Figure 3.

Sera from PPRV-immune sheep can mediate ADCC against PPRV infected cells. (A) Gating strategy for flow cytometry-based ADCC assays. FSC/SSC dot plots discriminated debris and allowed for event gating. Target cells (PKH67⁺ events) were then gated to perform the viability analysis with propidium iodide (PI) staining. Gating for dead cells was based on PI unstained samples. Maximum target cell death was measured by addition of 0.2% saponin. (B,C) Examples of cytometry dot-plots (duplicates from three independent experiments for each sera) used to measure specific lysis of target cells by effector cells. Representative dot plots of (B) BTV- or (C) PPRV-infected B cell enriched fraction (target cells) cultured in the absence of effector cells (spontaneous cell death) or in presence of effector cells (autologous NK/T cell fraction) after incubation with anti-MHC-I antibody as positive control, no serum, naïve serum, or PPRV-immune serum. (D) Mean (±SEM) specific lysis in three independent experiments of BTV-infected cells incubated with anti-MHC-I antibody as positive control, no serum, naïve, or BTV-immune serum at different effector to target cell ratio (E: T). ^***p < 0.001 Two-way ANOVA with Dunnett's post-test (anti-MHC-I vs. controls (no serum or naïve serum). (E) Mean (±SEM) specific lysis in three independent experiments against PPRV-infected cells incubated with anti-MHC-I antibody as positive control, no serum, PPRV-naïve serum A, B or C, or PPRV-immune serum A, B or C at different effector to target cell ratio (E: T). ^**p < 0.01; ^***p < 0.001 Two-way ANOVA with Dunnett's post-test [anti-MHC-I and immune serum vs. controls (no serum and naïve serum)].