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: 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: 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

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: TGF-β1 production of NK1.1-CD4+NKG2D+ cells upon various stimulations at different times. A. TGF-β1 of NK1.1-CD4+NKG2D+ cells detected by flow cytometry at 0.5-, 2-, 8-, 16-hour stimulation. B. Statistical analysis of TGF-β1 variations at different time points. C. TGF-β1 transcription of NK1.1-CD4+NKG2D+ cells detected by real-time PCR. D. Concentration of TGF-β1 in supernatants of NK1.1-CD4+NKG2D+ cells upon indicated stimulations measured by ELISA. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Activation of PI3K, MAPK, STAT3 and NF-κB signaling pathways. Western blot analysis of PI3K-p85, -p110, Akt, pAkt in NK1.1-CD4+NKG2D+ cells upon different stimulations at 2-hour (A) or 8-hour (C). Statistical analysis of expression variations of PI3K-p85, -p110, Akt, pAkt at 2-hour (B) or 8-hour (D). Analysis of JNK, Erk, p38 (E, F), NF-κB, STAT3 (G, H) and their phosphorylation in NK1.1-CD4+NKG2D+ cells upon stimulations for 8 hours. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Expressing, Phospho-proteomics

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of PI3K/JNK/AP-1 on TGF-β1 expression. NK1.1-CD4+NKG2D+ cells were treated with the PI3K inhibitor (LY294002) under different stimulations for 8 hours (A). (B) Statistical analysis of effects of LY294002 on TGF-β1 expression. (C, D) Effects on JNK/pJNK expression by the PI3K inhibitor (LY294002). Effects of JNK inhibitor (E, F), Erk or p38 inhibitor (G), and AP-1 inhibitor (H, I) on TGF-β1 expression of NK1.1-CD4+NKG2D+ cells. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of NF-κB on TGF-β1 expression. Effects of Bortezomib (NF-κB inhibitor) on TGF-β1 expression (A, B). Effects of LY294002 on NF-κB p65 activation in NK1.1-CD4+NKG2D+ cells for 8 hours (C, D). Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Activation Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Inhibition of STAT3 on TGF-β1 expression. Effects of STAT3 inhibitors on TGF-β1 expression in NK1.1-CD4+NKG2D+ cells for 8 hours (A). (B, C) Effects of LY294002 on STAT3-pY705 phosphorylation. (D, E) Effects of Bortezomib on STAT3-pY705 phosphorylation. (F) The predicted binding sites of the TGF-β promoter by STAT3. (G) STAT3 engagement measured by a ChIP assay. Each experiment was repeated at least thrice. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques: Inhibition, Expressing, Phospho-proteomics, Binding Assay

Journal: American Journal of Cancer Research

Article Title: TGF-β1 expression in regulatory NK1.1 - CD4 + NKG2D + T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways

doi:

Figure Lengend Snippet: Diagram of the TGF-β1 transcription in NK1.1-CD4+NKG2D+ cells regulated by AP-1, NF-κB, and STAT3.

Article Snippet: Splenic NK1.1 - CD4 + T cells were enriched from splenic single cell suspension by mouse CD4 + T lymphocyte enrichment set (BD Biosciences) following the manufacturer’s instructions, and then NKG2D + T cells were isolated by indirectly labeled the cells with PE-conjugated anti-mouse NKG2D antibody (CX5) and anti-PE MicroBeads (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany).

Techniques:

Journal: Cancers

Article Title: Multiparametric Characterization of the DSL-6A/C1 Pancreatic Cancer Model in Rats

doi: 10.3390/cancers16081535

Figure Lengend Snippet: List of antibodies used for the immunological analysis of the DSL-6A/C1 tumors.

Article Snippet: CD314 (NKG2D) , APC , REA471 , Human cell line , Miltenyi , 130-106-992.

Techniques:

Journal: Cell Reports Medicine

Article Title: High-dimensional profiling of pediatric immune responses to solid organ transplantation

doi: 10.1016/j.xcrm.2023.101147

Figure Lengend Snippet: Antibodies used for CyTOF staining

Article Snippet: anti-human NKG2D-166Er (ON72) , Standard Biotools , Cat. #3166016B; RRID: AB_2892110.

Techniques: