Short-range interactions between fibrocytes and CD8+ T cells in COPD bronchial inflammatory response
- Univ-Bordeaux, Centre de Recherche Cardio-thoracique de Bordeaux, U1045, Département de Pharmacologie, CIC1401, Proteomics Facility, France
- INSERM, Centre de Recherche Cardio-thoracique de Bordeaux, U1045, France
- Univ-Lorraine, Institut Elie Cartan de Lorraine, France
- Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Center for Infection Research (HZI), Germany
- CHU de Bordeaux, Service d’exploration fonctionnelle respiratoire, France
- Univ. Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, France
- Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, France
- CNRS, UMR5164 ImmunoConcEpT, Université de Bordeaux, France
- CHU de Bordeaux, Laboratoire d’Immunologie et Immunogénétique, France
Figures
Figure 1 with 4 supplements
Increased interactions between CD8+ T cells, CD45+ FSP1+ cells in distal airways of chronic obstructive pulmonary disease (COPD) patients.
(A, B) Representative stainings of CD8 (brown, A), CD45 (red, B), and FSP1 (green, B) in distal bronchial tissue specimens from a control subject (left) and a COPD patient (right). The yellow …
Figure 1—figure supplement 1
Detection of CD8+ T cells, CD45+ FSP1+ cells, and quantification of interacting cells, and the minimal distances between the two cell types.
(A) CD8 (brown) staining of a representative bronchus. (B) CD45 (red) and fibroblast-specific protein 1 (FSP1, green) stainings of the same bronchus. (C, D) The left panels show a higher …
Figure 1—figure supplement 2
Spatial distributions of CD8+ T cells and fibrocytes in the peribronchial area.
(A, B) Quantification of the mean minimal distances between CD8+ T cells (A) and between fibrocytes (B) in one specimen/patient. (C, D) Quantification of the mean minimal distances between CD8+ T …
Figure 1—figure supplement 3
Principle of the use of Delaunay triangulation for cluster analysis.
(A) Binary images for CD8 (left panel) and double CD45-FSP1 (right panel) stainings were obtained after segmentation. (B) Images after Delaunay triangulation, were performed on the centers of mass …
Figure 1—figure supplement 4
Relationships between the forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio, the density of fibrocytes, the density of interacting cells, the mean minimal distance between fibrocytes and CD8+ T cells, and the density of fibrocytes-CD8+ T cells clusters.
Relationships between the density of CD45+ FSP1+ cells (A), the density of interacting cells (B), the mean minimal distance between fibrocytes and CD8+ T cells (C), the density of mixed cell …
Figure 2 with 1 supplement
CD8+ T cells from chronic obstructive pulmonary disease (COPD) tissue have increased chemoattractive properties for fibrocytes.
(A) Heatmaps showing the expression of differentially expressed genes with p-value <0.05 of chemokines and chemokine receptors in resting tissular tissue-resident memory T-cells (TRM) and effector …
Figure 2—figure supplement 1
Transcript levels of adhesion genes in CD8+ T cells from chronic obstructive pulmonary disease (COPD) tissues compared to control tissues.
Heatmaps showing the expression of differentially expressed genes with p-value <0.05 of adhesion molecules and adhesion receptors in resting tissular tissue-resident memory T-cells (TRM) and …
Figure 3 with 5 supplements
CD8+ T cells repeatedly contacts fibrocytes and this contact greatly enhances CD8+ T cell proliferation.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A) Representative brightfield images of co-culture between CD8+ T cells and fibrocytes at the …
Figure 3—figure supplement 1
Direct contact between fibrocytes and CD8+ T cells preferentially increases proliferation of naïve CD8+ T cells subset.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, B, C, D) Representative gating strategy for identification of naïve CD8+CD45RA+ cells and memory …
Figure 3—figure supplement 2
CD4+ T cells death and proliferation after 6 days in direct co-culture with fibrocytes.
Prior to co-culture, CD4+ T cells have been either non-activated (‘CD4NA’) or activated (‘CD4A’). (A, B) Representative gating strategy for identification of CD4+ T cells without (w/o) fibrocytes (A)…
Figure 3—figure supplement 3
CD8+ T cells death after 6 days of co-culture with fibrocytes.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, C) Representative gating strategy for identification of dead CD8+ T cells without (w/o) …
Figure 3—figure supplement 4
Direct contact between fibrocytes and CD8+ T cells promotes phenotypic differences in CD8 expression.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, B, D, E) Representative gating strategy for identification of different CD8+ T cells sub-types …
Figure 3—figure supplement 5
The CD8low population appears in co-culture and is distinct from the CD45+ Collagen I+ population.
Prior to co-culture, CD8+ T cells have been activated. (A, B, C, D) Experiment design. Pure CD8+ T cells were characterized by flow cytometry for CD8 expression (A) before being were cultured either …
Figure 4 with 1 supplement
Fibrocytes act as a major promoter of CD8+ T cell proliferation in a CD54 and CD86-dependent manner.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, D, G, J) Representative gating strategy for identification of proliferating CD8+ T cells without …
Figure 4—figure supplement 1
LFA-1 or CD44 blockade is not sufficient to decrease CD8+ T cells proliferation induction.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, C, E, G) Representative gating strategy for identification of proliferating CD8+ T cells without …
Figure 5 with 1 supplement
Fibrocyte-CD8+ T cell interactions alter cytokine production.
Prior to co-culture, CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’). (A, C) Representative gating strategy for identification of CD8+ T cells expressing IFN- γ, TNF-α, …
Figure 5—figure supplement 1
Glucocorticoid drugs significantly decrease fibrocyte-induced TNF-α secretion by CD8+ T cells but not the proliferation induction.
Prior to co-culture, CD8+ T cells have been either non-activated or activated. (A, B, C, D) Comparison of quantifications of CD8+ T cells that have proliferated, removed from co-culture without …
Figure 6
Direct contact between fibrocytes and CD8+ T cells triggers CD8+ T cell cytotoxicity against primary bronchial basal epithelial cells.
(A) Experiment design: CD8+ T cells have been either non-activated (‘CD8NA’) or activated (‘CD8A’) before being co-cultured with fibrocytes. Six days after fibrocytes co-culture, CD8+ T cells were …
Figure 7
Direct contact between fibrocytes and CD8+ T cells favors the acquisition of fibrocyte immune properties.
(A) Experiment design: fibrocytes have been either cultured alone, or with CD8+ T cells that have been previously non-activated (‘CD8NA’) or activated (‘CD8A’). After 6 days of (co)-culture, …
Figure 8 with 2 supplements
A probabilistic cellular automata-type model captures the features of the normal and pathological patterns of cell organization observed in the tissues.
(A) Schematic representation of the probabilities associated with CD8+ T cells (left panel) and fibrocytes (right panel). For each CD8+ T cell, we define a ‘basal’ probability of dying, an …
Figure 8—figure supplement 1
Minimal intercellular distance distributions are similar between in situ analyses and simulations.
Mean frequency distributions of minimal distances (with 7 μm binning) between fibrocytes and CD8+ T cells for control subjects (white) and patients with chronic obstructive pulmonary disease (COPD) …
Figure 8—figure supplement 2
Spatial cellular repartition of cells obtained at the final state of simulations are distinct from random distributions.
Final states of the simulations obtained after 20 years of control dynamics and chronic obstructive pulmonary disease (COPD) dynamics are compared to random distributions with indicated fibrocyte …
Figure 9
The outcomes of therapeutic interventions are predicted by simulations.
(A) Schematic representation of the design used to test therapeutic strategies. Chronic obstructive pulmonary disease (COPD) states were first generated by applying COPD dynamics for 20 years (n=144 …
Figure 10
Proposed model of how fibrocytes interact with CD8+ T cells in the context of chronic obstructive pulmonary disease (COPD).
Fibrocyte chemotaxis towards CD8+ T cells is mainly due to an increased CXCL8 secretion by CD8+ T cells in COPD lungs, and promotes direct contact between both cell types. This interaction triggers …
Appendix 1—figure 1
The lamina propria forms two 2-dimensional crown shapes in the bronchial wall, between the bronchial epithelium and the smooth muscle layer.
Adapted from Dupin et al., 2023.
Appendix 1—figure 2
Schematic representation showing the neighborhood at the site s (shaded blue).
Appendix 1—figure 3
Cell death rules.
σ has to be taken equal to three neighbors. F and C cells are indicated by, respectively, green and pink squares. Adapted from Dupin et al., 2023.
Appendix 1—figure 4
The different cases for C cell proliferation rules.
λ has to be taken equal to three neighbors. F and C cells are indicated by, respectively, green and pink squares. Adapted from Dupin et al., 2023.
Appendix 1—figure 5
C and F cell infiltration rules at the stable state.
F and C cells are indicated by, respectively, green and purple squares. Adapted from Dupin et al., 2023.
Appendix 1—figure 6
Examples of cell infiltration, death, proliferation, and displacement of cells at each time step.
We consider the beginning of the time step . This period is divided into sub-time steps, where is the number of cells at the beginning of period . F and C cells are indicated by, …
Appendix 1—figure 7
F Cell displacement rules.
F and C cells are indicated by, respectively, green and pink squares. has been taken equal to 10–3.
Appendix 1—figure 8
C Cell displacement rules.
F and C cells are indicated by, respectively, green and pink squares. has been taken equal to 10–3. Adapted from Dupin et al., 2023.
Author response image 1
Expression of CXC chemokines in lung CD8+ CD103+ and CD8+ CD103- T cells from patients with COPD (n=18 independent samples) in comparison with healthy control subjects (n=29 independent samples) under resting conditions by Single-Cell RNA sequencing analysis (GEO accession GSE136831).
The heatmaps show the normalized expression of genes (horizontal axes) encoding CXC chemokines. PF4=CXCL4, PPBP = CXCL7.
Videos
Video 1
Two days after adding non-activated CD8+ T cells (bright round cells) on fibrocytes (adherent elongated cells), phase-contrast images of co-culture taken were recorded every 2 min.
A tracked lymphocyte is indicated by a blue dot and its trajectory is shown by a blue line dot (Manual Tracking plugin, Fiji software).
Video 2
Cell dynamics within the peribronchial area, 2 years after the initial time, with control dynamics.
Images of the simulations were recorded every 3 min for 24 hr. CD8+ T cells and fibrocytes are represented, respectively, by pink and green squares. control (resp. COPD) situation.
Video 3
Cell dynamics within the peribronchial area, 2 years after the initial time, with chronic obstructive pulmonary disease (COPD) dynamics.
Images of the simulations were recorded every 3 min for 24 hr. CD8+ T cells and fibrocytes are represented, respectively, by pink and green squares. control (resp. COPD) situation.
Video 4
Cell dynamics within the peribronchial area, 7 years after the initial time, with control dynamics.
Images of the simulations were recorded every 3 min for 24 hr. CD8+ T cells and fibrocytes are represented, respectively, by pink and green squares. control (resp. COPD) situation.
Video 5
Cell dynamics within the peribronchial area, 7 years after the initial time, with chronic obstructive pulmonary disease (COPD) dynamics.
Images of the simulations were recorded every 3 min for 24 hr. CD8+ T cells and fibrocytes are represented, respectively, by pink and green squares. control (resp. COPD) situation.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-CD8 (rabbit monoclonal) | Fisher Scientific | Cat. #:MA5-14548, RRID:AB_10984334 | IHC (1:200) |
| Antibody | Anti-rabbit-HRP (goat polyclonal) | Nichirei Biosciences | Cat. #:414141 F, RRID:N/A | IHC (1:200) |
| Antibody | Anti-CD45 (mouse monoclonal) | BD Biosciences | Cat. #:555480, RRID: AB_395872 | IHC (1:50) |
| Antibody | Anti-FSP1 (rabbit polyclonal) | Agilent | Cat. #:A5114, RRID: AB_2335679 | IHC (1:200) |
| Antibody | Anti-mouse-Alexa568 (goat polyclonal) | Fisher Scientific | Cat. #:A-11004, RRID:AB_2534072 | IHC (1:50) |
| Antibody | Anti-rabbit-Alexa488 (goat polyclonal) | Fisher Scientific | Cat. #:A-11008, RRID:AB_143165 | IHC (1:200) |
| Antibody | Anti-LFA-1 (mouse monoclonal) | BioLegend | Cat. #:301233, RRID:AB_2832576 | Blocking experiment (1 µg/mL) |
| Antibody | Anti-CD54 (mouse monoclonal) | Fisher Scientific | Cat. #:15247027, RRID:N/A | Blocking experiment (10 µg/mL) |
| Antibody | Anti-CD86 (mouse monoclonal) | Fisher Scientific | Cat. #:15297097, RRID:N/A | Blocking experiment (10 µg/mL) |
| Antibody | Anti-CD44 (rabbit monoclonal) | Fisher Scientific | Cat. #:15266957, RRID:N/A | Blocking experiment (10 µg/mL) |
| Antibody | Anti-CXCL8 (mouse monoclonal) | BioTechne | Cat. #:MAB208-100, RRID:N/A | Blocking experiment (1 µg/mL) |
| Antibody | Anti-CD4-PerCP-Vio700 (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-113-228, RRID:AB_2726039 | FC (1:50) |
| Antibody | Anti-CD8-PerCP-Vio700 (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-110-682, RRID:AB_2659249 | FC (1:50) |
| Antibody | Anti-CD45RA-FITC (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-113-365, RRID:AB_2726135 | FC (1:50) |
| Antibody | Anti-granzyme-APC (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-099-780, RRID:AB_2651900 | FC (1:20) |
| Antibody | Anti-TNF- α-PE (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-110-066, RRID:AB_2654213 | FC (1:20) |
| Antibody | Anti IFN- γ-APC (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-113-496, RRID:AB_2751119 | FC (1:20) |
| Antibody | Anti-IL-17-PE-Cy7 (mouse monoclonal) | Miltenyi Biotec | Cat. #:130-120-413, RRID:AB_2752086 | FC (1:20) |
| Antibody | Anti-IL-10-PE (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-112-728, RRID:AB_2652318 | FC (1:20) |
| Antibody | Anti-Collagen Type I-FITC (mouse monoclonal) | Sigma Aldrich | Cat. #:FCMAB412F, RRID:AB_11204160 | FC (1:50) |
| Antibody | Anti-CD45-APC (mouse monoclonal) | BD Pharmingen | Cat. #:555485, RRID:AB_398600 | FC (1:10) |
| Antibody | Anti-CXCR1-PE (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-115-879, RRID:AB_2727234 | FC (1:50) |
| Antibody | Anti-CXCR2-APC-Cy7 (human recombinant monoclonal) | Miltenyi Biotec | Cat. #:130-119-571, RRID:AB_2733103 | FC (1:50) |
Additional files
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Supplementary file 1
Association between the density of fibrocytes and clinical characteristics.
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; LFT, lung function test; RV, residual volume; TLCO, Transfer Lung capacity of Carbon monoxide, PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure; WA, mean wall area; LA, mean lumen area, WA%, mean wall area percentage; WT, wall thickness; LAA, low-attenuation area; MLA E or I, mean lung attenuation value during expiration or inspiration. MLA I-E, the difference between inspiratory and expiratory mean lung attenuation value. %CSA<5, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels less than 5 mm2; %CSA5–10, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels between 5 and 10 mm2; CSN<5, number of vessels less than 5 mm2 normalized by total lung area; CSN5-10, number of vessels between 5 and 10 mm2 normalized by total lung area; NR: not relevant. The correlation coefficient (r), 95% confidence interval, and significance level (p value), were obtained by using nonparametric Spearman analysis.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp1-v1.docx
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Supplementary file 2
Association between the density of CD8+ T cells and clinical characteristics.
FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; LFT, lung function test; RV, residual volume; TLCO, Transfer Lung capacity of Carbon monoxide, PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure; WA, mean wall area; LA, mean lumen area, WA%, mean wall area percentage; WT, wall thickness; LAA, low-attenuation area; MLA E or I, mean lung attenuation value during expiration or inspiration. MLA I-E, the difference between inspiratory and expiratory mean lung attenuation value. %CSA<5, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels less than 5 mm2; %CSA5–10, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels between 5 and 10 mm2; CSN<5, number of vessels less than 5 mm2 normalized by total lung area; CSN5-10, number of vessels between 5 and 10 mm2 normalized by total lung area; NR: not relevant. The correlation coefficient (r), 95% confidence interval, and significance level (p value), were obtained by using nonparametric Spearman analysis.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp2-v1.docx
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Supplementary file 3
Association between interacting cell density and clinical characteristics.
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; LFT, lung function test; RV, residual volume; TLCO, Transfer Lung capacity of Carbon monoxide, PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure; WA, mean wall area; LA, mean lumen area, WA%, mean wall area percentage; WT, wall thickness; LAA, low-attenuation area; MLA E or I, mean lung attenuation value during expiration or inspiration. MLA I-E, the difference between inspiratory and expiratory mean lung attenuation value. %CSA<5, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels less than 5 mm2; %CSA5–10, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels between 5 and 10 mm2; CSN<5, number of vessels less than 5 mm2 normalized by total lung area; CSN5-10, number of vessels between 5 and 10 mm2 normalized by total lung area; NR: not relevant. The correlation coefficient (r), 95% confidence interval, and significance level (p value), were obtained by using nonparametric Spearman analysis.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp3-v1.docx
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Supplementary file 4
Association between the mean minimal distance between fibrocytes and CD8+ T cells and clinical characteristics.
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; LFT, lung function test; RV, residual volume; TLCO, Transfer Lung capacity of Carbon monoxide, PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure; WA, mean wall area; LA, mean lumen area, WA%, mean wall area percentage; WT, wall thickness; LAA, low-attenuation area; MLA E or I, mean lung attenuation value during expiration or inspiration. MLA I-E, the difference between inspiratory and expiratory mean lung attenuation value. %CSA<5, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels less than 5 mm2; %CSA5–10, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels between 5 and 10 mm2; CSN<5, number of vessels less than 5 mm2 normalized by total lung area; CSN5-10, number of vessels between 5 and 10 mm2 normalized by total lung area; NR: not relevant. The correlation coefficient (r), 95% confidence interval, and significance level (p value), were obtained by using nonparametric Spearman analysis.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp4-v1.docx
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Supplementary file 5
Association between the density of mixed cell clusters and clinical characteristics.
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; LFT, lung function test; RV, residual volume; TLCO, Transfer Lung capacity of Carbon monoxide, PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure; WA, mean wall area; LA, mean lumen area, WA%, mean wall area percentage; WT, wall thickness; LAA, low-attenuation area; MLA E or I, mean lung attenuation value during expiration or inspiration. MLA I-E, the difference between inspiratory and expiratory mean lung attenuation value. %CSA<5, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels less than 5 mm2; %CSA5–10, percentage of total lung area taken up by the cross-sectional area of pulmonary vessels between 5 and 10 mm2; CSN<5, number of vessels less than 5 mm2 normalized by total lung area; CSN5-10, number of vessels between 5 and 10 mm2 normalized by total lung area; NR: not relevant. The correlation coefficient (r), 95% confidence interval, and significance level (p value), were obtained by using nonparametric Spearman analysis.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp5-v1.docx
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Supplementary file 6
Multivariate analysis of FEV1/FVC.
FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp6-v1.docx
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Supplementary file 7
Patient characteristics (for tissular CD8+ T cells purification).
Plus–minus values are means ± SD. PFT, pulmonary function test; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp7-v1.docx
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Supplementary file 8
Patient characteristics (for circulating CD8+/CD4+ T cells and fibrocyte precursors purification).
Plus–minus values are means ± SD. PFT, pulmonary function test; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; PaO2, partial arterial oxygen pressure, PaCO2, partial arterial carbon dioxide pressure.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp8-v1.docx
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Supplementary file 9
Patient characteristics (for basal bronchial epithelial cell purification).
Plus–minus values are means ± SD. PFT, pulmonary function test; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp9-v1.docx
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Supplementary file 10
Definition of the notations and parameters of the mathematical model.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp10-v1.docx
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Supplementary file 11
Numerical values of parameters depending in control and COPD situations.
- https://cdn.elifesciences.org/articles/85875/elife-85875-supp11-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85875/elife-85875-mdarchecklist1-v1.pdf
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Source code 1
Program to simulate CD8+ T cells and fibrocytes evolution in the peribronchial area.
- https://cdn.elifesciences.org/articles/85875/elife-85875-code1-v1.zip
