Fig 1: 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).