Fig 1: Evs derived from RIG-I stimulated cells express enhanced levels of the NKp30-ligand BAG6. (A) CFSE labeled EVs (EV protein amount: 10 µg/mL) induced by 3pRNA (RIG-I-EVs) versus ctrl RNA (ctrl-EVs) were incubated with PBMCs and 24 h later CFSE staining of NK cells (CD3 negative, CD56 positive) or CD3 positive lymphocytes (CD3+ cells) were determined by flow cytometry (n = 3). (B) D04mel cells were transfected with 3pRNA (RIG-I-EVs) or ctrl RNA (ctrl-EVs) and the expression of MIC A/B, ULBP 1/2/3, Vimentin, B7-H6 and BAG6 on EVs was analyzed by flow cytometry (filled gray: isotype, dashed: ctrl-EVs, black line: RIG-I-EVs). One representative of four independent experiments is shown. (C) EVs induced by 3pRNA (RIG-I-EVs) vs. ctrl RNA (ctrl-EVs) were analyzed for binding of NKp30-fc by flow cytometry. Histogram shows one representative experiment (left, filled gray: isotype, dashed: ctrl-EVs, black line: RIG-I-EVs) and graph (right) shows quantification of x-fold induction of the geometric mean normalized to CD9 (n = 4). (D) Expression level of BAG6 on D04mel cells (left) or D04mel derived EVs (right) after transfection with 3pRNA or ctrl RNA was determined by flow cytometry (n = 5). (E) Purified EVs from melanoma (Ma-Mel-86c) cells induced by 3pRNA (RIG-I-EVs) versus ctrl RNA (ctrl-EVs) were analyzed for BAG6 expression on the surface by flow cytometry. Graphs show % induction of the geometric mean normalized to CD9 and s.e.m. of at least four independent experiments. (F) Exosomes from cells with siRNA mediated control knock down (ctrl kd) or RIG-I knock down (RIG-I kd) were analyzed for BAG6 expression on the surface by flow cytometry in response to 3pRNA (RIG-I-EVs) vs. ctrl RNA (ctrl-EVs). Graph shows geometric mean of BAG6 relative to CD9 (n = 4). All error bars reflect mean ± s.d. *, ** and *** indicates p < 0.05, p < 0.01 and p < 0.001.
Fig 2: BAG6-positive RIG-I-EVs induce enhanced NK cell cytotoxicity in vitro. (A) PBMCs were incubated over night with RIG-I-EVs versus ctrl-EVs (EV protein amount: 10 µg/mL) and CD69 expression on NK cells (CD3 negative, CD56 positive) was determined by flow cytometry (n = 3). (B) Primary purified NK cells were left untreated (PBS) or incubated with 100 µg/mL (protein amount) RIG-I-EVs vs. ctrl-EVs for 36 h with (aNKp30) or without blocking (Isotype control antibody (AB)) of NKp30 (clone P30-15). Expression of CD69 (MFI) on NK cells after incubation with EVs was measured and normalized to PBS (n = 3). (C+D) Done as described in (B). (C) Cytotoxicity against untreated melanoma cells (D04mel) was assessed by europium release assay and results were normalized to 100% lysis by RIG-I-EVs in different E/T (n = 3). (D) Instead of NKp30 on NK cells, BAG6 was blocked on EVs with anti-BAG6 (aBAG6). Results were normalized to 100% lysis by RIG-I-EVs in an E/T-ratio of 5:1 (n = 3 for ctrl- and RIG-I-EVs + aBAG6, n = 2 for RIG-I-EVs + Isotype ctrl antibody). All error bars reflect mean ± s.d. *, ** and *** indicates p < 0.05, p < 0.01 and p < 0.001.
Supplier Page from R&D Systems, a Bio-Techne Brand for NKp30/NCR3 Fc Chimera Protein
Available conjugates: Sizes Available: 25 ug