Figure 4.
Platelet–T reg cell interactions are required for pulmonary T reg cell recruitment and activation. (A) Pneumonia was induced in WT mice by i.t. instillation of K. pneumoniae and sCD40L levels in the plasma were analyzed at the indicated time points (data pooled from two independent experiments with four to five mice in each group; Mann-Whitney test was used for statistical analysis). Isolated WT platelets were incubated with vehicle, sCD40L (20 ng/ml), or thrombin (0.1 U/ml) in vitro. (B and C) Surface P-selectin expression (B) and platelet fibrinogen binding (C) were analyzed by flow cytometry (data pooled from at least two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis). (D) Platelet shape change after sCD40L stimulation was analyzed by light microscopy (data pooled from three independent experiments with three samples in each group). Platelet spreading stages were identified as round (S1), filopodia formation (S2), filopodia and lamellipodia formation (S3), and fully spread (S4). Scale bar, 2 µm. (E) Aggregometry in murine PRP was analyzed in unstimulated samples or after stimulation with sCD40L (20 ng/ml) or thrombin (0.1 or 1.0 U/ml; exemplary graphs from three independent experiments). Isolated WT T reg cells and platelets were coincubated in vitro in the presence or absence of blocking antibodies against P-selectin (clone RB40.34, 5 µg/ml), PSGL-1 (clone 4RA10, 5 µg/ml), or CD40 (clone HM40-3, 5 µg/ml) and left unstimulated or stimulated with sCD40L (20 ng/ml) for 1 h at 37°C. (F) Platelet–T reg cell interactions were analyzed by flow cytometry. (G and H) The concentrations of the cytokines IL-10 (G) and TGFβ (H) in the supernatant from the platelet–T reg cell cocultures were analyzed by ELISA (data pooled from at least two independent experiments with five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Bacterial pneumonia was induced in WT mice and CD40L−/− mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse). Some WT mice received Fab fragments of blocking antibodies against P-selectin (clone RB40.34, 50 µg/mouse), CD40 (clone HM40-3, 50 µg/mouse i.v.), or isotype control 2 d after induction of pneumonia. (I and J) The number of T reg cells (I) and neutrophils (J) in the BAL fluid was analyzed by flow cytometry (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). To conduct RNaseq in the lung during resolution of pulmonary inflammation, single T reg cells, platelet–T reg cell aggregates, and macrophages were isolated from BAL fluid obtained at day 5 after induction of bacterial pneumonia from WT mice and platelet-depleted WT mice. (K and L) Heat map with hierarchical tree (K) and volcano plot (L) comparing differences of RNaseq-based gene expression values in T reg cells after physical binding to platelets.

Platelet–T reg cell interactions are required for pulmonary T reg cell recruitment and activation. (A) Pneumonia was induced in WT mice by i.t. instillation of K. pneumoniae and sCD40L levels in the plasma were analyzed at the indicated time points (data pooled from two independent experiments with four to five mice in each group; Mann-Whitney test was used for statistical analysis). Isolated WT platelets were incubated with vehicle, sCD40L (20 ng/ml), or thrombin (0.1 U/ml) in vitro. (B and C) Surface P-selectin expression (B) and platelet fibrinogen binding (C) were analyzed by flow cytometry (data pooled from at least two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis). (D) Platelet shape change after sCD40L stimulation was analyzed by light microscopy (data pooled from three independent experiments with three samples in each group). Platelet spreading stages were identified as round (S1), filopodia formation (S2), filopodia and lamellipodia formation (S3), and fully spread (S4). Scale bar, 2 µm. (E) Aggregometry in murine PRP was analyzed in unstimulated samples or after stimulation with sCD40L (20 ng/ml) or thrombin (0.1 or 1.0 U/ml; exemplary graphs from three independent experiments). Isolated WT T reg cells and platelets were coincubated in vitro in the presence or absence of blocking antibodies against P-selectin (clone RB40.34, 5 µg/ml), PSGL-1 (clone 4RA10, 5 µg/ml), or CD40 (clone HM40-3, 5 µg/ml) and left unstimulated or stimulated with sCD40L (20 ng/ml) for 1 h at 37°C. (F) Platelet–T reg cell interactions were analyzed by flow cytometry. (G and H) The concentrations of the cytokines IL-10 (G) and TGFβ (H) in the supernatant from the platelet–T reg cell cocultures were analyzed by ELISA (data pooled from at least two independent experiments with five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Bacterial pneumonia was induced in WT mice and CD40L−/− mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse). Some WT mice received Fab fragments of blocking antibodies against P-selectin (clone RB40.34, 50 µg/mouse), CD40 (clone HM40-3, 50 µg/mouse i.v.), or isotype control 2 d after induction of pneumonia. (I and J) The number of T reg cells (I) and neutrophils (J) in the BAL fluid was analyzed by flow cytometry (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). To conduct RNaseq in the lung during resolution of pulmonary inflammation, single T reg cells, platelet–T reg cell aggregates, and macrophages were isolated from BAL fluid obtained at day 5 after induction of bacterial pneumonia from WT mice and platelet-depleted WT mice. (K and L) Heat map with hierarchical tree (K) and volcano plot (L) comparing differences of RNaseq-based gene expression values in T reg cells after physical binding to platelets.

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