Increased interactions between CD8+ T cells, CD45+ FSP1+ cells in distal airways of 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 arrowheads indicate fibrocytes, defined as CD45+ FSP1+ cells. (C) Quantification of CD8+ T cells and fibrocyte densities (normalized by the sub-epithelial area) in one specimen/patient (n=20 control subjects, n=12 patients with COPD). (D) Merged segmented images for CD8 and CD45-FSP1 staining, showing CD8+ T cells and CD45+ FSP1+ cells respectively in magenta and yellow. The white arrows indicate interacting cells, detected by dilatation of CD8 positive particle. (E) Table showing the correspondence between dilatations in pixels and µm. F, Quantification of interacting cells densities (normalized by the sub-epithelial area) in one specimen/patient, using the different dilatations sizes (E). (G) Distance maps built from the binary image produced from CD8 staining, with FSP1+ CD45+ cells (blue outlines). (H) Quantification of the mean minimal distances between fibrocyte and CD8+ T cells in one specimen/patient. (I) Cluster analysis performed by Delaunay triangulation on segmented images for CD8 and CD45-FSP1 staining, followed by the application of a threshold value (40 μm) above which connections are not kept. CD8+ T cells and fibrocytes appear respectively with green and red dots, connections are shown in blue. (J) First row: densities of clusters containing exclusively CD8+ T cells (“CD8 clusters”), fibrocytes (“Fib clusters”) and both cell types (“CD8-Fib clusters”) normalized by the sub-epithelial area) in one specimen/patient. Second row: mean number of cells by cluster. (C, F, H, J) The medians are represented as horizontal lines, n=20 specimens from control subjects, n=12 specimens from patients with COPD. *: P<0.05, **:P<0.01; ***: P<0.001. unpaired t-tests or Mann Whitney tests.

CD8+ T cells from 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 memory T-cells (TEM) from patients with COPD (n=2 independent samples) in comparison with control subjects (n=2 independent samples) (GEO accession GSE61397). Expression values are expressed as Z-score transformed transcript count. (B) Migration experiment design. (C) Migration of fibrocytes from patients with COPD in response to CD8+ T cells supernatants from control subjects (CtlNS) or COPD patients (COPD). n=6 independent experiments (D) CCL2, CXCL2 and CXCL8 levels in CD8+ T cells supernatants from non-smoking control subjects (CtlNS) or patients with COPD (COPD) using BioPlex (CCL2, CXCL2) or ELISA (CXCL1, 3, 5, 6, 8). n=4 CtlNS samples, n=4 COPD samples. * P < 0.05, Mann–Whitney test. (E-F), Migration of fibrocytes from patients with COPD in response to CD8+ T cells supernatants from control subjects (CtlNS) or COPD patients (COPD), in the presence of 200nM Reparixin (+) or corresponding vehicle (-) (E), and in the presence of 1µg/mL blocking antibody for CXCL8 (+) or control antibody (-) (F). n=6 independent experiments * P < 0.05, Wilcoxon matched pairs test.

CD8+ T cell repeatedly contact 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 initial state of the acquisition (t0) and after 12 hours (t12h) in both conditions of activation. The orange arrowheads indicate CD8+ T cells (bright round-shaped cells) in contact with fibrocytes (elongated adherent cells). (B) Quantifications of the proportion of fibrocyte-interacting CD8+ T cells at t0 and t12h in both conditions of activation. (C) Top panel: typical CD8+ T cells trajectory (blue) relatively to a fibrocyte (elongated adherent cell) for a period of 140 min. Bottom panel: speed (µm/min) over time for the tracked CD8+ T cell. Short-lived (<12 min, n=4) and longer-lived (>32 min, n=1) contacts are represented respectively in light and dark orange. (D) Comparison of the mean speed of individual CD8+ T cells measured in the absence (“Vfree”) or presence (“Vcont”) of contact with fibrocytes in both conditions of activation. (E) Mean frequency distributions of contact time duration (with 4 min binning) between CD8+ T cells and fibrocytes for CD8NA (black) and CD8A (gray). Error bars indicate standard error of the mean. Two-way ANOVA (F-H) Dot plots representing spatiotemporal variables measured for each individual CD8+ T cell tracked over 12h. Each dot represents one cell. (F) Contact coefficient. (G) Mean speed of CD8+ T cells measured in the absence of contact with fibrocytes (“Mean free speed”). (H) Mean speed of CD8+ T cells measured in the presence of contact with fibrocytes (“Mean contact speed”). (A-H) n=2 independent experiments, n=10 movies by experiments, n=10 CD8+T cells tracked by movies. (I, J, M, N) Representative gating strategy for identification of CD8+ T cells without (w/o) fibrocytes (I, M) or with (w) fibrocytes (J, N) in indirect (I, J) or direct (M, N) co-culture. Left panels: dot plots represent representative CD8-PerCP-Cy5-5 fluorescence (y-axis) versus side scatter (SSC, x-axis) of non-adherent cells removed from the culture. Right panels: histograms represent representative cell count (y-axis) versus Cell Trace-Pacific Blue fluorescence (x-axis). The distinct fluorescence peaks correspond to the different generations of CD8+ T cells. The gate and the percentage indicate cells that have proliferated. (K, O) Comparison of manual count of non-adherent cells removed from co-culture without fibrocyte (“CD8”) and with fibrocyte (“CD8+F”). L, (P) Comparison of quantifications of CD8+ T cells that have proliferated, removed from co-culture without fibrocyte (“CD8”) and with fibrocyte (“CD8+F”). (I-P) n=6 independent experiments. (B, D, F, G, H, K, L, O, P) Medians are represented as horizontal lines. * P < 0.05, ** P < 0.01, *** P < 0.001. (B, D, K, L, O, P) Wilcoxon matched pairs test. (F, G, H) Mann Whitney tests.

Fibrocytes act as a major promoter on 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 (w/o) fibrocytes (A, G) or with (w) fibrocytes (D, J) using neutralizing CD54-mAb (A, D) or neutralizing CD86-mAb (G, J) and respective control isotype. Histograms represent representative cell count (y-axis) versus Cell Trace-Pacific Blue fluorescence (x-axis). The distinct fluorescence peaks correspond to the different generations of CD8+ T cells. The gate and the percentage indicate cells that have proliferated. (B, E, H, K) Comparison of manual count of non-adherent cells removed from co-culture treated with neutralizing CD54-mAb or control isotype (Iso) (B, E) and neutralizing CD86-mAb or control isotype (Iso) (H, K). (C, F, I, L) Comparison of quantifications of CD8+ T cells that have proliferated, removed from co-culture treated with neutralizing CD54-mAb (C, F) or neutralizing CD86-mAb (I, L) and respective control isotype. n=6 independent experiments. Medians are represented as horizontal lines. * P < 0.05, Wilcoxon matched paired tests.

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, granzyme, IL-10 and IL17 without (w/o) fibrocytes (left panel) or with (w) fibrocytes (right panel) in indirect (A) or direct (C) co-culture. (B, D) Quantifications of CD8+ T cells expressing IFN-γ, TNF-α, both, granzyme and IL-10 after co-culture without fibrocytes (CD8NA/CD8A) or with fibrocytes (CD8NA/CD8A +F) in indirect (B) or direct (D) co-culture. (E) TNF-α concentrations in supernatants from co-cultures without fibrocytes (CD8NA/CD8A), with fibrocyte (CD8NA/CD8A +F), and only with fibrocytes (F) as control, for direct co-cultures. n=6 independent experiments. * P < 0.05, Wilcoxon matched paired tests or Friedman tests.

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. 6 days after fibrocytes co-culture, CD8+ T cells were transferred and cultured with primary bronchial basal epithelial cells during 6 hours. (B, C) Representative gating strategy for identification of CD8 T cells expressing CD107a without (w/o) fibrocytes (left panels) or with (w) fibrocytes (right panels). Histograms represent representative cell count (y-axis) versus CD107a fluorescence (x-axis) for total (B) and extracellular expression (C). (D, E) Comparison of quantifications of CD8+ T cells expressing the CD107a, removed from co-culture without fibrocyte (“CD8”) and with fibrocyte (“CD8+F”) for total (D) and extracellular (E) expressions. (F) Representative brightfield images of primary basal epithelial cells co-cultured with CD8+ T cells for 6 hours, following their previous co-culture without (CD8) or with fibrocytes (CD8+F) for 6 days. (G) Representative gating strategy for identification of apoptotic primary bronchial epithelial basal cells exposed to CD8+ T cells which were previously co-cultured without (CD8, left panel) or with fibrocytes (CD8+F, right panel). Dot plots represents representative Propidium iodure fluorescence (y-axis) versus Annexin fluorescence (x-axis). (H) Comparison of quantifications of apoptotic primary bronchial epithelial basal cells exposed to CD8 T cells which were previously co-cultured without (CD8) or with fibrocytes (CD8+F). (D, E, H) Medians are represented as horizontal lines. * P < 0.05, Wilcoxon matched paired tests.

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, fibrocyte proteins have been extracted for proteomic analyses. (B) Top 10 Canonical Ingenuity Pathways significantly altered in fibrocytes co-cultured with non-activated CD8+ T cells or activated CD8+ T cells vs fibrocytes cultured alone (“FNA vs F”, left graph, “FA vs F”, right graph, respectively, n=4 for each condition), ranked by Z-score, obtained by Gene Set Enrichment Analysis. (C) Heatmaps of significantly differentially regulated proteins in FNA vs F and FA vs F, including proteins related to antigen presentation, co-stimulation and adhesion, remodeling, IFN-γ signaling and differentiation. The color scale indicates the log2 fold changes of abundance for each protein.

A probabilistic cellular automata type model captures the features of the normal and pathological patterns of cell organisation 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 pdc of dying, an increased probability pdc+ of dying when the CD8+ T cell has many other CD8+ T cells in its neighborhood, a “basal” probability pc of dividing, an increased probability pc/F of dividing when the CD8+ T cell has fibrocytes in its neighborhood, a probability Pc (s, s′) of moving from a site s to a neighboring site s’, a probability pistaF to be infiltrated at the stable state and a probability piexaC to be infiltrated during exacerbation. For each fibrocyte, we define a probability pdF of dying, a probability pF of dividing, a probability PF (s, s′) of moving from a site s to a neighboring site s’, a probability pistaF to be infiltrated at the stable state and a probability piexaF to be infiltrated during exacerbation. The ≠ symbol indicates parameters whose numerical value differs from control to COPD situation. (B) Selected representative pictures for initial state and final states after 20 years of control and COPD dynamics. Images surrounded by black squares: higher magnifications of peribronchial area. CD8+ T cells and fibrocytes are represented respectively by pink and green squares. (C) CD8+ T cells (left) and fibrocyte (right) densities. (D) Interacting cells densities of interacting cells. (E) Mean minimal distances between fibrocyte and CD8+ T cells. (F) CD8+ T cells-fibrocytes-containing clusters (“CD8-Fib clusters”) densities. (G) mean number of cells by CD8-Fib clusters. (C-G), n=160 simulations for each situation. The medians are represented as horizontal lines. The equivalent measurements measured on patient’s tissues are represented by gray boxes (25th to the 75th percentile) and whiskers (min to max). ***: P<0.001. unpaired t-tests or Mann-Whitney tests. (H, I) Mean kinetics of the populations of CD8+ T cells and fibrocytes in control and COPD situation in silico. Standard deviations are indicated in gray, n=160 simulations. Left panels: Nx−1,x(ic) and Nx−1,x(iF) are the number of CD8+ T cells (resp. fibrocytes) that have infiltrated the peribronchial area for the month x, relatively to the surface of interest. For fibrocytes, the infiltration at the stable state and during exacerbation are indicated respectively in green and light green. For control situation, there is no infiltration by exacerbation. Midde panels: Nx−1,x(pc) is the number of CD8+ T cells that have proliferated for the month x, relatively to the surface of interest. Basal duplication, fibrocyte-induced duplication and total duplication are indicated respectively in pink, light pink and black. Right panels: Nx−1,x(dc) and Nx−1,x(dF) are the number of CD8+ T cells (resp. fibrocytes) that have died for the month x, relatively to the surface of interest. For CD8+ T cells, basal death, T cell-induced death and total death are indicated respectively in pink, light pink and black. (J) Graphs showing the variations of the mean densities of CD8-Fib clusters over time in control (light purple) and COPD situation (dark purple).

The outcomes of therapeutic interventions are be predicted by simulations.

(A) Schematic representation of the design used to test therapeutic strategies. COPD states were first generated by applying COPD dynamics during 20 years (n=144 simulations). Then, different dynamics were applied during 7 years: a COPD dynamics (corresponding to the placebo condition), a control dynamics (corresponding to an ideal treatment able to restore all cellular processes, “Total inhibition”), and modified COPD dynamics (corresponding to CD54, CXCR1/2 and dual inhibitions with alterations of the probability pc/F of dividing when the CD8+ T cell has fibrocytes in its neighbourhood, the probability PF for a fibrocyte to move, and both, respectively). (B) CD8+ T cells (left), fibrocyte (middle) and CD8+ T cells-fibrocytes-containing clusters (“CD8-Fib clusters”, right) densities at the final state (t=7 years). The medians are represented as horizontal lines. One-way ANOVA with Tukey’s post-tests. P-values below 0.05 with Tukey’s post-tests are indicated on the graphs, except for the comparisons between the condition “Total inhibition” and the other conditions of inhibition, that are not indicated. (C-D) Mean kinetics of the populations of CD8+ T cells and fibrocytes in the different conditions (t=0 to 7 years). Left panels: Nx−1,x(ic) and Nx−1,x(iF) are the number of CD8+ T cells (resp. fibrocytes) that have infiltrated the peribronchial area for the month x, relatively to the surface of interest. For fibrocytes, the infiltration at the stable state and during exacerbation are indicated respectively in green and light green. For the total inhibition situation, there is no infiltration by exacerbation. Midde panels: Nx−1,x(pc) is the number of CD8+ T cells that have proliferated for the month x, relatively to the surface of interest. Basal duplication, fibrocyte-induced duplication and total duplication are indicated respectively in pink, light pink and black. Right panels: Nx−1,x(dc) and Nx−1,x(dF) are the number of CD8+ T cells (resp. fibrocytes) that have died for the month x, relatively to the surface of interest. For CD8+ T cells, basal death, T cell-induced death and total death are indicated respectively in pink, light pink and black. (E) Mean kinetics of the densities of CD8-Fib clusters in the different conditions (t=0 to 7 years). (C-E) Standard deviations are indicated in gray, n=144 simulations.

Proposed model of how fibrocytes interact with CD8+ T cells in the context of 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 CD8+ T cell proliferation, cytokine production and cytotoxic activity. The interaction and its consequences might be further increased by a reinforcement of IFN-γ signaling and expression of molecules belonging to the immune synpase, from the fibrocyte side.