Fig 2: Expansion of CD3-/NKp46+ cells on K562 mbIL-21 feeder cells with human versus canine IL-2. (A) Canine CD3-/NKp46+ cells expanded on K562 Clone 9.mbIL-21 feeder cells with canine IL-2 have significantly increased purity of CD3-/NKp46+ cells at day 21 compared to expansion with human IL-2 (n = 6) representative flow plot shown. Canine donors are depicted by connecting lines. (B) CD3-/NKp46+ cells expanded with canine IL-2 have an increase in yield of CD3-/NKp46+ cells at day 21.

Fig 3: 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 4: CD3-/NKp46+ cells expand rapidly on K562 mbIL-21 feeder cells and express NK-cell associated markers. (A) CD3-/NKp46+ cells expand on K562mbIL-21 feeder cells to make up on average 60.2% of total cells, with contaminating T-cells on average 31.8% (n = 16, median ± IQR shown). Expanded CD3-/NKp46+ cells are CD4- (median = 0.08%) and are significantly decreased in percentage of cells coexpressing CD5 (median = 0.4%, p = 0.037) (open histogram = negative control, black filled = CD3-/NKp46+) and increased in CD8 coexpression (median = 35%, p = 0.005, n = 7; median ± IQR shown). (B) CD3-/NKp46+ cells express mRNA for NKp46, NKp30, Perforin, Granzyme B, NKG2D, DNAM-1, NKp80, NKp44, Ly49, and CD16 (n = 5, all PCR reactions were conducted concurrently). (C) On average, 4.5 × 108 (median = 1.7e8) CD3-/NKp46+ cells are produced at the end of 3 weeks from an average 17,774 CD3-/NKp46+ (median = 7,000) cells at day 0 (shown is median ± IQR) representing a median 20,283-fold expansion. (D) Depletion of T-cells at day 0 significantly increases the purity of CD3-/NKp46+ cells at day 21 (95.8 versus 40.6%) and does not hinder fold expansion (157,281 versus 64,686, n = 3).

Fig 5: Function of CD3-/NKp46+ cells. (A) Sorted CD3-/NKp46+ cells have increased cytotoxicity against the canine osteosarcoma (Gray) cell line when compared to CD3+ T-cells (n = 6). (B) CD3-/NKp46+ cells uniformly kill OSCA78, Abrams, and MDCK. (C) Canine CD3-/NKp46+ cells have titratable, donor-dependent cytotoxicity against four different canine osteosarcoma cell lines: Gray (n = 2), OSCA8 (n = 3), and OSCA40 (n = 3), and the canine thyroid adenocarcinoma cell line (CTAC, n = 3) (mean ± SD for each donor depicted). (D) Expanded CD3-/NKp46+ cells secrete GM-CSF, IFN-?, KC-like, IL-8, IL-10, and TNF-a upon coculture with K562 Clone9.mbIL-21 feeder cells (n = 7, median depicted). When compared to CD3+ T-cells (n = 3) from the same donor and also expanded on K562 mbIL-21, canine CD3-/NKp46+ cells secrete similar cytokines at similar concentrations.