EMBO/soda-nel huggingface dataset:
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 2

Figure 2 │ NK cell degranulation is associated with increased TRAIL expression across multiple NK cell subsets

Comparison of CD107a and TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether.

(A) Relative frequency of CD107a+ cells (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells (n = 13 different donors).

(B) HIV-specific responses of predefined NK cell sub-populations (x-axis) displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 13 different donors).

(C) TRAIL expression levels (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as median fluorescence intensity (MdFI) (n = 13 different donors).

(D) Comparison of TRAIL expression levels (y-axis) between CD107a- and CD107a+ cells of predefined NK cell subsets. Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as MdFI. (n = 13 different donors).

(E) Fold-differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells of pre-defined NK cell sub-populations (x-axis). (n = 13 different donors).

Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 3

Figure 3 │ In vitro HIV-1-infected CD4 T cells display increased surface expression of the TRAIL receptors DR4/5

Primary human CD4 T cells from eight different donors (n = 8) were separately infected with lab-adapted and primary HIV-1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry four days after infection. Uninfected CD4 T cells were determined as CD4-positive and HIV-1 p24-negative, infected cells as CD4-negative and p24-positive.

(A) Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with either NL4-3, JR-CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity (x-axis).

(B) Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles) and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4-3: n = 8; JR-CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7-8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary-AB-only control (y-axis).

Data information Wilcoxon signed-rank test. Values for non-infected and infected cells of the same donor are connected with lines.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 4

Figure 4 │ Antibody-mediated blocking of TRAIL inhibits NK cell degranulation

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αTRAIL or αDR4/5.

(A) Comparison of CD107a expression after co-culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 10 different donors per condition). Each data point represents the mean of 2 technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis).

(B) Box plots display inhibition of degranulation (y-axis) after co-culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 10 different donors per condition).

(C) Comparison of CD107a expression after co-culture with autologous HIV-I-infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 9 different donors per condition) Each data point represents the mean of 2 technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis).

(D) Box plots display inhibition of degranulation (y-axis) after co-culture with autologous HIV-I-infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 9 different donors per condition).

(E) Representative histograms (overlay, left panel) and bar graphs (n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of 3 technical replicates.

(F) Comparison of CD107a expression after co-culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each), or 20 µg/ml isotype control (Iso) using flow cytometry (n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a+ NK cells (y-axis).

(G) Box plots display inhibition of degranulation after co-culture with 721.221 target cells in presence of either isotype or αDR4/5 as relative reduction compared to no antibody (n = 9 different donors per condition).

Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 5

Figure 5 │ TRAIL engagement directly induces NK cell degranulation

Degranulation of primary human NK cells after incubation with plate-coated antibodies or whole proteins.

(A) Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry (n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated antibodies of different concentrations (x-axis).

(B) Comparison of CD107a expression after incubation with plate-coated DR4 protein, DR5 protein or human IgG using flow cytometry (n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis).

(C) Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing Granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition).

(D) Correlation analysis between relative frequency of CD107a+ NK cells and granzyme B concentration (n = 53, data points obtained from A, B, and C, 11 different donors).

(E) Comparison of CD107a expression after incubation with plate-coated DcR1 protein, osteoprotegerin (OPG) or human IgG using flow cytometry (n = 9 different donors). Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis).

Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, C, E) Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 6

Figure 6 │ TRAIL engagement induces Interferon y production but does not promote cell adhesion

(A) Comparison of IFNγ production (y-axis) after incubation with plate-coated antibodies or proteins (10 µg/ml each) (x-axis). Box plots showing IFNγ concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition).

(B) Comparison of IFNγ levels in the supernatant after co-culture of NK cells with 721.221 target cells in the presence or absence of either 10 µg/ml αTRAIL or isotype control (Iso) using ELISA (n = 8 different donors per condition). Effector:target ratio was 1:1. Left panel: Box plots showing IFNγ levels (y-axis) for the described culture conditions (x-axis). Right panel: Box plots display inhibition of IFNγ production by NK cells (y-axis) in presence of either isotype or αTRAIL as relative reduction compared to no antibody.

(C) Representative contour plots showing conjugate formation between NK cells and 721.221 target cells as determined by flow cytometry. Plots showing three distinct populations at the beginning of the co-culture (0 min) and after 50 min: single 721.221 cells (CT Orange-positive), single NK cells (CT Violet-positive) and conjugates (double-positive). Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells.

(D) Comparison of conjugate formation after co-culture between NK cells and 722.221 cells after 0 min and after 50 min in the presence or absence of 10 µg/ml αTRAIL or isotype control (n = 8 different donors). Left panel: Box plots showing relative frequency of NK cells in conjugates (y-axis) for different time points and conditions (x-axis). Right panel: Box plots display inhibition of conjugate formation (y-axis) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody.

(E) Expression levels of phosphorylated signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk, p38 MAPK, Akt and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.

Data information: Wilcoxon signed-rank test. (A, B, D) Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 7

Figure 7 │ TRAIL contributes to NK-cell-mediated killing of target cells

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays.

(A) Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining (y-axis) after co-culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference of target cells remaining (y-axis) between αTRAIL and isotype conditions displayed as p.p. (n = 12 different donors). Each data point represents the mean of at least 2 technical replicates.

(B) Left panel: Representative contour plots showing the percentage of .221-DR4/5KO (control) and .221-Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221-DR4/5KO and .221-Cas9 cells (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221-Cas9 cells displayed as % (n = 12 different donors). Each data point represents the mean of at least 3 technical replicates.

(C) Left panel: Representative contour plots showing the percentage of Raji-pSIP (control) and Raji-DR5++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji-pSIP and Raji-DR5++ (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji-DR5++ cells displayed as % (n = 12 different donors). Each data point represents the mean of at least three technical replicates.

Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.

EXPANDED VIEW FIGURE LEGENDS
The mechanosensitive TRPV2 calcium channel promotes melanoma invasiveness and metastatic potential. (2023)
Figure 2

Fig 2. TRPV2 expression, plasma membrane targeting and contribution to basal Ca2+ influx, increase with melanoma cell aggressiveness.

(A) Analysis for total and plasma membrane (PM) TRPV2 expression in melanoma cell lines. β-actin was used as loading and cell integrity control. PM fractions were isolated using the surface protein biotinylation technique. The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel (Barnhill et al, 2004).

(B) Constitutive Ca2+ influx comparison between resting 501mel (Light blue), WM266.4 (Blue) and 451Lu cells (Dark blue) assessed using the Fura-2 Mn2+ quenching assay. Representative traces of Fura-2 quenching kinetics are shown on the top panel (each point represents the mean of technical quadruplicates normalized to the baseline). The bar graph summarizes the quantification of the measured quenching rates and is presented as mean ± SEM (n>3 biological replicates). **P =0.0032 and ****P < 0.0001, ordinary one-way ANOVA followed by Dunnett's multiple comparisons post-test.

(C) TRPV2 overexpression in the non-invasive 501mel cell line (GFP-TRPV2) or downregulation by lentiviral-delivery of TRPV2 specific shRNAs (shRNA V2-1 and V2-2) in the WM266.4 and 451Lu metastatic melanoma cell lines assessed by western-blot. β-actin was used as a loading control.

(D) Representative traces of Fura-2 Mn2+ quenching rates in control (GFP) or GFP-TRPV2 overexpressing 501mel cells, or in control (shRNA Ctrl) or TRPV2 repressed (shRNA V2) WM266.4 and 451Lu cells (each point represents the mean of technical quadruplicates normalized to the baseline).

(E) Average normalized quenching rates (as in D) presented as mean ± SEM (n>3 biological replicates). **P = 0.0095, the Mann-Whitney test (left panel); *P = 0.0399-0.0336 (middle panel) and *P = 0.0438, **P = 0.0033 (right panel) the Kruskal-Wallis test followed by Dunn's multiple comparisons test.
The mechanosensitive TRPV2 calcium channel promotes melanoma invasiveness and metastatic potential. (2023)
Figure 3

Fig 3. TRPV2 is essential for melanoma tumor cell migration and invasion.

(A) Impact of TRPV2 genetic manipulation in 501mel, WM266.4 and 451Lu on serum-induced migration and matrigel invasion. Representative pictures show crystal violet-stained cells that have migrated through 8 µm pore size membranes. Histograms illustrate the average numbers of migrating/invading cells normalized to control (presented as mean ± SEM of n=3 independent experiments). For 501mel the Mann-Whitney test was used for statistical analysis (**P = 0.0022); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; ***P < 0.001; ****P < 0.0001 ; See appendix Table S2 for exact P values).

(B) Tracks comparison between fibronectin-plated shRNA Ctrl or shRNA V2 WM266.4 cells migrating toward a serum gradient over a 12 h period (n=10 and 12 cells, respectively) (See also appendix Fig S3B).

(C) Velocity analysis of 2D migration experiments described in B (dot, single cell, n=19-21). Scatter plots show mean ± SD. ****P <0.0001, the Mann-Whitney test.

(D) TRPV2-silencing effect on 3D invasion. Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266.4 cells expressing either shRNA ctrl or TRPV2-targeting shRNAs (scale bar=0.5 mm).

(E) Quantification of collagen invasion. For each spheroid, the cell-covered area at day 2 was normalized to the starting area of the collagen-embedded spheroids. Histograms represent the invasion relative to control WM266.4 spheroids (shRNA Ctrl) from at least 12 spheroids from three independent experiments (See also appendix Fig S3C). M. ****P <0.0001 and **P = 0.0012 the Kruskal-Wallis test followed by Dunn's multiple comparisons test.
The mechanosensitive TRPV2 calcium channel promotes melanoma invasiveness and metastatic potential. (2023)
Figure 4

Fig 4. TRPV2 specifically associates with paxillin, a marker of nascent adhesive structures.

(A) Representative confocal images of low confluency WM266.4 and 451Lu metastatic melanoma cells seeded on fibronectin-coated coverslips. Cell nuclei are depicted with DAPI in blue, TRPV2 in green and indicated proteins (actin, paxillin, vinculin or pY397-FAK) in red (scale bar = 20 µm). Insets are magnifications of the indicated area. Arrows indicate sites of colocalization.

(B) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity-ligation assays (PLA). Low confluency WM266.4 and 451Lu metastatic melanoma cells plated on fibronectin-coated coverslips were stained for F-actin (green), cell nuclei (blue) and PLA reaction using antibodies specific of the indicated proteins (red) (scale bar = 20 µm ; identical for all pictures). Red fluorescent spots indicate the association of the two proteins of interest, close to 40 nm. Scatter plots represent the quantification of the number of PLA spots per cell (bars indicate the medians) between TRPV2 and a control antibody (IgG), paxillin (PAX) or vinculin (VCL). (n=29-51 cells from at least three independent experiments).

(C) Super-resolution (dSTORM) imaging of the clustering of TRPV2 channels with paxillin, but not with vinculin, in migrating WM266.4 cells (Scale bar = 5µm). a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co-clusters.
The mechanosensitive TRPV2 calcium channel promotes melanoma invasiveness and metastatic potential. (2023)
Figure 5

Fig 5. TRPV2 activity controls the calpain-dependent mechanical maturation of focal adhesions.

(A) Quantification of vinculin-stained focal adhesion sites per cell. Confocal images taken on low confluency melanoma cells seeded on fibronectin-coated coverslips were analyzed using the imageJ software to count the number of vinculin clusters per cell. The total number of cells counted for each cell line is indicated in the boxes. Data are presented as whisker-box plots (Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the horizontal line in each box is plotted at the median). **P = 0.0066, the Mann-Whitney test (501mel); ****P <0.0001 the Kruskal-Wallis test followed by Dunn's multiple comparisons tests (WM266.4 and 451Lu).

(B) Comparison of calpain activity in control (GFP) or overexpressing TRPV2 (GFP-TRPV2) 501mel cells, and in control (shRNA Ctrl) or TRPV2-silenced (shRNA V2-1, -2) WM266.4 cells or 451Lu cells. Bar graphs show mean normalized calpain activity ± SEM (n=3 biological replicates). For 501mel the Mann-Whitney test was used for statistical analysis (*P = 0.05); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; See appendix Table S2 for exact P values).

(C) Representative immunoblots showing the full-length (230 kDa ; Black Arrows) and the calpain-mediated degradation product (190 kDa ; Grey Arrows) of talin after overexpression or repression of TRPV2, in the corresponding cell lines. Tubulin was used as a loading control.

(D) Densitometric analysis of the cleaved-talin ratios from 3 independent experiments (as described in C) normalized to control. Data are presented as mean ± SEM. For 501mel the Mann-Whitney test was used for statistical analysis (ns P = 0.2); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (*P < 0.05; ***P < 0.001; See appendix Table S2 for exact P values).
The mechanosensitive TRPV2 calcium channel promotes melanoma invasiveness and metastatic potential. (2023)
Figure 6

Fig 6. TRPV2 expression level dictates the in vivo metastatic potential of human melanoma tumor cells xenografted in mice.

A-D Representative bioluminescence imaging (BLI) data of mice injected intravenously with the non-metastatic melanoma cell line 501mel-Luc transfected with either GFP-TRPV2 or GFP control (A-B), or with the invasive 451Lu-Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA (C-D). Tumor growth and metastasis formation were monitored for 64 days (501mel-Luc) or 35 days (451Lu-Luc) after injection. Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point (ns P = 0.0903 (A) and **P = 0.0064 (C), the Mann-Withney test). Graphs presented in B and D show the number of metastatic foci per animal (B) or per lungs (D) counted at necropsy (*P = 0.0397 (B) and ns P = 0.0691 (D), unpaired t-tests). For (D) Representative ex vivo BLI images of lung metastasis are shown (Scale bar = 5mm).

Data information: Boxes extend from the 25th to 75th percentiles, whiskers from min to max, the horizontal line in each box is plotted at the median and each dot correspond to a single mouse (n=6-7).