Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A ) Overlay histograms of LFA-1 diffusion on monocytes (4578 trajectories), resting mDCs (26756 trajectories) and CCL21 activated mDCs (4213 trajectories). ( B ) Percentage of total mobile LFA-1 population (normalized to 100%) displaying slow and fast diffusion on monocytes, mDCs and 2 min CCL21 activated mDCs. ( C ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on monocytes, mDCs and 2 min CCL21 activated mDCs. ( D ) Stationary fraction of LFA-1 on monocytes, mDCs and CCL21 activated mDCs, displayed as the difference from the total stationary fraction on mDCs, which serve here as the default. Data from A–D on CCL21 activated mDCs is based on 22 cells in independent experiments from 3 different donors. ( E ) Percentage of the stationary, slow and fast diffusing LFA-1 molecules at different time points after CCL21 activation. ( F ) D values for the total mobile, and slow and fast fractions of LFA-1 at different time points after CCL21 activation. Data from E and F is based on 11 cells, 11 independent samples and around 2000 trajectories per time point. A – F Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns (P >0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Activation Assay, Comparison

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A,B ) ICAM-1 (either monomeric: ICAMm, or as nano-aggregates: ICAMagg) was added to resting mDCs and mobility was measured before, and between 1 and 5 min after addition. ( A ) Stationary fraction of LFA-1 molecules on resting mDCs and mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs, which serve here as the default. ( B ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs and after addition of either monomeric ICAM-1 or ICAM-1 nano-aggregates. 30 cells divided over 2 independent experiments (3393 trajectories) were imaged for the ICAMm condition and 10 cells (684 trajectories) for ICAMagg. ( C, D ) ICAM-1 (either monomeric or nano-aggregates) was added together with CCL21 to mDCs and mobility was measured before, and 2 minutes after addition. ( C ) Stationary fraction of LFA-1 molecules on resting mDCs (serving as reference control), CCL21 activated mDCs and CCL21 activated mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs. ( D ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs, CCL21 activated mDCs and after simultaneous addition of CCL21 and either monomeric ICAM-1 or ICAM-1 nano-aggregates. 16 cells (8423 trajectories) from 2 different donors divided over 5 independent samples (ICAMm) and 7 cells (314 trajectories) from 2 different donors divided over 7 independent samples (ICAMagg) were imaged. ( E ) Quantification of fluorescent ICAM-1 dimers (monomers bound together due to antibody labelling) and nano-aggregates binding in resting and CCL21 activated mDCs, normalized to the area quantified and to the background signal outside of the cell. For this, regions of the cell in between the obvious fluorescent ICAM-1 aggregates were selected, the fluorescent intensity was measured using ImageJ, and used to compare the baseline fluorescent signal across all 4 conditions. 20 cells per condition were imaged. ( A–E ) Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05), ** (P<0.001) and *** (P<0.0001). ( F ) Quantification of bound ICAM-1 nano-aggregates to resting and CCL21 activated mDCs. After applying a threshold of 25% of the fluorescent signal, all visible fluorescent spots per cell were counted. 20 cells per donor and 3 different donors were imaged. Each data point represents the mean value for 1 donor. Means ± SEM and individual data points are depicted, and dotted lines connecting datapoint of the same experiment indicate that not just in average, but in each individual experiment using a different donor, an increase of ICAM-1 nano-aggregate binding is observed after CCL21 activation. The paired two-tailed Student T-test was used to determine significant differences between means. ( G–I ) Representative examples of confocal images of ICAM-1 binding to mDCs: ( G ) dimeric ICAM-1 to resting cells, ( H ) nano-aggregates of ICAM-1 to resting cells and ( I ) nano-aggregates to CCL21 activated cells. Arrows in H and I point to the binding of individual ICAM-1 nano-aggregates to LFA-1.

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Control, Binding Assay, Comparison, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A, B ) Representative images of ( A ) a monocyte seeded on a TS2/4 pattern, ( B ) a resting mDC on irrelevant IgG1 pattern, ( C ) a resting mDC seeded on a TS2/4 pattern and ( D ) a CCL21 activated mDC seeded on a ICAM-1 pattern. Green corresponds to Talin1 and red to the location of IgG1, TS2/4 or ICAM-1 positive squares. Cells are delineated by white lines. ( E ) Quantification of the degree of Talin1 enhancement to the positive areas in monocytes in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( F ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in monocytes. An enhancement factor of ≥1.5 was considered significantly enhanced, since 95% of the control sample (monocytes on IgG1) showed an enhancement factor below this value. ( G ) Quantification of the degree of Talin1 enhancement to the positive areas in mDCs in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( H ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in mDCs. Around 60 cells of 3 different donors were analyzed per condition. Monocytes contained 10 positive areas on average per cell, while mDCs contained around 50 positive areas. Means ± SEM are depicted. The Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used to determine significant differences between means in E and G. The One-way ANOVA followed by the Tukey’s multiple comparison test were used to determine significant differences between means in F and H. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Control, Comparison

Journal: Oncotarget

Article Title: Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization

doi: 10.18632/oncotarget.16311

Figure Lengend Snippet: A . Expression of the CCL21 receptor (CCR7) on NKL1, NKL2 and NKL3 cells surface. Results represent Δ MFI (mean fluorescence intensity) from flow cytometry analysis. Values marked with a star vary significantly (p<0.05, N=5) from the NKL1 level of expression. B . Quantification of adhered NK cells counted from ten representative fields. Values marked with a star vary significantly (p<0.05, N=3) from NKL1 and NKL2 adhesion levels. C . Flow adhesion data of NKL cell lines on HPLNEC.B3 cells NKL1 (a, b); NKL2 (c, d); (e, f).

Article Snippet: Mouse IgG2A anti human CCR7 fluorescein-coupled antibodies, mouse IgG2a fluorescein-coupled isotypic control, mouse IgG2A anti human CCR7 neutralizing antibodies, recombinant human CCL21 and human CCL21 ELISA kits were purchased from R&D Systems (UK).

Techniques: Expressing, Fluorescence, Flow Cytometry

Journal: Oncotarget

Article Title: Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization

doi: 10.18632/oncotarget.16311

Figure Lengend Snippet: A . Hypoxia vs normoxia CCL21 production by HPLNEC.B3 cells measured by ELISA in cell lysates. Concentration of CCL21 protein produced under normoxia was set as 100%. Values marked with a star vary significantly (p<0.05, N=3). B . Quantification of the NKL3 adhesion to HPLNEC.B3 as number of NKL3 cells counted on the surface of HPLNEC.B3 cells (ten representative fields were counted) in normoxia and in hypoxia showing a reduction before as well as after treatment with CCL21. NK cells were labelled by the PKH26 red fluorescent cell linker kit. NK cells were injected on the HPLNEC.B3 cells monolayer (1.10 6 NK cells/ml) at a fixed flow rate of 0, 2 dyn/cm 2 for 5 minutes and washing by OptiMEM at 0, 6 dyn/cm 2 for 10 min. Adhered NK cells amount was quantified. *** p<0.001, N=3, ### p<0.001, N=3.

Article Snippet: Mouse IgG2A anti human CCR7 fluorescein-coupled antibodies, mouse IgG2a fluorescein-coupled isotypic control, mouse IgG2A anti human CCR7 neutralizing antibodies, recombinant human CCL21 and human CCL21 ELISA kits were purchased from R&D Systems (UK).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Produced, Injection

Journal: Oncotarget

Article Title: Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization

doi: 10.18632/oncotarget.16311

Figure Lengend Snippet: A . NK cells labelled with FTC-anti-CCR7 antibodies, at 4°C and fixed with 4% PFA, capping does not occur. B . Patching upon warming the cells from 4°C to 25°C for 5 min. at 25°C. C . Warming for 15 mins, capping occurs. D . Quantification of the NKL3 cells adhesion to HPLNEC.B3 as number of NKL3 cells counted on the surface of HPLNEC.B3 cells monolayer (ten representative fields were counted), NKL3 were either maintained at room temperature or capping was induced (4-25°C) before and after preincubation with CCL21 *p<0.05, **p<0,01, N=3, # p<0.05, ## p<0,01 N=3.

Article Snippet: Mouse IgG2A anti human CCR7 fluorescein-coupled antibodies, mouse IgG2a fluorescein-coupled isotypic control, mouse IgG2A anti human CCR7 neutralizing antibodies, recombinant human CCL21 and human CCL21 ELISA kits were purchased from R&D Systems (UK).

Techniques:

Journal: Oncotarget

Article Title: Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization

doi: 10.18632/oncotarget.16311

Figure Lengend Snippet: A . Impact of CCL21 presentation by GAGs on CCR7+ NK cells adhesion to endothelial cells. Quantification of the NKL3 adhesion to HPLNEC.B3 as number of NKL3 cells counted on the surface of HPLNEC.B3 cells (ten representative fields were counted) in normoxia (A) and in hypoxia B . showing an increase in adhered NKL3 after treatment with CCL21 and decrease after treatment with CCR7 neutralizing antibody and chondroitin sulfate in both conditions. NK cells were labelled by PKH26 red fluorescent cell linker kit then, treated or not with CCR7 neutralizing antibodies (10 μg/ml), or ChSE (0,6 μg/ml) for 1 hour at 4°C. Then, NK cells were injected on the HPLNEC.B3 cells monolayer (1.10 6 NK cells/ml in OptiMEM) at a fixed flow rate of 0, 2 dyn/cm 2 for 5 minutes and washing by OptiMEM at 0, 6 dyn/cm 2 for 10 min. Adhered NK cells amount was quantified. * p<0.05, N=3, # p<0.05, N=3; ** p<0.005, N=3, ## p<0.005, N=3. C . Plasmon resonance assessment of the affinity of CCL21 chemokine for various glycosaminoglycans. CCL21 was injected over the GAG surface in a range of concentrations (from 0 to 300 nM) to produce sensorgrams for association and dissociation phases analysis. Binding kinetics were evaluated with a 1:1 Langmuir model using the BiaEvaluation software (Biacore). D . Inhibition experiments by co-injection of CCL21 chemokine with various concentrations of GAGs on the immobilized ChSD surface. Relative inhibition was preferential for chondroitin sulphate E compared to ChS B, D and C.

Article Snippet: Mouse IgG2A anti human CCR7 fluorescein-coupled antibodies, mouse IgG2a fluorescein-coupled isotypic control, mouse IgG2A anti human CCR7 neutralizing antibodies, recombinant human CCL21 and human CCL21 ELISA kits were purchased from R&D Systems (UK).

Techniques: Injection, Binding Assay, Software, Inhibition

Journal: Oncotarget

Article Title: Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization

doi: 10.18632/oncotarget.16311

Figure Lengend Snippet: A . Quantification of podoplanin expression at mRNA level in hypoxia vs normoxia. B . PDPN protein detection by immunoblotting. C . PDPN protein detection (b) in cells by immunocytochemistry with AlexaFuor-murine IgG anti human PDPN (green), nuclei are labelled with DRAG-5 (red). Control (a) is MSU1.1 NC cells (transduced with empty vector). D . PDPN mRNA expression level in hypoxia vs normoxia in MSU1.1 PDPN. E . PDPN protein expression level in hypoxia vs normoxia in MSU1.1 PDPN. F . Quantification of the MDA-MB-231 cells adhesion to MSU1.1 and MSU1.1 PDPN cells surface. MDA-MB-231 cells were counted on the surface of MSU1.1 NC or MSU1.1 PDPN cells (ten representative fields were counted) after flow adhesion in normoxia. * p<0.05, N=3 (mean from ten representative fields). G . Effect of CCL21/CCR7 interaction on MDA-MB-231 cell adhesion onto MSU 1.1 PDPN cells in normoxia. H . Effect of CCL21/CCR7 interaction on MDA-MB-231 cell adhesion on MSU1.1 PDPN cells in hypoxia. I . Effect of CCL21/CCR7 interaction on MDA-MB-231 cell adhesion on MSU1.1 NC cells in normoxia. J . Reduction of adhesion of NKL3 cells to MSU1.1 PDPN vs MSU1.1 NC and MSU 1.1.

Article Snippet: Mouse IgG2A anti human CCR7 fluorescein-coupled antibodies, mouse IgG2a fluorescein-coupled isotypic control, mouse IgG2A anti human CCR7 neutralizing antibodies, recombinant human CCL21 and human CCL21 ELISA kits were purchased from R&D Systems (UK).

Techniques: Expressing, Western Blot, Immunocytochemistry, Control, Transduction, Plasmid Preparation