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: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa1-6 cells were stained with anti-EphA2 D4A2 (blue, left panel) or anti-CD81 MT81 (blue, right panel) followed by Alexa Fluor-488 conjugated secondary antibodies, and analyzed by FACS. The grey histograms represent cells stained with secondary antibody only (negative control). The vertical line represents the threshold used to distinguish EphA2 low and EphA2 high cells B. Hepa1-6 cells were stained with anti-EphA2 D4A2 (rabbit) and anti-CD81 MT81 (rat) primary antibodies, followed by Alexa Fluor-594 (anti-rabbit) and Alexa Fluor-488 (anti-rat) secondary antibodies, and analyzed by FACS (dot plot on the left). As a control, cells were stained with secondary antibodies only (dot plot on the left). There was no correlation between EphA2 and CD81 fluorescence intensities (Pearson coefficient R 2 = 0,263). C. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2. 48 hours after transfection, surface (left panel) or total (right panel) EphA2 expression was analyzed by FACS in control (blue) and siEphA2-treated (orange) cells. The grey histograms correspond to controls cells stained with secondary antibodies only. D. HepG2 cells were transfected with siRNA oligonucleotides targeting human EphA2. 48 hours after transfection, surface (left panel) or total (right panel) EphA2 expression was analyzed by FACS in control (blue) and siEphA2-treated (orange) cells. The grey histograms correspond to controls cells stained with secondary antibodies only. E. HepG2/CD81 cells were transfected with siRNA oligonucleotides targeting human EphA2 or CD81, or electroporated without siRNA (mock). Cells were lysed 48 hours post-transfection and protein extracts were analyzed by Western blot, using antibodies specific for EphA2 or CD81. F . Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or with a control siRNA (siCtrl). Cells were lysed 48 hours post-transfection and protein extracts were analyzed by Western blot, using antibodies specific for EphA2 or GAPDH. Among three siRNA oligonucleotides tested against mouse EphA2, siRNA #2 was the most efficient and was used throughout the study.

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Staining, Negative Control, Fluorescence, Transfection, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or electroporated without siRNA (mock). Cells were infected with PyGFP sporozoites 48 hours after siRNA transfection, and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy after UIS4 staining. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). B. HepG2/CD81cells were transfected with siRNA oligonucleotides targeting human EphA2 or human CD81, or with a control siRNA (siCtrl). Cells were infected with PyGFP sporozoites 48 hours after siRNA transfection, and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). C. HepG2/CD81 cells were transfected with siRNA targeting EphA2 or with a control siRNA (siCtrl) and infected 48 hours later with PyGFP sporozoites. Infected cultures were fixed 24 hours post-infection and stained with anti-UIS4 antibodies (red) and the nuclear stain Hoechst 33342 (blue). The images show PyGFP EEFs (green) surrounded by a UIS4-positive PVM (red). Scale bar, 10 μm. D. Hepa1-6 cells were incubated with PyGFP sporozoites in the presence of increasing concentrations of the EphA2-blocking antibody D4A2, of a control antibody (DA1E, IgG Ctrl, used at the same dilution as D4A2 highest concentration) or without antibody (Ctrl). The number of EEFs was determined 24 hours post-infection by fluorescence microscopy after UIS4 staining. NS, non-significant (Kruskal-Wallis with Dunns post-test).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Infection, Fluorescence, Microscopy, Staining, Incubation, Blocking Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. HepG2 cells were transfected with siRNA oligonucleotides targeting human EphA2 or SR-BI, or with a control siRNA (siCtrl). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). B. HepG2/CD81 cells were transfected with siRNA oligonucleotides targeting human EphA2 or with a control siRNA (siCtrl). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, in the absence or presence of a CD81-blocking antibody (1D6, 20 μg/ml) and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. C. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or electroporated without siRNA (mock). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy after UIS4 staining. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). D. Hepa1-6 cells were incubated with PbGFP sporozoites in the presence of the EphA2-blocking antibody D4A2 (5 μg/ml) or a control antibody (IgG Ctrl), and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy. NS, non-significant (Mann-Whitney U Test, two-tailed).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Infection, Blocking Assay, Fluorescence, Microscopy, Staining, Incubation, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-EphA2 primary antibody (D4A2) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish EphA2 low and EphA2 high cells. B. The percentage of EphA2 low and EphA2 high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. C. Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish CD81 low and CD81 high cells. This threshold was arbitrarily defined to obtain a majority of CD81 low population after siRNA silencing of CD81. D. The percentage of CD81 low and CD81 high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. E. Hepa1-6 cells were transfected with siRNA targeting CD81 and infected with PbGFP sporozoites. Cells were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (orange histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only, and the blue histogram corresponds to the control cells labeled with CD81 antibodies, as in C. The vertical line indicates the same threshold as in C, used to distinguish CD81 low and CD81 high cells. F. The percentage of CD81 low and CD81 high cells was determined in non-infected (GFP-negative) and infected (GFP-positive) CD81 siRNA-treated Hepa1-6 cells.

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Infection, Staining, Labeling, Transfection

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A-B . Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 (siEphA2) or with a control siRNA (siCtrl). 48 hours after siRNA transfection, cells were incubated for 1.5 hours with PyGFP ( A ) or PbGFP ( B ) sporozoites in the presence of rhodamine-labeled dextran, and the number of traversed (dextran-positive) cells was determined by FACS. NS, non-significant (Mann-Whitney U Test, two-tailed).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Incubation, Labeling, MANN-WHITNEY, Two Tailed Test