Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD314 , REA1228 , 50 , 130-124-341 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Nature Communications

Article Title: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer

doi: 10.1038/s41467-020-17644-0

Figure Lengend Snippet: a , b Tumor-free survival (TFS), overall survival (OS) and time-to-death (TTD) of WT C57BL/6 mice ( a , b ) and Rag2 − / − Il2rg − / − mice ( a ) or Ifng − / − mice ( b ) subjected to M/D-driven oncogenesis. Number of mice, hazard ratio (HR) and p values (two-sided log-rank) are reported. c – e . Variations in TFS ( c ), OS ( d ) and TTD ( e ) imposed to M/D-driven oncogenesis in mice by the indicated genotype or immunomodulatory interventions. Results are means ± SEM plus individual data points. Number of mice, HR and p values (two-sided log-rank and one way-ANOVA plus Fisher LSD, calculated with respect to individual control experiments) are reported. Green dots indicate mice that were free of disease ( c ) and alive ( d ) at the end of the experiment. f – h TFS and OS of WT C57BL/6 ( f – h ) mice and Rag2 − / − BALB/c mice ( f ), C57BL/6 mice receiving NKG2D-depleting antibodies ( g ), or C57BL/6 mice receiving CD4- and CD8-depleting antibodies ( h ). Number of mice, HR and p values (two-sided log-rank) are reported.

Article Snippet: All antibodies blocking or neutralizing specific immune cell populations, cytokines or cytokine receptors and their corresponding isotype controls were acquired from BioXCell (West Lebanon): CD4 (clone GK1.5, #BE0003-1), CD8α (clone 2.43, #BE0061), NKG2D (clone HMG2D, #BE0111), NK1.1 (clone PK136, #BE0036), PD-1 (clone RMP1-14, #BE0146), IFNγ (clone R4-6A2, #BE0054), IFNAR1 (clone MAR1-5A3, #BE0241), IL-17A (clone 17F3, #BE0173).

Techniques: Control

Journal: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) Heatmaps generated from eve smFISH data showing the number of active transcription sites across the expression domain at progressive time points during nc14 (underlying data in Fig. S1A). (B) Maximum projection of an smFISH image of early mid eve stripe 2, with mRNAs shown in white and DAPI in blue. Scale bars: 5 μm (main image) and 1 μm (inset regions). Green boxes mark cells displayed in the inset regions in the centre and the anterior and posterior edges. Active transcription sites were identified within the nuclear compartment in Z and are marked with a green arrow. (C) As in B for stripe 2 in late mid nc14. (D) (i) Confocal images of fixed nc14 embryos stained with smFISH probes for eve (magenta) and Me31B-GFP P-bodies (green). Scale bar: 5 μm. Images are maximum projections of 7 slices; individual mRNAs (magenta arrowheads), P-bodies (green arrowheads), and colocalised mRNA and P-body signals (white arrowheads) are highlighted. (E) P-body colocalisation index of eve mRNAs in early mid and late mid nc14; n = 3 embryos, p = 0.0034, unpaired t-test used to determine significance with α = 0.05. (F) Schematic illustrating anterior-posterior (AP) binning of a region across stripe 2 used in (G). (G) P-body colocalisation data across stripe 2 for 3 timepoints from mid nc14. Data over the AP axis are fit with a gaussian process (GP). The green shaded region marks the 95% confidence interval. Heatmaps above display the average mRNA per cell over the corresponding AP bins.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques: Generated, Expressing, Staining

Journal: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) smFISH image of P-bodies (green arrowheads) marked by Me31B-GFP in wt and pcm - mCherry-BLID blue light treated nc14 embryos. (B) (i) P-body volumes in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP embryos, n = 3 embryos, p = 0.0041, nested t-test used to determine significance with α = 0.05. (ii) P-body numbers in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos, n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. (C) smFISH images of eve mRNAs (magenta arrowheads), P-bodies (green arrowheads) and colocalised mRNAs (white arrowheads) in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos. (D) Number of mRNAs per cell in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos, n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. (E) Percentage of mRNAs localised to P-bodies in Me31B-GFP and pcm - mCherry-BLID; Me31B-GFP blue light treated embryos; n = 3 embryos, unpaired t-test used to determine significance with α = 0.05. Scale bars: 2 μm.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques:

Journal: bioRxiv

Article Title: Spatially regulated mRNA decay sharpens expression patterns in the Drosophila embryo

doi: 10.1101/2025.02.08.637258

Figure Lengend Snippet: (A) smFISH images of eve mRNAs in early nc14 in control ( nos-Cas9 and Me31B-GFP ) and degron ( pcm - mCherry-BLID and pcm-mCherry-BLID; Me31B-GFP ) embryos. All embryos have membrane ingression < 5 μm. (B) Quantitation of wild-type and mutant eve patterns in control ( nos-Cas9 and Me31B-GFP ) degron ( pcm - mCherry-BLID and pcm - mCherry-BLID; Me31B-GFP ) and pcm mutant embryos ( pcm 15 / FM7 and pcm 15 / FM6; Me31B-GFP ) from eve RISH ( n = 106, 26, 57,11, 63 and 94). (C) . Confocal images of mutant eve expression patterns in pcm 15 /FM6; Me31B-GFP embryos. Scale bars: 50 μm.

Article Snippet: A Rabbit anti-Me31B antibody (1:400, biorbyt, orb1147725) was used to immunostain embryos in and Fig. S5. smFISH staining in , and S1-4 was carried out in the Me31B-GFP line (BDSC Stock #51530).

Techniques: Control, Membrane, Quantitation Assay, Mutagenesis, Expressing

Journal: OncoImmunology

Article Title: NKG2D- and T-cell receptor-dependent lysis of malignant glioma cell lines by human γδ T cells: Modulation by temozolomide and A disintegrin and metalloproteases 10 and 17 inhibitors

doi: 10.1080/2162402x.2015.1093276

Figure Lengend Snippet: Figure 6. Involvement of NKG2D and TCR in T cell-mediated lysis of GBM

Article Snippet: Where indicated, effector cells were pre-incubated for 1 h with 10 μg/ml anti TCR-V9 mAb 7A5,29 anti-NKG2D mAb (#MAB139, clone 149810, R&D systems) or mouse isotype control IgG1 (#18443, clone MOPC-21, Abcam) before the addition of Calcein AM-labelled tumor target cells.

Techniques: Lysis

Journal: Experimental and therapeutic medicine

Article Title: Role of natural killer cells in liver transplantation treatment of liver cancer.

doi: 10.3892/etm.2017.4748

Figure Lengend Snippet: Figure 2. Activation of liver NK cells was detected using flow cytometry. (A) Number of NK cells in 105 CD45+ cells was compared between L‑IFN and H‑IFN groups. Expression of activation markers (B) NKG2D, (C) CD69 and (D) IFN‑γ in NK cells. ***P<0.001. NK, natural killer; CD, cluster if differentiation; IFN‑γ, interferon‑γ; L‑IFN, low in IFN group; H‑IFN, high in IFN group; NKG2d, NK group 2D.

Article Snippet: The antibodies used were as follows: Fluorescein isothiocyanate-conjugated NK1.1 (cat. no. FAB22252P), phycoerythrin (PE)-conjugated CD69 (cat. no. FAB23591P), IFN-γ (cat. no. 285-IF) or NK group 2D (NKG2D, cat. no. 1299-NK), PE-cy5.5-conjugated CD3 (cat. no. MAB100) (all from R&D Systems, Inc.).

Techniques: Activation Assay, Flow Cytometry, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization

doi: 10.4049/jimmunol.2300834

Figure Lengend Snippet: TGF-β1 and IL-15 induce CD103 and CD49a expression on NK cells. PBMCs were stimulated with TGF-β1 and IL-15 or with IL-15 alone for 7 d in vitro and stained for CD103. (A) Contour plot showing percentage of CD56+Lin− (CD3−CD14−CD19−CD20−CD34−CD123−CD303−FCεRIα−TCRαβ−TCRγδ−) NK cells expressing CD103. U/S PB NK cells are also shown as a control condition. A total of five experiments across six PB donors were conducted. (B and C) Percentage of CD56+Lin− NK cells expressing CD103 after NK cells were stimulated with different cytokine combinations, including (B) IL-2, IL-7, IL-12, IL-18, and IL-21 in combination with TGF-β1 and (C) IL-15 in combination with different cytokines. U/S represents U/S CD56+Lin− NK cells. Data are representative of two independent experiments with two different donors. (D) Representative contour plots from PB NK cells stimulated with TGF-β1 and IL-15, IL-15 only, or U/S showing expression of CD103 versus CD49a or CD103 versus CD69. Figures are representative of four separate experiments conducted across five PB donors. Two-way ANOVA (main effect of TGF-β1, p < 0.0001) (C) and Welch’s t test (B) were used for statistical analysis. ***p < 0.001; ****p < 0.0001. Variances are all displayed as SEM unless otherwise specified.

Article Snippet: In some experiments, cells were treated with or without anti-TGF-β neutralizing Ab (10 μg/ml, catalog no. BP0057, 1D11.16.8, Bio X Cell).

Techniques: Expressing, In Vitro, Staining, Control

Journal: The Journal of Immunology Author Choice

Article Title: Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization

doi: 10.4049/jimmunol.2300834

Figure Lengend Snippet: Ascites-derived TGF-β induces expression of CD103 and CD49a on PB NK cells. (A) To determine whether the TME induces regulatory features in NK cells, PB NK cells from healthy donors were cultured with 50% supernatant of ascites from patients with EOC and IL-15 for 7 d. (B) Contour plots of a sample where PB CD56+Lin− NK cells were stimulated with 50% ascites supernatant and IL-15 versus U/S. Expression of CD103 with either CD49a (top panels) or CD69 (bottom panels) are shown. Patient-matched ascites CD56+ NK cells are also shown (right panels). Coexpression analysis was done across two ascites samples (i.e., ASC641, ASC888). (C) Dot plots showing expression of CD103, CD49a, or CD69 in U/S PB NK cells compared with paired PB NK cells stimulated with 50% ascites supernatant and IL-15 from multiple patients (n = 4–5; i.e., ASC591, ASC635, ASC640, ASC641, ASC888). Paired Wilcoxon test or Student t test was performed. (D) Ascites-stimulated PB CD56+ ILCs treated with or without anti-TGF-β neutralizing Abs for 7 d. Supernatant from six ascites samples was used (i.e., ASC736, ASC743, ASC748, ASC769, ASC775, ASC777). The percentage of cells positive for CD103 is shown. Two-way ANOVA (F[1,12] = 76.80) and multiple comparisons with Dunn-Šidák correction was performed. Data included two or three technical replicates. Data with supernatant ASC777 was repeated in a separate experiment. (E) Summary plot and pairwise comparisons of (D) with lines representing matched ascites supernatants. Variance displayed as min/max, and unpaired Student t test was used for statistical significance. TME, tumor microenvironment. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Article Snippet: In some experiments, cells were treated with or without anti-TGF-β neutralizing Ab (10 μg/ml, catalog no. BP0057, 1D11.16.8, Bio X Cell).

Techniques: Derivative Assay, Expressing, Cell Culture

Journal: The Journal of Immunology Author Choice

Article Title: Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization

doi: 10.4049/jimmunol.2300834

Figure Lengend Snippet: TGF-β1/IL-15–induced NK-like cells express markers similar to intratumoral CD103+CD56+ ILCs. (A) Time kinetics of surface markers and transcription factors expressed by PB CD56+Lin− ILCs that were stimulated with IL-15 and TGF-β1 for 1, 3, and 7 d. Circle plots show proportion of PB CD56+Lin− NK cells coexpressing select markers throughout time averaged across two technical replicates. Numbers in square brackets represent the number of markers coexpressed. Findings are representative of two separate experiments across three different PB donors. (B) Circle plots comparing coexpression of markers by CD56+ NK/ILCs from PB from healthy donor, ascites from EOC patients, or EOC tumors. Each circle plot represents an individual sample. Representative contour plots of CD56+Lin− NK/ILCs from healthy PB donors, tumor, or PB NK cells cultured with IL-15 and TGF-β1 for 7 d (C and D). Expression of CD103 in combination with CD49a or CD69 (C), or CD101 or GITR (D), are shown. Findings for IL-15/TGF-β1 stimulation are representative of three separate experiments.

Article Snippet: In some experiments, cells were treated with or without anti-TGF-β neutralizing Ab (10 μg/ml, catalog no. BP0057, 1D11.16.8, Bio X Cell).

Techniques: Cell Culture, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization

doi: 10.4049/jimmunol.2300834

Figure Lengend Snippet: TGF-β1/IL-15–induced CD103+ NK-like cells suppressed autologous CD4+ T cells in vitro. To determine whether TGF-β1/IL-15–induced CD103+ NK-like cells are functionally inhibitory, TGF-β1/IL-15–induced CD103+ NK-like cells were cocultured with autologous CD4+ T cells. (A) Schematic diagram of suppression assay using TGF-β1/IL-15–induced CD103+ NK-like cells. PB CD56+ NK cells were stimulated with IL-15 and TGF-β1 for 7 d. TGF-β1/IL-15–induced CD103+CD56+Lin− (CD3−CD14−CD19−) NK-like cells were FACS sorted and cocultured in 1:1 ratio with autologous CD4+ T cells stimulated with Dynabeads (coated with αCD3/αCD28) for 4 d. These assays have two or three technical replicates, and data are representative of three independent experiments. (B) Absolute numbers of live CD4+ T cells that remained after coculture with either TGF-β1/IL-15–induced CD103+ NK-like cells, U/S NK cells, or T cell only in vitro with different cytokine conditions (i.e., IL-2, IL-15, or no cytokines). Two-way ANOVA with Dunn-Šidák multiple comparisons test was performed for CD4+ T cells (main effect of cells cocultured; F[2,17] = 85.79, p < 0.0001). (C) Percentage suppression was calculated using absolute CD4+ T cell numbers after exposure to either U/S NK cells or TGF-β1/IL-15–induced CD103+ NK-like cells, normalized to T cell only conditions. Suppression assay shown has a decreasing ratio of suppressors to responder cells from 1:1 to 1:8. (D) Proliferation of live CD4+ T cells with either TGF-β1/IL-15–induced CD103+ NK-like cells, U/S NK cells, or T cell only using cell trace proliferation dye. Expression of activation marker CD25 is shown from CD4+ T cells at a 1:1 ratio (E) or a downward titration (F). (G) Percentage of dead CD4+ T cells measured using fixable viability dye. (H) Supernatant from cocultures at day 4 were collected for CBA assay and concentration of perforin is shown. Data are representative of two separate experiments. Two-way ANOVA with Dunn-Šidák multiple comparisons test was performed for (C) (main effect of cells cocultured, F[1,11] = 107, p < 0.0001), (F) (main effect of cells cocultured, F[1,8] = 128.4, p < 0.0001), and (H) (main effector of cells cocultured, F[1,8] = 184.5, p < 0.0001). One-way ANOVA with Dunnett’s multiple comparison test was performed for (E) (F[2,6] = 8.403, p = 0.0182) and (G) (F[2,5] = 120.7, p < 0.0001). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; variance displayed as SEM. CBA, cytometric bead array.

Article Snippet: In some experiments, cells were treated with or without anti-TGF-β neutralizing Ab (10 μg/ml, catalog no. BP0057, 1D11.16.8, Bio X Cell).

Techniques: In Vitro, Suppression Assay, Expressing, Activation Assay, Marker, Titration, Concentration Assay, Comparison

Journal: iScience

Article Title: Hepatic iNKT cells facilitate colorectal cancer metastasis by inducing a fibrotic niche in the liver

doi: 10.1016/j.isci.2025.112364

Figure Lengend Snippet:

Article Snippet: Rat Monoclonal NKG2D (CX5) , BioXCell , BE0334; RRID: AB_2894754.

Techniques: Recombinant, Membrane, Infection, Transfection, Plasmid Preparation, Microscopy, In Vivo, SYBR Green Assay, Amplification, Multiplexing, Staining, Reverse Transcription, RNA Sequencing, Software, Injection, Control, Ointment, Imaging