Figure 3. Engagement of SLAMF7 triggers an inflammatory program.

A-C) Macrophages were treated with siRNA control or siRNA targeting SLAMF7. They were then potentiated with IFN-γ (10 ng/ml) for 24 hours. A) TNF or B) SLAMF7 expression was quantified relative to macrophages treated with control siRNA only by RT-PCR. C) After potentiation with IFN-γ, macrophages were stimulated with r-SLAMF7 (100 ng/ml) for 2.5h, and TNF was quantified relative to macrophages treated with control siRNA and IFN-γ only by RT-PCR. Data represent mean ± SD of triplicate samples in an experiment representative of at least 2 independent experiments. D-I) Macrophages were potentiated with IFN-γ (10 ng/ml) for 24 hours prior to treatment with a-SLAMF7 (10 μg/ml) or r-SLAMF7 (1 μg/ml) for 4h. D) Differential gene expression for macrophages incubated with a-SLAMF7 (n=3 donors) compared to macrophages treated with only IFN-γ (n=4 donors). E) Gene set enrichment analysis for macrophages after stimulation with a-SLAMF7 or r-SLAMF7 showing the top and bottom 5 GO categories for macrophages after SLAMF7 engagement. F) Heatmap showing z-scores for gene expression values of differentially expressed genes in the msigdb GO:cytokine activity gene set for IFN-γ pre-treated macrophages without additional stimulation (n=4 donors), stimulated with a-SLAMF7 (n=3 donors), or with r-SLAMF7 (n=4 donors). G) Secreted TNF-α, and H) secreted IL-6 after macrophage incubation with a-SLAMF7 or r-SLAMF7 (n=7 donors). I) Release of IL-1β after macrophage incubation with a-SLAMF7 or r-SLAMF7 for 4h followed by treatment with nigericin (10 μM) for 30m (n=5 donors). Data represent mean ± SD. Statistics were calculated with the one-way ANOVA, using Dunnett’s multiple comparisons test. Unstim, unstimulated; a-SLAMF7, anti-SLAMF7 antibody; r-SLAMF7, recombinant SLAMF7 protein; si-Ctrl, control siRNA; si-SLAMF7, siRNA against SLAMF7; NES, normalized expression score; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.