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: 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: The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition

doi: 10.3389/fimmu.2021.714799

Figure Lengend Snippet: Expression of three NKG2D ligands is affected during HHV-6A infection. (A) Detection of surface expression of MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA class I on the T cell lines HSB-2 (upper panel) or J-Jhan (lower panel), either on uninfected cells (black histograms) or on HHV-6A infected cells at 72 hpi (red histograms). The grey shaded histogram is staining of an IgG isotype control on uninfected cells; the background of infected cells was virtually identical. Due to the lack of ULBP3 expression on HSB-2 cells, HSB-2-ULBP3 overexpression transfectants were stained as well. One representative experiment out of at least four is shown. Quantification of surface expression of MICB, ULBP1 and ULBP3 in uninfected cells (grey bars) and at 72 hpi (red bars) is shown on the right. Averages using median fluorescence intensity of at least 4 experiments were used, *p < 0.005 in student’s t-test. (B) Surface expression of MICB, ULBP1 and ULBP3 kinetics in J-Jhan cells presented as median fluorescence intensity over time. (C) NK cell degranulation towards uninfected (grey bars) or HHV-6A infected or J-Jhan cells at 72 hpi (red bars) in presence of an isotype IgG antibody (left) or a NKG2D blocking antibody (right). Data is merged from four independent experiments and standard error of means is shown, *p < 0.05 in student’s t test; ns = not significant. (D) qRT-PCR analysis of mRNA levels of MICA, MICB, ULBP1, ULBP2 and ULBP3 in J-Jhan cells (black bars) and HSB-2 cells (grey bars). Shown are relative values of HHV-6A infected cells at 72 hpi compared to uninfected cells (=1, grey line) normalized to HPRT. GAPDH was used as additional internal control and yielded similar results. ULBP3 mRNA levels in HSB-2 cells were insufficient to yield reliable results in qRT-PCR, n.d. = not detected. Averaged data out of at least three experiments including standard error of the mean is shown. (E) Western Blot analysis of MICB levels in HSB-2 cells as well as ULBP1 or ULBP3 in J-Jhan. Cells were infected for 72 hours prior to sample preparation. One representative experiment out of three is shown. Quantification was statistically analyzed using a single sample t-test, *p (MICB) = 0.01, *p (ULBP1) = 0.04, *p (ULBP3) = 0.03. (F) ELISA for soluble NKG2D ligands in supernatants of uninfected or HHV-6A infected J-Jhan cells using an NKG2D-Fc fusion protein. Merged data from three independent infections is shown. BJAB cell supernatants served as positive control. Differences between uninfected and infected cells were not significant (ns) according to the student’s t-test.

Article Snippet: Then, following antibodies, all diluted in 5% BSA in PBS, were used to detect the proteins: rabbit-anti-vinculin (1:1000, ab129003, Abcam), mouse-anti-human MICB (1:375, MAB1599, R&D systems), rabbit-anti-human ULBP1 (1:500, H-46, Santa Cruz Biotechnology), mouse-anti-human ULBP3 (1:500, MAB15171, R&D systems), anti-FLAG (DYKDDDDK) (1:1000, L5, Biolegend).

Techniques: Expressing, Infection, Staining, Control, Over Expression, Fluorescence, Blocking Assay, Quantitative RT-PCR, Western Blot, Sample Prep, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Frontiers in Immunology

Article Title: The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition

doi: 10.3389/fimmu.2021.714799

Figure Lengend Snippet: Viral U20 and U21 proteins suppress expression of ULBP1 and ULBP3, respectively. (A) Surface staining of MICB, ULBP1 and ULBP3 on J-Jhan transfectants that overexpress the viral U20 (red) or U21 (blue). The black histogram depicts staining for these ligands on an empty vector control transfectant, the grey shaded histogram depicts an IgG isotype staining on these control cells. Staining of this isotype on the transfectants expressing viral genes was virtually identical. (B) Quantification of ULBP1 and ULBP3 levels on J-Jhan, statistical analysis was performed using ANOVA with post hoc Tukey Honestly Significant Difference (HSD) test, *p < 0.05. (C) Surface staining of MICB, ULBP1 and ULBP3 on 293T transfectants that overexpress the viral U20 (red) or U21 (blue). The black histogram depicts staining for these ligands on an empty vector control transfectant, the grey shaded histogram depicts an IgG isotype staining on these control cells. Staining of this isotype on the transfectants expressing viral genes was virtually identical. (D) Quantification of ULBP1 and ULBP3 levels on 293T, statistical analysis was performed using ANOVA with post hoc Tukey HSD test, *p < 0.05. (E) Comparison of U20 and U21 RNA levels between cells after HHV-6A infection of J-Jhan cells, and J-Jhan cells overexpressing U20 or U21, respectively, by qRT-PCR. GAPDH was used for normalization. (F) NK cell degranulation towards transfected U20-293T (red bar), U21-293T (blue bar) and controls (grey) measured in flow cytometry by CD107a. Data is representative from two independent donors. U20 and U21 were found significantly changed by One-way ANOVA with post-hoc Tukey Honestly Significant Difference Test, no significant differences were observed when NK cells blocked with an antibody blocking NKG2D was used (ns). (G) J-Jhan cells transfected with an empty control vector (EV), a sgRNA expressing an irrelevant guide RNA (ctrl sgRNA) or each two sgRNAs targeting U20 (left) or U21 (right) stained for ULBP1 or ULBP3 respectively after three days of infection with HHV-6A. Restoration was significant (*p < 0.05 in student’s t test) both compared to EV and the control guide RNA. Merged data of at least 3 experiments (2 for ctrl guide RNA in ULBP1 staining) with averages and standard error of the means is shown.

Article Snippet: Then, following antibodies, all diluted in 5% BSA in PBS, were used to detect the proteins: rabbit-anti-vinculin (1:1000, ab129003, Abcam), mouse-anti-human MICB (1:375, MAB1599, R&D systems), rabbit-anti-human ULBP1 (1:500, H-46, Santa Cruz Biotechnology), mouse-anti-human ULBP3 (1:500, MAB15171, R&D systems), anti-FLAG (DYKDDDDK) (1:1000, L5, Biolegend).

Techniques: Expressing, Staining, Plasmid Preparation, Control, Transfection, Comparison, Infection, Quantitative RT-PCR, Flow Cytometry, Blocking Assay

Journal: Frontiers in Immunology

Article Title: The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition

doi: 10.3389/fimmu.2021.714799

Figure Lengend Snippet: Primer pairs for human and viral genes used in qPCR.

Article Snippet: Then, following antibodies, all diluted in 5% BSA in PBS, were used to detect the proteins: rabbit-anti-vinculin (1:1000, ab129003, Abcam), mouse-anti-human MICB (1:375, MAB1599, R&D systems), rabbit-anti-human ULBP1 (1:500, H-46, Santa Cruz Biotechnology), mouse-anti-human ULBP3 (1:500, MAB15171, R&D systems), anti-FLAG (DYKDDDDK) (1:1000, L5, Biolegend).

Techniques: Sequencing

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