Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) PD-1 binding capacity of IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC was measured using a functional ELISA, where mean OD was measured at varying concentrations of IgG1-FC, rhPD-L1-FC or sec-PD-L1-long-FC protein. Kd values are displayed. The corresponding Scatchard graph displaying the differences in slope (−1/Kd) of rhPD-L1-FC (medium gray) or sec-PD-L1-long-FC (light gray) is shown as an inset. Data is from two separate experiments. b) The ability of PD-L1 and PD-1 neutralizing antibodies (10ug/ml) to block PD-1 binding to rhPD-L1-FC and sec-PD-L1-long-FC was tested with a functional ELISA. An IgG1 isotype control antibody was used as a negative control. Normalized mean OD values compared to incubation with the appropriate IgG1 isotype control conditions are plotted. Data is from two separate experiments. A two-way ANOVA was used. The mean and SEM are plotted. c, d) Primary human T cell blasts were incubated with IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC at either 20ug/ml or 40ug/ml in the presence of anti-CD3 for 24 hours, and media was harvested to quantify c) IL-2 and d) IFNg with ELISA. Samples were run in duplicate. N=5. Normalized protein levels compared to the appropriate IgG1-Fc control are plotted. A one-way ANOVA was used. The mean and SEM are plotted. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: For PD-L1 surface staining of cancer cell lines, cells were stained as described with the following antibodies: anti-human CD274-APC (1:100, clone 29E.2A3, mouse IgG2b, κ, Biolegend, #329707) or isotype control (1:100, clone MPC-11, mouse mouse IgG2b, κ, Biolegend, #400319.

Techniques: Binding Assay, Negative Control, Positive Control, Functional Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation

Journal: Pharmacological research

Article Title: TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats.

doi: 10.1016/j.phrs.2021.105877

Figure Lengend Snippet: Fig. 1. Progression of MAP elevation in SHRs is dependent upon AT1R and TLR4. Final indirect tail-cuff mean arterial pressure (MAP; mmHg) measure ments of Losartan cohort (n = 6/group) (A) and TAK-242 cohort (n = 11 WKY, 13 SHR, 17 SHR-TAK) (B) at conclusion of respective treatment periods; final direct MAP (mmHg) of TAK-242 sub-cohort (n = 5 WKY, 6 SHR, 10 SHR-TAK; C). Data evaluated by one-way ANOVA with Tukey post-hoc analysis; data shown as mean±SEM; ****p < 0.0001 vs. WKY; +++p < 0.001 vs. SHR; ++++p < 0.0001 vs. SHR.

Article Snippet: Sections were incubated in PBST (0.01 M PBS, 0.1% Triton, 0.04% NaN3) with 5% normal donkey serum and primary antibodies against TLR4 (mouse monoclonal IgG2bκ, 1:250, Novus Biologicals, USA, NB100–56567, lot CJU03–11 and IMG-5031A), ionized calcium-binding adaptor molecule 1 (IBA1; microglial marker [rabbit polyclonal, 1:1000, Wako Chemicals, USA, 019–19742, lot WDK2121 and WDF6884; goat polyclonal, 1:500, Abcam, UK, ab5076, lot GR3195324–1; or rabbit polyclonal, 1:2000, EnCor Biotechnology Inc., USA, RPCA-IBA1, lot 040119]), TNF-α (mouse monoclonal IgG1, 1:100, Santa Cruz Biotechnology, USA, sc-52746, lot C0119), and/or IL-6 (mouse monoclonal IgG2a, 1:100, Santa Cruz Biotechnology, USA, sc32296, lot I2818).

Techniques:

Journal: Pharmacological research

Article Title: TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats.

doi: 10.1016/j.phrs.2021.105877

Figure Lengend Snippet: Fig. 2. TLR4 protein expression within CNS cardioregulatory nuclei. Example confocal maximum projection images of PVN vaso pressin (VP; red) and TLR4 (white) from WKY, SHR, SHR-Los, and SHR-TAK (n = 6/group) (A). Percent change in TLR4 staining (% area) compared to WKY in the PVN (B), RVLM (C), and NTS (D). Data evaluated by one-way ANOVA with Tukey post-hoc analysis; shown as mean±SEM; * ** * p < 0.0001 vs. WKY; + ++ + p < 0.0001 vs. SHR; scale bars: 100 µm; 3 V: third ventricle.

Article Snippet: Sections were incubated in PBST (0.01 M PBS, 0.1% Triton, 0.04% NaN3) with 5% normal donkey serum and primary antibodies against TLR4 (mouse monoclonal IgG2bκ, 1:250, Novus Biologicals, USA, NB100–56567, lot CJU03–11 and IMG-5031A), ionized calcium-binding adaptor molecule 1 (IBA1; microglial marker [rabbit polyclonal, 1:1000, Wako Chemicals, USA, 019–19742, lot WDK2121 and WDF6884; goat polyclonal, 1:500, Abcam, UK, ab5076, lot GR3195324–1; or rabbit polyclonal, 1:2000, EnCor Biotechnology Inc., USA, RPCA-IBA1, lot 040119]), TNF-α (mouse monoclonal IgG1, 1:100, Santa Cruz Biotechnology, USA, sc-52746, lot C0119), and/or IL-6 (mouse monoclonal IgG2a, 1:100, Santa Cruz Biotechnology, USA, sc32296, lot I2818).

Techniques: Expressing, Staining

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Evaluation of the mSA2 CAR-T cell specificity and efficacy in additional primary GB cell lines. (a) mSA2 CAR-T cell activation after co-culture with S24 or T269 cells, measured by flow cytometry. Data gated on live single CD3 + /EGFP + cells. A one-way ANOVA test with a post-hoc Šídák test for multiple comparisons was used. b) Confocal microscopy images after O/N co-culture of S24 cells labeled with an anti-CD276 biotinylated antibody with mSA2 CAR-T cells. Indicative photos from N = 3 biological replicate co-cultures per construct. N = 1 T-Cell donor. (c) Quantification of Annexin-V signal from (b). d) mSA2 CAR-T cell activation after co-culture with S24, P3 and T269 tumor cells, after incubation with targeting, or a non-targeting biotinylated isotype control antibody, measured by flow cytometry. Data gated on live single CD3 + /EGFP + cells. For (c and d), a one-way ANOVA test with a post-hoc Dunnett’s test for multiple comparisons was used. For (a and d), N = 3 biological replicates (three independent co-cultures) per group and N = 3 independent T-Cell donors. Indicative data from N = 2 experimental repeats. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: Activation Assay, Co-Culture Assay, Flow Cytometry, Confocal Microscopy, Labeling, Construct, Incubation, Control

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vivo histological investigation of the anti-GB mSA2 CAR-T cell killing functionality. (a) In vivo pipeline. (b) Evaluation of TIM-3, LAG-3 and PD-1 levels on mSA2 CAR-T cells before in vivo treatment by flow cytometry. Isotype control antibodies were used to determine gating. Data gated on live single CD3 + /EGFP + cells. (c) Tumor signal one day before and 5 days after mSA2_h28z CAR-T cell treatment, measured by BLI. N ≥ 5 animals per group. (d) Tumor signal quantification from (c). A paired t-test (mSA2_h28z, mSA2_h28z + a-CD70) and a Wilcoxon matched-pairs signed-rank test (mSA2_h28z + a-CD276) were used to assess significance. (e) Tumor signal fold-change quantification from (c). Each dot represents an animal. A one-way ANOVA test with a post-hoc Dunnett test for multiple comparisons was performed. (f) IF analysis of brains of treated animals. Representative images from two animals per treatment group. (g) Quantification of CAR-T and cleaved caspase-3 signal in stained sections from (f). Each dot represents an image from different tumor regions of a given animal. A Kruskal-Wallis test with a post-hoc Dunn’s multiple comparisons test was performed to evaluate significance. For (b and c), mSA2 CAR-T cells were produced from N = 1 T-Cell donor. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant. No data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: In Vivo, Flow Cytometry, Control, Staining, Produced

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vivo evaluation of the potential mSA2 CAR-T cell interaction with endogenous biotin. (a) Screening of mouse brain single cell suspensions for extracellular biotin presence by flow cytometry. Data gated on live single cells. N = 5 mice. In vitro biotinylated or non-biotinylated HEK293 cells were used as positive (blue histogram) and negative (red histogram) control samples respectively. (b) Analysis of CAR-T cell activation after co-culture of mSA2_h28z cells from N = 1 donor with the mouse brain single cell suspensions from (a) by flow cytometry. Data gated on live single CD3 + /EGFP + cells. Representative plots from N = 3 biological replicates (three independent co-cultures) per mouse. Isotype control antibodies were used to determine gating. (c) Quantification of CD137 positive signal from (b). (d) Granzyme-B and IFN-γ secretion levels in the co-culture SN from (b), determined by ELISA. For (b, c and d), P3 cells (CD276 + ) labeled or unlabeled with an anti-CD276 biotinylated antibody and co-cultured with mSA2_h28z cells were used as positive and negative activation control samples respectively. For (b, c and d), a one-way ANOVA test with a post-hoc Dunnett test for multiple comparisons was performed. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant; n.d., not detected. No data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec) or 10 μg/mL of a biotinylated non-targeting isotype control antibody (#13–4714–85, Thermo Fisher Scientific) in PBS for 30 min at 4 °C.

Techniques: In Vivo, Flow Cytometry, In Vitro, Control, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture