Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A ) Scheme of EphA2-CAR constructs. ( B ) Summary plot of %tCD19 + T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( C ) Summary plot of %F(ab′) 2 -positive T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( D ) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone ( n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). ( E ) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 ( n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). ( F ) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Transformation Assay, Incubation, MTS Assay

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: T cells were cocultured with tumor cells at a 2:1 ratio with weekly restimulation against fresh tumor cells until they lost their effector function and no longer killed all the tumor cells. ( A ) Average expansion of CAR T cells against EphA2-positive (U373 and LM7) and U373 EphA2 KO cell line (mean ± SEM, LM7: n = 4; U373: n = 8 [NT, CD28, MC], n = 4 [41BB]; U373 KO: n = 8 [NT, CD28, MC], n = 6 [41BB]). ( B ) Summary of the maximum expansion CAR T cells from individual donors achieved against EphA2-positive tumor cells and the maximum number of times CAR T cells were able to kill fresh EphA2-positive tumor cells ( n = 12 [NT, CD28, and MC], n = 8 [41BB]; median and quartiles, 1-way ANOVA with Tukey’s test for multiple comparisons). T cells were phenotyped 7 days after stimulation with U373. ( C ) Summary plot of CD4/CD8 composition after stimulation with U373 ( n = 3, mean ± SEM). ( D ) Scheme for phenotyping T cells and representative flow cytometry plots of CCR7 and CD45RA expression on CAR T cells after stimulation with U373. ( E ) Summary plot of T cell phenotype after stimulation with U373 ( n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A – D ) NSG mice were injected with 1 × 10 6 LM7-ffLuc i.p. One week later, mice were injected with 1 × 10 4 or 1 × 10 5 CD28-, 41BB-, or MC-CAR T cells. PBS and 1 × 10 5 Delta-CAR T cells were used as controls. ( A ) Total flux from tumor cells in all mice treated with 1 × 10 4 EphA2 CAR T cells (PBS: n = 5; CD28, 41BB, MC: n = 10). ( B ) Total flux from tumor cells in all mice treated with 1 × 10 5 EphA2 CAR T cells (Delta: n = 5, CD28: n = 8, 41BB: n = 9, MC: n = 10). ( C and D ) Kaplan-Meier survival analysis of mice treated with 1 × 10 4 ( C ) or 1 × 10 5 ( D ) EphA2 CAR T cells (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons; * P < 0.05; ** P < 0.01; *** P < 0.001). Experiments were repeated twice with CAR T cells generated from 2 different healthy donors.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Injection, Generated

Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P<0.001 by one-way ANOVA. (C–D)PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.5 μg/ml ephrin-A 5Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. (C) The histogram shows the average area of the cells normalized to the value obtained for the Fc-treated cells. Error bars represent standard errors from three wells. The average cell areas in cells treated with ephrin-A1 Fc and compound were compared to that in cells treated only with ephrin-A1 Fc by one-way ANOVA and Bonferroni’s post test, showing that 76D10 significantly (***P<0.001) inhibits ephrin-A1-dependent cell retraction at concentrations between 100 and 25 μM. The effect of ephrin-A1 was reverted completely by 100 μM 76D10 and partially by 50 and 25 μM (comparison between Fc and ephrin-A1 Fc treated samples at each compound concentration yielded P values of >0.05 for 100μM, <0.05 for 50 μM and <0.001 for 25μM 76D10. (D) Representative images of cells stained with rhodamine-phalloidin to label actin filaments (red) and DAPI to label nuclei (blue). Scale bar = 50 μm.

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Activation Assay, Control, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Comparison, Concentration Assay, Staining

Journal: Chemical biology & drug design

Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

doi: 10.1111/j.1747-0285.2011.01199.x

Figure Lengend Snippet: Compound 76D10 inhibits ephrin- and TNFα-induced tyrosine phosphorylation and capillary-like tube formation in HUVE cells. (A) Cells plated on Matrigel were treated with the indicated concentrations of 76D10 or DMSO and imaged 18 hours later. The number of polygons present in each picture and the average tube length were quantified. The histograms show averages from 4 independent experiments and the error bars represent the standard errors. **P<0.01 and ***P<0.001 by one-way ANOVA and Dunnett’s post test. (B) HUVE cells were left unstimulated or stimulated with 20 nM TNFα for 2 hours in the presence of the indicated concentrations of 76D10. EphA2 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphA2. (C) MTT assay to determine the number of viable HUVE cells after growth in the presence of the indicated concentrations of 76D10 for 1 or 3 days. Only DMSO was used in the “0 μM” sample, as a control. The histogram shows average absorbance at 570 nm in the presence of 76D10 normalized to the absorbance in the absence of the compound. Error bars represent standard error from 3 measurements in each of two experiments. *P<0.05 by one-way ANOVA and Dunnett’s post test for the comparison to cells not treated with compound (0 μM).

Article Snippet: Polystyrene high binding capacity plates (Corning, Corning, NY) were incubated overnight at 4°C with 4 μg/ml goat anti-EphA2 antibody (directed to the extracellular region of the receptor; R&D Systems, Minneapolis, MN) diluted in phosphate buffered saline (PBS), and then incubated for 2 hours at room temperature with cell lysate diluted in RIPA or ELISA lysis buffer.

Techniques: Phospho-proteomics, MTT Assay, Control, Comparison

Journal: International Journal of Cancer

Article Title: Up-regulation of ephrin-a1 during melanoma progression

doi: 10.1002/(sici)1097-0215(19991022)84:5<494::aid-ijc8>3.0.co;2-o

Figure Lengend Snippet: FIGURE 1 – Northern blot analysis of total RNA from human metastatic melanoma biopsies (15 µg/lane). L and S, samples derived from lymph node and s.c. metastases, respectively; GRM is a metastatic melanoma cell line. Hybridization was to ephrin-A1, EPH-A2 and GAPDH probes. Durations of exposure were 2, 4 and 1 days, respectively.

Article Snippet: Affinity-purified rabbit polyclonal antibodies for EPH-A2 and ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for blotting, followed by a peroxidase-conjugated goat anti-rabbit secondary antibody.

Techniques: Northern Blot, Derivative Assay, Hybridization

Journal: International Journal of Cancer

Article Title: Up-regulation of ephrin-a1 during melanoma progression

doi: 10.1002/(sici)1097-0215(19991022)84:5<494::aid-ijc8>3.0.co;2-o

Figure Lengend Snippet: FIGURE 2 – Immunoblot analysis of melanoma cell lines and normal melanocytes using (a) anti-EPH-A2 and (b) anti-ephrin-A1 polyclonal antibodies. Cell lines WM39 and WM115 were from primary melano- mas; WM46, WM164 and WM1232 were from metastases. FM lysates were from normal foreskin melanocytes. (a) A doublet at around 130 kDa was detected for 1205Lu cells with the anti-EPH-A2 polyclonal antibody; (b) a band of 36 kDa was detected with the anti-ephrin-A1 polyclonal antibody in all melanoma cell lines. Following prolonged chemiluminescence, signals were also detected for the normal melano- cytes. Left scale, apparent molecular mass in kDa.

Article Snippet: Affinity-purified rabbit polyclonal antibodies for EPH-A2 and ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for blotting, followed by a peroxidase-conjugated goat anti-rabbit secondary antibody.

Techniques: Western Blot

Journal: International Journal of Cancer

Article Title: Up-regulation of ephrin-a1 during melanoma progression

doi: 10.1002/(sici)1097-0215(19991022)84:5<494::aid-ijc8>3.0.co;2-o

Figure Lengend Snippet: FIGURE 3 – Immuno-histochemistry of EPH-A2 and ephrin-A1 in sections of pigmented lesions. (a) Normal skin, ephrin-A1. The reaction is intense in epidermal keratinocytes and appendages such as the hair follicle at lower left; endothelial cells are weakly stained. (b) Normal skin, EPH-A2. A reaction is seen in the granular layer of the epidermis, and hair follicle keratinocytes appear weakly positive. (c) Melanocytic dysplasia, ephrin-A1. Dysplastic melanocytes show little or no immunoreactive ephrin-A1, though surrounding epidermal keratinocytes are again strongly reactive. (d–f) MMIS, sections of the same lesion. (d) Hematoxylin and eosin stain for comparison with (e) and (f). (e) Ephrin-A1. Most or all of the abnormal melanocytes in this MMIS are positive. (f) EPH-A2. Again, the melanocytes are reactive, indicating co-localization of receptor and ligand. (g) VGP primary melanoma, ephrin-A1. Most or all nests of malignant melanoma cells are reactive; surrounding stromal cells are negative. (h) Metastatic melanoma, ephrin-A1. Tumor cells are reactive for ephrin-A1, while surrounding lymphocytes are unstained. Scale bars: 50 µm in (a–f) and (h), 25 µm in (g).

Article Snippet: Affinity-purified rabbit polyclonal antibodies for EPH-A2 and ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for blotting, followed by a peroxidase-conjugated goat anti-rabbit secondary antibody.

Techniques: Immunohistochemistry, Staining, H&E Stain, Comparison

Journal: International Journal of Cancer

Article Title: Up-regulation of ephrin-a1 during melanoma progression

doi: 10.1002/(sici)1097-0215(19991022)84:5<494::aid-ijc8>3.0.co;2-o

Figure Lengend Snippet: FIGURE 4 – Effects of cytokines on expression of EPH-A2 and ephrin-A1 in metastatic melanoma cell lines (DX3, LT5.1 and RPMI7932). Indicated lines were grown with cytokines, as shown, for 4 hr before Northern blot analysis using poly(A)1-enriched RNA (5 µg/lane). Hybridization was to ephrin-A1, EPH-A2 and GAPDH probes. (a) Autoradiography. Durations of exposure were 4, 8 and 1 hr. respectively; the control lane for DX3 is overloaded but remains within the linear phase for quantitation and is suitable for densitometry. (b) Histogram of RNA indices of ephrin-A1 expression, means and SEs shown. Densitometric readings: initially, all values for ephrin-A1 were normalized to GAPDH; in turn, these values were normalized to untreated cells (which were defined as 100). For each cell line, the results are from 3 independent experiments, each in triplicate.

Article Snippet: Affinity-purified rabbit polyclonal antibodies for EPH-A2 and ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for blotting, followed by a peroxidase-conjugated goat anti-rabbit secondary antibody.

Techniques: Expressing, Northern Blot, Hybridization, Autoradiography, Control, Quantitation Assay