Figure 3.

Fas-induced secretome mediates immunosuppression and monocyte chemotaxis via PGE2 activity

(A) CellTrace-stained CD3 T cells were treated with varying amount of Fas-CM or Ut-CM at 37°C, 5% CO₂ for 3 days. CD3/28 Dynabeads were used to stimulated CD3 T cells. Percentage of relative proliferation was calculated by the following formula: (% of V450^low population of samples/% of V450^low population of positive control: CD3 with beads only) × 100%. The amount of (B) IL-2 and (C) IFN-γ in the cell culture medium was assessed using ELISA kits. (D–F) Different amounts of anti-PGE2 neutralizing antibodies (clone 2B5) were added to the Fas-CM and incubated for 30 min at 37°C, 5% CO₂ before adding to stimulated CD3 T cells. Likewise, the amount of (E) IL-2 and (F) IFN-γ in the cell culture medium was assessed. (G) Isolated human monocytes were first resuspended in transwells and incubated for 10 min at 37°C, 5% CO₂. Fas-CM and Ut-CM was diluted 50-fold with complete RPMI and then then added to the 24-well for another 2-h incubation at 37°C, 5% CO₂. Migrated monocyte concentration (cell/μL) was then calculated based on the manufacturer’s manual of CountBright Absolute Counting Beads. Different amount of anti-CCL2/MCP-1, anti-CCL20, and anti-CXCL8/IL-8 neutralizing antibodies (all mouse IgG1) were added to the culture medium and incubated for 20 min at 37°C, 5% CO₂ before adding to the 24-well plates. The same amount of PBS was added as vehicle controls. (H) MSCs were stimulated with anti-Fas CH11 in the presence of caspase inhibitor Z-VAD-FMK (50μM), NF-κB inhibitor BAY 11–7085 (30 μM), or selective COX2 inhibitor NS-398 (100 μM). The level of CCL2 in the cell culture medium was assessed after 24 h treatment using ELISA kit. All results shown are representative of at least three independent experiments. Experimental data were expressed as mean ± SD. One-way ANOVA and post hoc Tukey test were used to compare the mean differences among the samples (∗p values <0.05, ∗∗p values <0.01, ∗∗∗p values <0.001, ∗∗∗∗p values <0.0001, n.s, not significant).