Fig 2: NKp46- NK cells in canines and humans are CD16low/-, retain NK cell phenotype and function, and NKp46 is inducible. CD3-/CD21-/CD14-, NKp46- (Null) cells are present in canine PBMC (A) as described for expanded cells in Figure 3A. Null cells consist of on average 5.1% of lymphocytes in PBMC, and 5.7% of expanded cells (median ± IQR shown, day 0 n = 8, day 21 n = 12). (B) The addition of null cells to fold expansion calculation decreases mean fold expansion by 50% (n = 8, median ± IQR shown). (C) Null cells have negligible expression of CD4, CD5, and CD8 in PBMC, but increase CD8 expression with expansion similar to CD3-/NKp46+ cells (3.1–52.4%, n = 7, median ± IQR shown). (D) NKp46 can be induced in Null cells after 5 days coculture with K562 Clone 9.mbIL-21 feeder cells, while NKp46 expression is constant in sorted CD3-/NKp46+ cells (n = 7). (E) Expanded Null cells express similar levels of DNAM-1, Perforin, and Granzyme B, but decreased expression of NKp46, NKG2D, NKp44, and CD16, compared to CD3-/NKp46+ cells (mean ± SD shown). (F) Expanded Null cells have decreased cytotoxicity against Gray and melanoma-12 [n = 9, mel-12 (in black) = 4, Gray = 6 (in blue), individual donors designated by unique symbols, median ± IQR shown], but similar cytokine secretion (n = 3) compared to CD3-/NKp46+ cells (n = 7, median depicted). (G) NKp46- NK cells are present in primary (mean = 21%) and K562 Clone 9.mbIL-21-expanded (mean = 21.7%) human NK cells (n = 4, median ± IQR shown). (H) Spade clustering analysis of primary and mbIL-21 expanded human NK cells for CD56, NKp46, NKp30, NKp44, NKG2C, NKG2D, and NKG2A revealed that NKp46 and CD16 cluster together (outlining delineates cells that stain positive for the indicated marker) and (I) NKp46- cells have decreased expression of CD16, NKp44, DNAM-1, and NKG2D (n = 4, median ± IQR shown, whiskers are minimum to maximum).

Fig 3: 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 4: 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.