EMBO/soda-nel huggingface dataset:
TOP3A amplification and ATRX inactivation are mutually exclusive events in pediatric osteosarcomas using ALT (2022)
Figure 7

Figure 7. TOP3A depletion affects BLM localization at APBs and disrupts telomeric DNA synthesis

(A) FISH-IF of telomeres (in red) and BLM (in green) in TOP3A-depleted cells BLM foci at telomeres are indicated by white arrows. Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s. = non-significant, **p < 0.01, Mann-Whitney test. Scale bars are 5 μm.

(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere in red, EdU in purple) in PML (in green) according to TOP3A KD. EdU signal at APB (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.

Expanded View Figure Legends
Unexpected PD-L1 Immune Evasion mechanism in TNBC, Ovarian and other Solid Tumors by DR5 Agonists (2020)
Figure 1

Figure 1. DR5 agonist antibodies surface stabilizes PD-L1 on solid cancer cells and tumors

(A, B, C) Total PD-L1, PARP, cleaved caspase-3 from colon (Colo-205), lung (A549), pancreatic (PANK1), triple negative breast cancer cell (MDA-MB-436) and ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, and tigatuzumab. GAPDH is loading control

(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control. See also EV1A for data using additional cell line.

(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH. Representative blots are in Appendix S2A.

(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1). See also EV1B for additional cell line data

(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included. (See Appendix S1 for additional plots)

(H) Relative surface PD-L1 % cells, after DR5 agonist treatments (See Appendix Fig S1). Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).

(I) 0.5x106-2x106 indicated tumor cells were injected subcutaneously in NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ animals with Matrigel in PBS. When tumors appeared on animals (3-4 weeks), animals were i.p. injected with indicated DR5 agonists (4-6 doses), followed by tumor extraction and preparation of single cell suspension isolation from tumors after indicated antibody treatments.

(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments (See Appendix Fig S3). Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)

(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.

(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC). For additional images see Appendix Fig S2C. Scale bar indicates 50μm.

(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr). For DR5 resistant cell generation and additional data see Fig EV2A-C.

Data information: Error bars represent SD. In (E), (H) and (J) unpaired Welch's t-test was used to determine p values. (*p <0.05, **p <0.005, ***p <0.0001)
Unexpected PD-L1 Immune Evasion mechanism in TNBC, Ovarian and other Solid Tumors by DR5 Agonists (2020)
Figure 2

Figure 2. PD-L1 stabilization is CSN5 independent but protease dependent

(A) Model of CD3 activation that induces luciferase in PD-1 effector jurkat reporter cells. Also see Appendix Fig S4.

(B) Tumor cell-Jurkat cell co-culture model: Upon DR5 activation in tumor cells, surface mobilized PD-L1 engages PD-1 on jurkat reporter cells leading to loss of luciferase activity. Also see Appendix Fig S4.

(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3).

(D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture.

(E-G) MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65, total p65, cleaved caspase-3 and PARP. GAPDH is loading control in E and G. Tubulin is loading control in F. Additional data from OVCAR-3 is shown in Fig EV2F.

(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).

(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132. Additional data from MDA-MB-436 is shown in Fig EV2G.

(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.

(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.

(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.

(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).

(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies. See also Appendix Fig S5B.

(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.

(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.

Blue arrow indicate decreased S5a levels and red brackets in corresponding lanes (below) shows overall increased ubiquitin signal in lysates.

Data information: Error bars in (C), (D) and (M) represent SD. Unpaired Welch's t-test was used to determine p values for (C) and (D). Paired t test was used for M. (**p <0.005, ***p <0.0001)
Unexpected PD-L1 Immune Evasion mechanism in TNBC, Ovarian and other Solid Tumors by DR5 Agonists (2020)
Figure 3

Figure 3. ApoEVs and non-apoptotic caspase-8 help stabilize PD-L1 in DR5 insensitive tumor cells

(A) Schematic showing presence of tumor cells capable of optimal and non-optimal apoptotic activation by DR5 agonists constitute heterogeneous tumors.

(B-D) ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots. ApoEVs isolated after IgG1 and tigatuzumab (C) and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.

(E) Details of experimental data described in (F)

(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation (3rd plot, See Methods for more details) from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.

(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs (See also Appendix Fig S6).

(H) Same as (G) except after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).

(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.

(J) Same as H and I except ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal (n=3) (See Fig 2).

(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band

(L) Relative caspase-8 and caspase-3 activity assays from K. Similar to cleaved caspase-8 profile in (K), caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).

(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) similar to K, L, cells were allowed to grow 24 hrs, followed by cell viability analysis.

Data information: Error bars in (J) represent SD. Error bars in (L), (M) represent SEM. For statistical significance t-test was used for (J), (L) and (M). (**p <0.005, ***p <0.0001, ****p <0.00005)
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 1

Fig. 1 Expression of CD80 and PD-L1 in cis inhibits PD-L1 detection.

A-C. CHO cells expressing PD-L1 alone (A) or co-expressing CD80 (B) or CD86 (C) were stained with CD80 Ig, PD-1 Ig, CTLA-4 Ig or anti-PD-L1 Ab clones MIH1, MIH3, 29E.2A3) at 1µg/ml for 30 min at 37°C. Representative FACS plots show staining plotted against total PD-L1 mCherry levels.

D. Integrated data showing PD-L1 detection as determined by the indicated 3 aPD-L1 Ab clones (mean ± SEM, 2 repeats from 3 independent experiments, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test).

E. Western blot analysis of an immunoprecipitation by anti-mCherry antibody (PDL1) from indicated DG-75 cell lysates, with and without the BS3 crosslinker, and probed for mCherry (PD-L1) or GFP (CD80).
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 2

Fig. 2 Transendocytosis of CD80 is not inhibited by PD-L1 co-expression

A. Transendocytosis assays were carried out overnight (as detailed in EV2) using CHO donor cells (CD80GFP alone or CD80GFP with PD-L1mCherry co-expression) incubated with CTLA-4 recipient cells at a ratio of 1:1. Recipient cells either lacked CTLA-4 (No CTLA-4) or expressed CTLA-4. FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA-4+ recipients.

B. Assays as in A but showing PD-L1 expression following CD80 transendocytosis (red shaded quadrant).

C-F. Transendocytosis assays were performed at a ratio of 2 donor:1 CTLA-4 recipient for the times indicated. (C), Representative FACS plots showing CD80GFP levels and (D) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA-4 control (mean ± SEM, 3 independent experiments, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test). (E) Representative FACS plots showing PD-L1mCherry level at time points indicated. (F) Full kinetic analysis of PD-L1mCherry level on donor cells (mean ± SEM, 3 independent experiments).
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 3

Fig. 3 The CTLA-4 cytoplasmic domain promotes efficient transendocytosis of CD80 and CD86

A-B. CTV-labelled B cells (DG-75) co-expressing PD-L1mCherry and CD80GFP (A) or PD-L1mCherry and CD86GFP (B) were incubated with Jurkat cells expressing no CTLA-4, CTLA-4 WT or a mutant CTLA-4 (Del36) lacking the cytoplasmic tail. Transendocytosis was carried out at a 1:1 ratio for the indicated times and analysed by flow cytometry. FACS plots show GFP ligand loss from donor B cells (upper left quadrants) and acquisition by Jurkat recipients (lower right quadrants).

C. Levels of CD80GFP and PD-L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA-4 WT or Del36, plotted as a percentage relative to no CTLA-4 control (mean ± SEM, 6 independent experiments). ****P≤0.0001, RM one-way ANOVA.

D. Levels of CD86GFP and PD-L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA-4 WT or Del36 plotted relative to no CTLA-4 control (mean ± SEM, 6 independent experiments). ****P≤0.0001, RM one-way ANOVA.

E. Comparison of CTLA-4 surface expression levels in Jurkat cells expressing CTLA-4 WT or CTLA-4 Del36 as determined by anti-CTLA-4 (clone BNI3 at 1:100 dilution) stain on ice and analysed by flow cytometry. Graph shows mean ± SEM from 4 independent experiments, **P≤0.01, Student's two-tailed independent samples t-test.
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 4

Fig. 4 Efficient removal of CD80 by transendocytosis liberates free PD-L1 and restores PD-1 Ig binding

A-B. APC-labelled PD-1 Ig (0.75µg/ml) was used to detect PD-L1 in DG-75 cells co-expressing CD80GFP (A) or CD86GFP (B). Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA-4, CTLA-4 WT or CTLA-4 Del36 (at a ratio of 1:1) for the indicated durations. (A) shows representative FACS plots of CD80GFP vs. PD-1 Ig at the time points indicated, with full kinetic data plotted below (mean ± SEM, 3 independent experiments, ***P≤0.001, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test). (B) As for (A) except using CD86-PD-L1 expressing cells.
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 5

Fig. 5 A 1:1 stoichiometry between PD-L1 and CD80 prevents PD-1 binding

Molecular ratios of CD80GFP and PD-L1mCherry were determined using Quantum™ Simply Cellular® beads and ratios of PD-L1:CD80 calculated.

A. Staining of free PD-L1 with APC-conjugated PD-1 Ig in DG-75 cells expressing only PD-L1mCherry, using cell lines with increasing ratios of CD80GFP: PD-L1mCherry (as calculated in EV6) compared to CD80GFP only. FACS plots are representative of 4 independent experiments.

B. Representative FACS plot of a single DG-75 cell line co-expressing CD80GFP and PD-L1mCherry. Heatmap colours indicate levels of PD-L1 stained by PD-1 Ig as a function of CD80GFP expression. GFP levels of each cell line were gated into fractions (indicated by dotted lines) representing different GFP expression levels and the ratio of CD80: PD-L1 calculated and plotted against the respective APC-conjugated PD-1 Ig bound.

C. Integrated data showing PD1-Ig detection as a function of the calculated ratio of CD80: PD-L1 (mean ± SEM, 4 independent experiments).
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 6

Fig. 6 CD80/CD86 and PD-L1 are co-localised at the immune synapse during transendocytosis.

A. DG-75 B cells expressing CD80GFP or CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat cells (nuclei shown in grey) expressing CTLA-4 WT or mutant CTLA-4 Del36 for the indicated durations and analysed by confocal microscopy. 2 hours prior to assay endpoint, APC-labelled PD-1 Ig (0.75µg/ml) was added. CD80/CD86 and PD-L1 co-localisation at the immune synapse is shown in yellow with PD-1 Ig binding shown in purple. Scale bar, 10µm.

B. Fluorescence intensity of PD-1 Ig-APC binding to CD80/PD-L1 co-expressing B cells quantified after 3 and 24 hours transendocytosis (mean ± SEM of a minimum of 44 cells per condition, ****P≤0.0001, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test).
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 7

Fig. 7 CTLA-4 mediated trans-endocytosis of CD80 rescues PD-L1: PD-1inhibition.

A. Schematic of an in vitro assay to monitor CD80 transendocytosis and subsequent PD-1 mediated inhibition. CTFR-stained DG-75 cells expressing CD80 and PD-L1 were loaded with 200ng/ml of SEE superantigen and co-incubated for 24h with WT CTLA-4+ Jurkat T-cells (Jreg) or control CTLA-4- Jurkat cells. CTV-stained PD-1+ve Jurkat T-cells were used as responder cells to measure functional PD-L1-PD-1 interactions.

B. Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg cells used, and the anti-PD-1 status is indicated above the plot.

C. Flow cytometry plots of CD28 levels indicating contact between the CD80+ APC and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28 MFI in each condition is shown on the right.

D. Flow cytometry of CD69 levels on responder Jurkat T-cells and graphical representation of CD69 MFI in each condition.

E. Flow cytometry of CD69 levels on CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed.

F. as in E, but showing MFI of CD69+ cells on responder Jurkat T-cells with titration of SEE superantigen.

Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.
The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80 (2022)
Figure 8

Fig. 8 CTLA-4 Ig (Abatacept) modulates anti-PD-L1 antibody binding but fails to confer PD-1 Ig detection.

A. Concatenated flow cytometry plots for Abatacept-APC binding to DG-75 cells expressing CD80 or PD-L1/CD80.

B. Titration of Abatacept staining (left hand panel) and EC50 values of Abatacept (right hand panel) for CD80 only or PD-L1/CD80 co-expressing DG-75.

C. -Concatenated plot of a 1 in 5 serial dilution of Abatacept (starting at 10µg/ml) followed by PD-L1 detection using antibody (MIH3 or 29E.2A3 clone) or using PD-1 Ig, all at 1µg/ml. DG-75 with no ligand or PD-L1 alone (far right) are shown as staining controls in the presence of 10µg/ml of abatacept.

D. Comparison of PD-L1 detection using PD-1-Ig or anti-PD-L1 antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C).

E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept incubation periods (30mins, 3h or 6h).

Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B) or RM one-way ANOVA (D).

Extended view data
Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites (2020)
Figure 2

Figure 2: Characterization of NF54, NF135 and NF175

A: Molecular characterization by PCR using established markers: MSP2 IC1, GLURP, MSP1 K1, MSP2 FC27, MSP1 R033, and MSP1 MAD20.

B: Markers as in A as well as the sizes for the three parasite strains.

C: Expression of typical liver stage protein markers: Circumsporozoite protein (CSP) top left; Exported Protein 2 (EXP2) top right; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bottom left; and Merozoite surface protein 1 (MSP1) bottom right. Scale bar is 25 µm.
Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites (2020)
Figure 4

Figure 4: Distribution of human glutamine synthetase (hGS) in different Pf strains

A-C) hGS interaction with NF135 (A), NF175 (B) and NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.

D) Median sizes (+/- s.d.) of NF135 (green), NF175 (purple) and NF54 (blue) schizonts divided based on the presence (solid line) or absence (dashed line) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates. A two-way RM ANOVA was performed on the median followed by a Tukey's multiple comparisons test. The significant differences between hGS+ and hGS- population within a strain is showed with asterisks (* p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). See Appendix Fig S1 for the raw measurements.
Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites (2020)
Figure 5

Figure 5: Correlation between intra-parasitic hGS levels and schizont size on day 3 and 5 post invasion.

A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont.

C) The slope of the linear regression of the A) and B) of NF135 and NF175 on day 3 (circle) and day 5 (square) p.i..

D) The R squared value of the predicted linear relationship of hGS levels versus schizont size on day 3 and day 5 p.i..

E) The Spearman's correlation coefficient between hGS levels and schizont size of NF135 and NF175 on day 3 and day 5 p.i..

(For C-E: Each dot represents one biological replicate. See Appendix Fig S2A-D for raw data).