Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Rational design and construction of the Anti-MICB-CAR. ( A ) Schematic of the Anti-MICB chimeric antigen receptor (Anti-MICB-CAR). ( B ) The plasmid structure of the Anti-MICB chimeric antigen receptor lentiviral vector. ( C ) The expression of Anti-MICB-CAR in NK cells was analyzed via flow cytometry. ( D ) The secretion of IL-15 by Anti-MICB-CAR-NK cells was detected by ELISA. NC: supernatant of 1 × 10 6 NK cells, Anti-MICB–CAR–NK: supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells. Supernatants of Anti-MICB-CAR-NK cells were significantly elevated up to 48.88 pg/mL compared to the control NK cell supernatants. p = 0.0024. ( E ) The supernatant of 1 × 10 6 Anti-MICB-CAR-NK cells was filtered and incubated with PANC-1 for 1 h. MICB Protein hFc and IgG1 Fc FITC antibody staining were performed and free Anti-MICB-scFv was detected by flow cytometry. Statistical analysis was performed using a two-tailed Student’s t -test; ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Incubation, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells demonstrate potent in vitro activity against tumors with high expression of MICB. ( A ) Western blot detection of MICB expression in PANC-1, A549, HepG2, BxPC-3, and AsPC-1 human tumor cell lines. ( B ) The qPCR results showed in the MICB gene expression compared to the five tumor cell lines. ( C ) The Effect-to-Target Ratio (E/T Ratio) of Anti-MICB-CAR-NK cells on tumor cell PANC-1. ( D ) The mortality rate of five types of tumor cells treated with Anti-MICB-CAR-NK cells after 24 h with CCK-8 Assay, PANC-1(71.37%), A549(31.78%), HepG2(53.83%), BxPC-3(62.26%), and AsPC-1(39.86%), E/T Ratio 1:1. ( E ) Anti-MICB-CAR-NK treatment of AsPC-1 and PANC–1 tumor cells with differential MICB expression for 24 h by flow cytometry, E/T Ratio 1:1. The results showed that the viability of AsPC-1 was 68.9% and the viability of PANC-1 was 54.6%. ( F – H ) PANC-1, HepG2, and A549 tumor cells treated with NK cells, Anti-MICB-CAR-NK cells, and Anti-MICB-CAR-NK supernatant + NK cells for 24 h, E/T Ratio 1:1; CCK8 detects mortality rate. Anti-MICB-CAR-NK cells significantly enhanced the anti-tumor ability compared to NK cells. PANC-1: 68.37%, p = 0.0005. HepG2: 57.3%, p = 0.0027. A549: 33.18%, p = 0.0342. Supernatant of Anti-MICB-CAR-NK cells co-treated tumor cells with NK cells also promoted tumor cell killing compared to NK. PANC-1: 57.66%, p = 0.0006. HepG2: 48.21%, p = 0.0037. A549: 31.55%, p = 0.0446. ( I ) NC-NK cells, non-transduced NK(NT-NK), NT-NK supernatant + NK cells, Anti-MICB-CAR-NK supernatant + NK cells, Anti-MICB-CAR-NK cells treated with PANC-1 tumor cells for 24 h, E/T Ratio 1:1. The viability of each group was detected by flow cytometry and the results showed that NK cells: 89.4%, non-transduced NK(NT-NK): 86.6%, NT-NK supernatant + NK cells: 88.7%, Anti-MICB-CAR-NK supernatant + NK cells: 62.6%, and Anti-MICB-CAR-NK cells: 55.5%. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: In Vitro, Activity Assay, Expressing, Western Blot, Gene Expression, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: The anti-tumor mechanism induced by Anti-MICB-CAR-NK cells. ( A – D ) The release of perforin, granzyme B, TNF-α, and IFN-γ from PANC-1, HepG2, and A549 cells. NK cells and Anti-MICB-CAR-NK cells were treated with tumor cells for 24 h, with an E/T ratio of 1:1. Control represents complete medium cultivation. The supernatant was collected and detected by ELISA. Compared to the NK group, the Anti-MICB-CAR-NK group exhibited significantly higher levels of TNF-α expression (PANC-1, p = 0.0072; AsPC-1, p = 0.035; HepG2, p = 0.0076; A549, p = 0.0066) and IFN-γ expression (PANC-1, p = 0.0072; AsPC-1, p = 0.0162; HepG2, p = 0.002; A549, p = 0.0006) following treatment with PANC-1, AsPC-1, HepG2, and A549 tumor cells. Similarly, Granzyme B expression levels were significantly elevated in the Anti-MICB-CAR-NK group (PANC-1, p = 0.0007; HepG2, p = 0.0023; A549, p = 0.0004), as were Perforin expression levels (PANC-1, p = 0.0021; HepG2, p = 0.042; A549, p = 0.0022) after treatment with PANC-1, HepG2, and A549 tumor cells. Additionally, the Anti-MICB-CAR-NK group demonstrated significantly increased IL-15 expression following treatment with PANC-1 and A549 tumor cells (PANC-1, p = 0.0012; A549, p = 0.0025) compared to the NK group. ( E , F ) Elisa and flow cytometry were used to detect the levels of IL-15 and Anti-MICB-scFv in the tumor cells’ supernatants treated with Anti-MICB-CAR-NK cells for 24 h. ( G ) Study on MICB expression in PANC-1 treated with Anti-MICB-CAR-NK cells for 24 h using Western blot technique. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel MICB-Targeting CAR-NK Cells for the Treatment of Pancreatic Cancer

doi: 10.3390/ijms27010500

Figure Lengend Snippet: Anti-MICB-CAR-NK cells exhibit tumor regression in the PANC-1 Xenograft Model. ( A ) Flow chart of Anti-MICB-CAR-NK cells treatment for PANC-1 Transplanted Tumors. On the 0th day, PANC-1 cells were inoculated and on the 13th day, PBS, NK cells, and Anti-MICB-CAR-NK cells were injected into mice via tail vein at a dose of 5 × 10 6 cells per mouse. Record tumor growth observed every other day. ( B ) Growth curve of mouse transplanted tumor. ( C , D ) Resected tumors from each group were imaged and Weighed at the end of the experiment. NC: 834.31 ± 197.26 (mg), NK: 606.69 ± 141.37 (mg), Anti-MICB-CAR-NK: 447.32 ± 136.13 (mg). ( E ) Anti-tumor rate of Anti-MICB-CAR-NK cells against PANC-1 transplanted tumors. The tumor inhibition rate of NK compared to NC was 27.28%, p = 0.07. The tumor inhibition rate of Anti-MICB-CAR-NK compared to NC was 46.38%, p = 0.007. ( F ) Mouse serum was collected at the end of the experiment and the amounts of IL-15, IFN-γ, and TNF-α were measured by ELISA. The IL-15, IFN—γ, and TNF—α in the Anti-MICB-CAR-NK group were significantly increased compared to the NK group, with a p -value of 0.0013 for IL-15, 0.013 for IFN—γ, and 0.003 for TNF—α. ( G , H ) Mouse tumor tissues were analyzed by immunohistochemistry for MICB expression and NK cell infiltration within the tumor tissue. Statistical analysis was performed using a two-tailed Student’s t -test; * p < 0.05, ** p < 0.01.

Article Snippet: 10 6 NK cells centrifugation, resuspend in 1 mL PBS, 20 μL of serum for 45 min, 20 μL of anti-human CD56 Antibody APC (Biolegend, Cat: 362503, San Diego, CA, USA), and 20 μL of FITC anti-human CD3 antibody (Biolegend, Cat: 300305, San Diego, CA, USA), and incubate in the dark for 1.5 h. Anti-MICB-CAR expression on gene-modified NK cells using flow cytometry analyzed with an Anti-MICB-CAR Detection Reagent, containing a recombinantly expressed fusion protein consisting of the human MICB extracellular domains and a specifically mutated human lgG1 Fc region (Sino Biological, Recombinant Human MICB Protein-His & hFc Tag, Cat:10759-H03H, Beijing, China) and secondary addition of anti-Human IgG1 Fc-FITC-antibody (Invitrogen, anti-Human IgG1 Fc Secondary Antibody, Cat: A-10631, Carlsbad, CA, USA).

Techniques: Injection, Inhibition, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Expression of MICA, MICB and NKG2D in human leukemic myelomonocytic and cervical cancer cells

doi: 10.1186/1756-9966-30-37

Figure Lengend Snippet: Cervical cancer cell lines express MICA, MICB and NKG2D . CALO and INBL cells ( 1 × 10 7 ) were lysed proteins immunoprecipitated and equal amounts of protein from total lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The blots were developed with either polyclonal anti-MIC antibodies (A) or monoclonal anti-NKG2D antibodies (B) and an appropriate secondary antibody conjugated to HRP for chemiluminescence detection. Flow cytometric analysis of NKG2D expression in cervical carcinoma cell lines after 72 h induction with 10 ng MICB (C). We used only MICB to induce the expression of NKG2D because we previously obtained that MICB was a better inducer of myelomonocytic cell proliferation than MICA. Graphs show NKG2D levels (solid line) and isotype controls (dotted line).

Article Snippet: Polyclonal antibody against MICA/MICB and murine monoclonal anti-MICA, anti-MICB and anti-NKG2D antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation, SDS Page, 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: 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