Assay, Cell Culture, Isolation, Control, Staining

Journal:

Article Title: Positive T cell co-stimulation by TLR7/8 ligands is dependent on the cellular environment 1

doi: 10.1016/j.imbio.2010.03.011

Figure Lengend Snippet: A, PBMCs were depleted of CD14+ monocytes by magnetic bead separation. Depleted PBMCs or non-depleted PBMCs, as control, were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of six donors. B, PBMCs were stimulated for 24h with the indicated ligands with 0.1μM ORNs formulated with 1.25μg/ml DOTAP or 1μM ORN formulated with 12.5μg/ml DOTAP. PBMCs were stained with a cocktail of anti-CD19, anti-CD56, anti-CD3 antibodies together with anti-PD-L1 antibody. Monocytes (as CD56−CD3−CD19− population) were analyzed for PD-L1 expression after 24h. Data shown represent mean ± SEM of six donors (two independent experiments). Significance of differences was determined by Student’s t-test. C, PBMCs were stained with CFSE (2.5μM) and stimulated for three days with anti-CD3 antibody (2μg/ml) and the indicated TLR ligands. ORNs were formulated with DOTAP (2μM ORN with 25μg/ml DOTAP). Proliferation of CD4+ and CD8+ T cells was measured after three days of culture. Data shown are mean ± SEM of three donors. D, Splenocytes from C57BL/6 wt mice were isolated and stained with CFSE (2.5μM), and stimulated with the indicated TLR ligands. ORNs were formulated with DOTAP (4μM ORN with 50μg/ml DOTAP). Data shown represent mean ± SEM of four wt C57BL/6 mice.

Article Snippet: For the isolation of CD3 depleted PBMCs the RosetteSepTM Human CD3 + Depletion Kit (Stemcell Technologies) was used according to the manufacturer’s instructions.

Techniques: Control, Staining, Expressing, Isolation

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: