Journal: Med (New York, N.Y.)

Article Title: A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

doi: 10.1016/j.medj.2023.04.004

Figure Lengend Snippet: Figure 1. 3MICA/B CAR targets the conserved alpha-3 domains of MICA/B stress ligands and is resistant to binding competition by shed MICA/B A novel MICA/B-targeting synthetic receptor capable of high-affinity pan-allelic binding, surface MICA/B density augmentation, and resistance to inhibition via shed MICA and MICB peptides. (A) Domain-specific amino acid sequence similarity was scored across the 21 most prevalent MICA and MICB alleles using the BLOSUM method highlighting greater sequence conservation within the membrane-proximal a3 domain relative to the membrane-distal a1-2 domains. (B) Pan-allelic reactivity of 3MICA/B mAb, an a1-2 MICA/B-reactive mAb (6D4), and NKG2D:Fc fusion protein was assessed across an extended panel of 28 prevalent MICA alleles. (C) MM1-R cells, pre-treated with trypsin to cleave baseline surface-expressed MICA/B ligands, were incubated with 3MICA/B mAb, or relevant isotype, and surface MICA/B expression kinetics was measured using a commercial mAb (6D4) over a 96-h time course. In parallel, MM1.R cells were incubated

Article Snippet: The concentration of shed MICA/B was measured using a human MICA ELISA (Ab59569, Abcam) or human MICB DuoSet ELISA kit (DY1599, R&D Systems), following the manufacture’s recommendation.

Techniques: Binding Assay, Inhibition, Sequencing, Membrane, Incubation, Expressing

Journal: Scientific Reports

Article Title: All-trans retinoic acid enhances cytotoxicity of CIK cells against human lung adenocarcinoma by upregulating MICA and IL-2 secretion

doi: 10.1038/s41598-017-16745-z

Figure Lengend Snippet: Effects of CIK and ATRA, alone or in combination, on the expression of MICA and MICB. Representative flow cytometric profiles of cell surface MICA ( A ) and MICB ( B ). The black profile indicates a control profile of A549 or NCI-H520 cells incubated with mouse IgG 2b . ( C ) Effects of CIK and ATRA, alone or in combination, on the secretion of soluble MICA. Cells were treated for 48 h and the concentrations of soluble MICA in the medium were determined. The data presented are mean ± SD from at least three independent experiments.

Article Snippet: After treatment for 48 h, the cells in each group were harvested and incubated with either a phycoerythrin (PE)-labeled mouse anti-human MICA mAb (clone number 159227, R&D systems, USA) and a allophycocyanin (APC)-labeled mouse anti-human MICB mAb (clone number 236511, R&D systems, USA) on ice for 30 min. As isotype controls, cells were incubated with PE- or APC- labeled mouse IgG 2b antibodies.

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Tandem CAR T-cells targeting CD19 and NKG2DL can overcome CD19 antigen escape in B-ALL

doi: 10.3389/fimmu.2025.1557405

Figure Lengend Snippet: Cytokine secretion, cytolytic activity and proliferation of CD19/NKG2DL tandem CAR T-cells co-cultured with CD19+ and CD19- Nalm-6 cells. (A) Expression of CD19 and NKG2DL in Nalm-6 and Nalm-6 CD19 KO cells. (B) Expression of individual NKG2DL using antibodies directed against MICA, MICB, ULBP1, ULBP3 or ULBP2-5-6 (grey histograms) vs autofluorescence (white histograms). (C) Secretion of IFN-γ, TNF-α and IL-2 cytokines after a 24-hour co-culture at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells. (D) Cytolytic activity of CAR T-cells against Nalm-6 and Nalm-6 CD19 KO cells at 1:1 and 0.1:1 E:T ratio. Results are expressed as percentage of remaining cancer cells normalized to t0h timepoint. (E) Representative experiment showing CTV histograms of CAR T-cells after 4 days of co-culture without cancer cells or at 1:1 E:T ratio with Nalm-6 and Nalm-6 CD19 KO cells (4 individual experiments were performed on 4 different donors). Adjusted P values (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were determined by one-way ANOVA with Dunnet’s correction for multiple comparisons. Data presented as means (SD) of n=5. Each symbol denotes a different PBMC donor.

Article Snippet: NKG2DL were detected using antibodies directed against human MICA (FAB1300P, R&D systems), MICB (FAB1599P, R&D systems), ULBP1 (FAB1380P, R&D systems), ULBP3 (FAB1517P, R&D systems), ULBP2-5-6 (FAB1298P, R&D systems) or using a recombinant human NKG2D (rhNKG2D-Fc, 1299-NK-050, R&D systems) detected with an anti-human IgG-Fc antibody (12-4998-82, ThermoFisher).

Techniques: Activity Assay, Cell Culture, Expressing, Co-Culture Assay

Journal: OncoTargets and Therapy

Article Title: Potential therapeutic strategy for gastric cancer peritoneal metastasis by NKG2D ligands-specific T cells

doi: 10.2147/ott.s91122

Figure Lengend Snippet: Figure 2 ChNKG2D receptor-modified T cells released Th1 cytokines and CCL3 chemokine in response to NKG2D ligand-expressing human gastric cancer cell lines. Notes: (A) Expression of NKG2D ligands in gastric cancer cell lines. Two human gastric cancer cell lines were stained with specific antibodies recognizing MICA, MICB, ULBP1, ULBP2, or ULBP3, or matched isotype controls (filled histogram), and analyzed by flow cytometry. A murine cell line TC-1 was used as negative control. (B) Th1/ Th2 cytokine panel determination using cytometric bead array assay. ChNKG2D receptor-modified T cells were cultured with gastric cancer cell lines or TC-1 control cells for 20 hours. Supernatants were harvested and assayed to determine the concentration of six different cytokines. The beads were conjugated with antibodies against corresponding cytokines. Secondary antibody conjugated with the fluorescent dye PE was used as a detector. The dots from top to bottom represent IL-2, IL-4, CCL3, IL-10, TNF-α, and IFN-γ, respectively. Abbreviations: L, CD8α Leader; 2A, cleavable 2A-like peptide sequence; CBA, cytometric bead array; ChNKG2D, chimeric NKG2D; PE, phycoerythrin; IL, interleukin; CCL3, chemokine (C-C motif) ligand 3; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon-gamma.

Article Snippet: NKG2DL expression was analyzed with allophycocyanin (APC)-conjugated anti-human MICA (clone 159227), APC anti-human MICB (clone 236511), phycoerythrin (PE)conjugated anti-human ULBP1 (clone 170818), PE-antihuman ULBP2 (clone 165903), and APC-anti-human ULBP3 (clone 166510) (all R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Modification, Expressing, Staining, Flow Cytometry, Negative Control, Cell Culture, Control, Concentration Assay, Sequencing