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: 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: 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: Archivum Immunologiae et Therapiae Experimentalis

Article Title: Detection and Significance of Cytotoxic Cell Subsets in Biopsies of HCV-Infected Human Livers

doi: 10.1007/s00005-013-0258-6

Figure Lengend Snippet: Primary antibodies used

Article Snippet: NKG2D/CD314 , Mouse , Monoclonal , 1D11 , Novus Biologicals , NB100-65956.

Techniques:

Journal: bioRxiv

Article Title: Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer

doi: 10.1101/2025.08.19.671055

Figure Lengend Snippet: Following the pipeline described in scheme 1, γδ and αβ T cell subsets from Patient Tumor-infiltrating Lymphocytes (PAT pTILs), Patient Lamina propria Lymphocytes (PAT LPLs), and both Patient (PAT) and healthy donor (HD) Peripheral Blood Lymphocytes (PBLs). Different T cell populations were expanded in vitro through the Rapid Expansion Protocol (REP), and subsequently isolated using FACS bulk sorting. Afterwards, isolated T cell populations from the different comparments were expanded for various cycles of REP to produce sufficient cell numbers for co-receptor expression analysis and functional testing. Co-receptor expression patterns were determined by flow cytometry for (A) the Vδ1 - /2 - HD PBLsγδ and PAT PBLsγδ/LPLsγδ/pTILsγδ subset, (B) Vδ1 + HD PBLsγδ and PAT PBLsγδ/LPLsγδ/pTILsγδ subset and (C) the Vδ2 + HD PBLsγδ and PAT PBLsγδ/LPLsγδ/pTILsγδ subset. (D) IFNγ release by either Vδ1 - /Vδ2 - , Vδ1 + , or Vδ2 + , from the different sources, HD PBLsγδ and PAT PBLsγδ/LPLsγδ/pTILsγδ, in response to co-culture with HT-29 and Caco-2 cell lines. Tumor reactivity was assessed after a 24-hour co-culture of T cells with CRC cell lines at a 1:3 ratio, normalized to T cell only conditions, using an IFNγ ELISpot assay. (E) IFNγ release by either pTILsαβ or Vδ1+ pTILsγδ bulks in response to co-culture with autologous organoids. Tumor reactivity was assessed after a 24-hour co-culture of T cells with CRC organoids at a 1:3 ratio, T cell only condition has been shown as a negative control, using an IFNγ ELISpot assay. (F) Linear regression between the NKG2D expression of the expanded Vδ1+ pTILsγδ and IFNγ release against HT-29. (G) IFNγ release of the expanded Vδ1+ pTILsγδ as performed in (E) but with or without a one hour pre-incubation with an NKG2D blocking antibody (10ug/mL). (H) Luciferase based killing by Vδ1+ LPLsγδ and pTILsγδ with or without a one hour pre-incubation with either an NKG2D blocking antibody (10ug/mL) targeting the CRC cell line HT-29 at multiple E:T ratios, normalized to the target only condition. Mean values ± SEM are represented with error bars. Statistical significance was determined by either a one-way ANOVA (A-C) , two-way ANOVA (D) , or one-way t-test (G,H).

Article Snippet: In case of blocking experiments, T-cells were pre-incubated for 2h 37C with 10μg/mL, the neutralizing antibody NKG2D (MAB139, R&D systems) or IgG isotype as a negative control (110-HG-100, R&D system).

Techniques: In Vitro, Isolation, Expressing, Functional Assay, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunospot, Negative Control, Incubation, Blocking Assay, Luciferase

Journal: bioRxiv

Article Title: Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer

doi: 10.1101/2025.08.19.671055

Figure Lengend Snippet: MFI of NKG2D surface expression on tumor reactive Vδ1+ pTILs γδ derived from 5 patients measured by FACS (Fortessa BD) on the same day of functional tests.

Article Snippet: In case of blocking experiments, T-cells were pre-incubated for 2h 37C with 10μg/mL, the neutralizing antibody NKG2D (MAB139, R&D systems) or IgG isotype as a negative control (110-HG-100, R&D system).

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: bioRxiv

Article Title: Functional γδT-omics pipeline reveals compartmentalization of Vδ1⁺ T cell migration, tumor-reactivity, and clonality in human colorectal cancer

doi: 10.1101/2025.08.19.671055

Figure Lengend Snippet: Following the experimental pipeline described in scheme 1, single γδ T cell subsets from Patient Tumor-infiltrating Lymphocytes (PAT pTILsγδ), Patient Lamina propria Lymphocytes (PAT LPLsγδ), and both Patient (PAT) and healthy donor (HD) Peripheral Blood Lymphocytes (PBLsγδ) were single cell sorted, and expanded for various cycles of REP to produce sufficient cell numbers for co-receptor expression analysis and functional testing. (A) Frequency of tumor-reactive Vδ1-/ Vδ2 or (B) Vδ1+ T cell clones derived from the different biological compartment, PBLsγδ/LPLsγδ/pTILsγδ. Tumor reactivity was determined by the production of 30 IFNγ spots after a 24-hour co-culture of T cells with one of the CRC cell lines at a 1:3 ratio, normalized to T cell only conditions. (C) IFNγ release by either Vδ1+ or Vδ1-/Vδ2-expanded γδT cell bulks derived from the different sources, in response to co-culture with HT-29 and Caco-2 cell lines. Tumor reactivity was assessed after a 24-hour co-culture of T cells with CRC cell lines at a 1:3 ratio, normalized to T cell only conditions, using an IFNγ ELISpot assay. Each dot represents an individual clone. (D) Linear regression between the NKG2D expression of the expanded Vδ1+ pTILsγδ clones and IFN-γ release against HT-29. Individual data points indicate values from individual CRC patients of HDs ( A,B) or individual T cell clones (C,D) . Not all patients had paired samples. Mean values ± SEM are represented with error bars. Statistical significance was determined by ordinary one-way ANOVA for the different subsets.

Article Snippet: In case of blocking experiments, T-cells were pre-incubated for 2h 37C with 10μg/mL, the neutralizing antibody NKG2D (MAB139, R&D systems) or IgG isotype as a negative control (110-HG-100, R&D system).

Techniques: Expressing, Functional Assay, Clone Assay, Derivative Assay, Co-Culture Assay, Enzyme-linked Immunospot

Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: Surface expression of human NKG2D ligands (ULBP-2/5/6, MIC A/B) and human ErbB2 (HER2/neu) on tumor cell lines

Article Snippet: Blocking of NKG2D was achieved by adding 30 μg of human NKG2D (CD314) antibody (R&D Systems, Minneapolis, MN, USA) per 4 x 10 6 CIK cells.

Techniques: Expressing

Journal: Oncotarget

Article Title: Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children

doi: 10.18632/oncotarget.19821

Figure Lengend Snippet: As a proof of concept, the NKG2D- and ErbB2-mediated cytotoxic capacity of WT and genetically modified (ErbB2-CAR and mock-vector) CIK cells was analyzed by europium release assay against (A) ErbB2-negative, NKG2D ligand-positive acute myeloid leukemia target cell line THP-1, (B) ErbB2-overexpressing, NKG2D ligand-positive breast carcinoma cell line MDA-MB-453, and (C) ErbB2-negative, NKG2D ligand-negative allogeneic PBMCs. The NKG2D-restricted cytotoxicity of CIK cells was not impaired by the genetic modification, shown by the effective cytolysis of ErbB2-negative, NKG2D ligand-positive THP-1 cells by WT, mock- vector, and ErbB2-CAR CIK cells. As expected, the cytotoxic capacity of ErbB2-CAR CIK cells against ErbB2-overexpressing target cells was significantly increased compared with mock-vector and WT CIK cells, and was low against non-malignant ErbB2-negtive, NKG2D ligand-negative target cells. (D) Stably transfected mouse renal carcinoma (Renca) cells Renca-lacZ and Renca-lacZ/erbB-2 were used for evaluation of specific ErbB2-targeting by the killer cells. WT CIK cells did not lyse Renca-lacZ and Renca-lacZ/erbB-2 cells. ErbB2-CAR-CIK cells also showed no cytotoxic capacity against Renca-lacZ cells, but displayed highly significant increased cytotoxicity against Renca-lacZ/erbB-2. (E) NKG2D mediated cytotoxicity on the cytotoxic capacity of ErbB2-CAR CIK cells against ErbB2 expressing cell line TE671 was evaluated by europium release assay after blocking of the NKG2D receptor on ErbB2-CAR CIK cells. Blocking significantly reduced the efficacy of CAR-modified CIK cells against the tumor target, suggesting that the combination of NKG2D-mediated and ErbB2-specific killing in CAR-CIK cells may reduce the impact of tumor escape mechanisms and increase killing of tumors.

Article Snippet: Blocking of NKG2D was achieved by adding 30 μg of human NKG2D (CD314) antibody (R&D Systems, Minneapolis, MN, USA) per 4 x 10 6 CIK cells.

Techniques: Genetically Modified, Plasmid Preparation, Release Assay, Modification, Stable Transfection, Transfection, Expressing, Blocking Assay