Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity
- INSERM U932 & PSL Research University, France
- Université Paris-Diderot and CNRS, France
- Sorbonne Universités, UPMC Université Paris, France
- Institut Cochin & UMR8104, CNRS & Université Paris Descartes, France
Figures
Figure 1 with 1 supplement
T cell migratory properties and morphology are modulated by substrate stiffness.
(A) Mean instantaneous velocities of migrating T cells on ICAM-1-coated PA-gels of varying stiffness (ncells: 50–100 for each condition from nDonors: 4). (B) Maximum distance travelled by T cells on PA-gels of varying stiffness for a duration of 5 min (ncells: 50–100 for each condition from nDonors: 4). Boxes and whiskers for minimum and maximum are shown. For statistical analysis, unpaired parametric t-tests were performed: ****p-value<0.0001, ***p-value<0.001, **p-value<0.01. (C) Percentage of arrested cells on aCD3+aCD28+ICAM-1 coated PA-gels of varying stiffness. T cell response on glass coated with aCD3+aCD28+ICAM-1 is shown for comparison. Mean values and number of cells per condition are shown (nDonors: 2). (D) Scanning electron microscopy pictures of T cells (representative of ncells: 5 per condition from nDonors: 2) on aCD3+aCD28+ICAM-1-coated substrates for two magnifications (5000x and 20000x). Black scale bars: 2 μm, white scale bars: 500 nm.
Figure 1—figure supplement 1
Characterization of PA-gels and additionnal data on migration.
(A) Measurement of the elastic modulus G´ of a PA-gel containing 5% acrylamide and 0.5% bis-acrylamide. The value associated to a given sample corresponds to the maximum of G´ as a function of the distance between the rheometer plates. The dotted line shows the mean value of the elastic shear modulus G´ = 2212 ± 79 Pa for n = 15 different samples of PA-gels at 5% acrylamide and 0.5% bis-acrylamide. (B) Coating of PA-gels and glass coverslips by biotinylated (b-fibronectin) or non biotinylated fibronectin quantified by immunofluorescence labeling. (C) Biotinylated antibody coating on streptavidin containing PA-gels of varying stiffness and neutravidin-coated glass coverslips quantified by immunofluorescence labeling (nsamples: 14). (D) 5 min tracks of individual CD4+ T lymphoblasts on the 100 kPa gels coated with aCD3+aCD28+ICAM-1. Arrows indicate migrating cells and the arrowhead indicates an arrested cell. Scale bar: 10 μm. (E) Mean instantaneous velocities of migrating T cells on PA-gels of varying stiffness, coated with either ICAM-1 or aCD3+aCD28+ICAM-1, for a duration of 5 min (ncells: 50–100 for each condition from nDonors: 4). Boxes and whiskers for minimum and maximum are shown. For statistical analysis, unpaired parametric t-tests were performed: ****p-value<0.0001.
Figure 2
Gene expression of CD4+ T cells shows a graded response to stiffness.
(A) Principal component analysis reveals that gene expression is modulated by T cell substrate stiffness only in presence of aCD3 (nDonors: 4). (B) Number of genes that displayed differential expression between the conditions with and without aCD3 on PA-gels of varying stiffness. ‘Exclusive’ indicates the genes that are found Up- or Down-regulated only at a given stiffness value. (C) Relative expression of T cell related genes following Affymetrix microarray analysis. Asterisks indicate the presence of these genes in the differential analysis: *** for 0.5 to 100 kPa, ** for 6.4 to 100 kPa, * for 100 kPa only.
Figure 3 with 1 supplement
Stiffness potentiates TCR/CD3-induced transcriptional response.
(A) Enrichment of gene sets on PA-gels of varying stiffness for p-values lower than 0.05, false discovery rates lower than 0.25 and NES values higher than 1.75. (B) Pathway analysis with the GO – BP database for differentially expressed genes between the conditions with and without aCD3 on PA gels of varying stiffness. The number of different pathways (pie-chart) and the top 2 hits of the enriched pathways, along with their negative log adjusted p-value (table), are shown. (C) K-means clustering of genes with different expression profiles on PA-gels of varying stiffness demonstrates three different clusters: one with strong up-regulation in the presence of aCD3 (containing 1022 probes), one with weak down-regulation (containing 4412 probes) and one with weak or no up-regulation (containing 5928 probes). (D) Comparison of the relative changes in gene expression in the presence of aCD3 for the strong up-regulation cluster. The x-axis shows the difference in gene expression for the transition of 0.5 to 6.4 kPa, the y-axis for the transition of 6.4 to 100 kPa and the colour gradient for the transition of 0.5 to 100 kPa.
Figure 3—figure supplement 1
Pathway analysis with the KEGG database for differentially expressed genes between the conditions with and without aCD3 on PA gels of varying stiffness.
https://doi.org/10.7554/eLife.23190.014
Figure 4 with 1 supplement
Cytokine production is sensitive to a wide range of stiffness.
Production of (A) IFNγ (nDonors: 13) and (B) TNFα (nDonors: 10) on PA-gels of varying stiffness. In the presence of aCD3, the aCD3:aCD28 coating ratio was 1:10. (C) FACS plot of CD25 staining. A representative experiment is shown. (D) Mean fluorescence intensity of CD25-stained cells (nDonors: 5). Mean values with standard error are shown. For statistical analysis, paired parametric t-tests were performed: ***p-value<0.001, **p-value<0.01, *p-value<0.05.
Figure 4—figure supplement 1
Cytokine production on PA-gels: additionnal data.
(A) Production of IFNγ on PA-gels of varying stiffness; in the presence of aCD3, the aCD3:aCD28 coating ratio was 1:100 (nDonors: 5).
(B) Production of IFNγ on PA-gels of varying stiffness for non-biotinylated soluble aCD3+aCD28 antibodies at concentrations of 1 + 10 μg/mL respectively (nDonors: 4). (C) Production of IFNγ and TNFα on PA-gels of varying stiffness coated with aCD3+aCD28 only. The aCD3:aCD28 coating ratio was 1:10 (nDonors: 5). (D) Production of IFNγ and IL-2 and percentage of CD69+ cells for memory CD4+ T cells cultured on PA-gels of varying stiffness (nDonors: 3). Mean values with standard error are shown. For statistical analysis, paired parametric t-tests were performed: *p-value<0.05.
Figure 5 with 1 supplement
T cell metabolism is modulated by increased stiffness.
(A) Lactate production in the supernatant of T cell cultures on PA-gels of varying stiffness. (nDonors: 3). (B) Percentage of phospho-rpS6+ T cells cultured on PA-gels of varying stiffness (nDonors: 4). (C) Overall glycolytic capacity of T cells cultured on PA-gels of varying stiffness for 48 hr. The extracellular acidification rate is normalized to the number of cells per well. Mean values with standard error are shown (nDonors: 3). (D) Maximal mitochondrial respiration of T cells following culture on PA-gels of varying stiffness for 48 hr. The oxygen consumption rate is normalized to the number of cells per well. Mean values with standard error are shown (nDonors: 3). For statistical analysis, paired parametric t-tests were performed: **p-value<0.01, *p-value<0.05.
Figure 5—figure supplement 1
Phospho-rpS6 and metabolism: additionnal data.
(A) Representative FACS analysis of phospho-rpS6+ T cells at different culture times on aCD3+aCD28+ICAM-1 coated PA-gels of varying stiffness. (B) Overall glycolytic capacity of T cells following culture on PA-gels of varying stiffness for 24 hr. The extracellular acidification rate is normalized to the number of cells per well. Mean values with standard error are shown (nDonors: 3). (C) Maximal mitochondrial respiration of T cells following culture on PA-gels of varying stiffness for 24 hr. The oxygen consumption rate is normalized to the number of the cells per well. Mean values with standard error are shown (nDonors: 3).
Figure 6 with 1 supplement
Proliferation and cell cycle progression are potentiated by stiffness in response to TCR/CD3 induced activation.
The percentages of cells in G0/G1, S phase and G2/M are shown for (A) 24 hr (nDonors: 4) and (B) 72 hr (nDonors: 4). (C) Percentage of proliferating T cells following 72 hr culture on PA-gels of varying stiffness. (nDonors: 4). Mean values with standard error are shown. For statistical analysis, paired parametric t-tests were performed: **p-value<0.01, *p-value<0.05.
Figure 6—figure supplement 1
Cell cycle and proliferation: additionnal data.
(A) Representative FACS analysis of propidium iodide staining of T cells following 24 hr culture on PA-gels in the presence of aCD3. (B) Representative dot plot of T cell proliferation following 72 hr culture on PA-gels in the presence of aCD3.
Figure 7 with 1 supplement
T cell activation is potentiated by APC mechanical properties.
(A) HeLa-CIITA cells were grown at confluence on fibronectin coated PDMS gels of varying stiffness and were stained with phalloidin (F-Actin, in red) and cell trace violet (CTV, in cyan) (scale bar: 10 μm). (B) Area of HeLa-CIITA cells cultured on 1.5 kPa (453 ± 14 μm2, ncells = 254) and 28 kPa (569 ± 25 μm2, ncells = 215) PDMS gels. Boxes and whiskers for minimum and maximum are shown. For statistical analysis, unpaired t-tests with Welch's correction were performed: ****p-value<0.0001. (C) Young’s modulus of HeLa-CIITA cells cultured on 1.5 kPa (1.43 ± 0.15 kPa, ncells = 13) and 28 kPa (1.72 ± 0.2 kPa, ncells = 15) PDMS gels. Boxes and whiskers for minimum and maximum are shown. Production of D) IFNγ and E) TNFα by T cells interacting with HeLa-CIITA on PDMS gels of varying stiffness in the presence of different TSST-1 superantigen concentrations. The response for individual donors is shown (nDonors: 8). For statistical analysis, paired parametric t-tests were performed: *p-value<0.05.
Figure 7—figure supplement 1
Characterization of APC mechanical properties and T cell activation: additionnal data.
(A) Young’s modulus measurement of HeLa-CIITA cells. Left: principle of measurement and image of the HeLa cell monolayer with the glass probe on top of it (scale bar: 10 μm). When the probe is lowered by a distance D, its tip indents the top of the cell leading to a tip displacement d < D. Thus, the probe is deflected and exerts an elastic force F=k×D-d), where k is the calibrated probe stiffness. Right: force-indentation curve for an individual HeLa cell from a confluent layer, fitted following the Hertz model. (B) FACS plots of HeLa-CIITA cells stained for the APC markers HLA-DR and ICAM-1. Dotted lines display isotype antibody. (C) Mean fluorescence intensity of CD25 stained cells, which were activated for 24 hr by HeLa-CIITA cells grown at confluence on PDMS of varying stiffness in the presence of TSST-1 superantigen (1 ng/mL). The response for individual donors is shown (nDonors: 8). (D) Area of HeLa-CIITA cells cultured on 1.5 kPa (453 ± 14 μm2, ncells = 254), 28 kPa (569 ± 25 μm2, ncells = 215) PDMS gels or glass (473 ± 19 μm2, ncells = 140). Boxes and whiskers for minimum and maximum are shown. (E) Production of IFNγ by T cells interacting with HeLa-CIITA on PDMS gels of varying stiffness or glass in the presence of different TSST-1 superantigen concentrations. The response for individual donors is shown (nDonors: 4). For statistical analysis, paired parametric t-tests were performed: *p-value<0.05.
Author response image 1
Scanning electron microscopy picture of a T cell with migrating morphology on a 0.5 kPa PA-gel coated with aCD3+aCD28+ICAM-1.
https://doi.org/10.7554/eLife.23190.028Videos
Video 1
Live microscopy video of T lymphoblasts on a PA-gel of 0.5 kPa coated with ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.006
Video 2
Live microscopy video of T lymphoblasts on a PA-gel of 6.4 kPa coated with ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.007
Video 3
Live microscopy video of T lymphoblasts on a PA-gel of 100 kPa coated with ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.008
Video 4
Live microscopy video of T lymphoblasts on a PA-gel of 0.5 kPa coated with aCD3+aCD28+ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.009
Video 5
Live microscopy video of T lymphoblasts on a PA-gel of 6.4 kPa coated with aCD3+aCD28+ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.010
Video 6
Live microscopy video of T lymphoblasts on a PA-gel of 100 kPa coated with aCD3+aCD28+ICAM-1-Fc.
https://doi.org/10.7554/eLife.23190.011Tables
Table 1
Composition of PA-gels and equivalent elastic modulus values (mean values with standard error).
| Acrylamide (% w/v) | bis-Acrylamide (% w/v) | Young’s modulus E (Pa) |
|---|---|---|
| 3 | 0.2 | 513 ± 48 |
| 5 | 0.5 | 6416 ± 228 |
| 18 | 0.38 | 100,000 * |
-
*(reported in Trappmann et al., 2012)
Additional files
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Supplementary files 1
Differential Analysis_aCD3 Presence vs absence.
Lists of genes with differential expression comparing absence and presence of aCD3 stimulation on PA-gels of varying stiffness and Venn analysis of the lists.
- https://doi.org/10.7554/eLife.23190.023
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Supplementary files 2
Gene Set Enrichment Analysis_aCD3 Presence vs Absence.
Gene set enrichment analysis results with GO-BP and KEGG databases comparing absence and presence of aCD3 stimulation on PA-gels of varying stiffness and Venn analysis of the results.
- https://doi.org/10.7554/eLife.23190.024
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Supplementary files 3
Differential Analysis_GO-BP.
Pathway analysis results for differentially expressed genes using GO-BP database and Venn analysis of the results.
- https://doi.org/10.7554/eLife.23190.025
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Supplementary files 4
Differential Analysis_KEGG.
Pathway analysis results for differentially expressed genes using KEGG database and Venn analysis of the results.
- https://doi.org/10.7554/eLife.23190.026
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Supplementary files 5
Up-regulated Cluster.
List of genes in the strongly up-regulated cluster and pathway analysis results with GO-BP and KEGG databases.
- https://doi.org/10.7554/eLife.23190.027
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Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity
eLife 6:e23190.
https://doi.org/10.7554/eLife.23190
