Canine Lymphokine Activated Killer Cells Respond to Human Cytokines and Can Target Dog Osteosarcoma Cells. Dog PBMCs were obtained from healthy dogs and laboratory beagles. Adherent lymphocytes were isolated by standard techniques and cultured with short term rhIL-12/15/18 for 24 h followed by co-culture with low dose rhIL-2 (100 IU/mL) for 7 days. Cells were assessed for expansion, viability, and cytotoxicity at various time points. A. From 4 donors, the mean number of ALAKs at day 0 was 12 × 106 ALAKs. After 7 days in culture, the mean number of recovered ALAKs was 23 ± 9.8 × 106 cells. B. After 7 days in culture, the mean fold expansion of ALAKs was 1.8 ± 0.3. C. Mean viability decreased from 97.7 ± 1.8% on day 0 to 92.3 ± 4.7% on day 7. D. Using PBMCs from a 4-year old healthy unknown breed, we observed that cytotoxicity against OSA-1 targets at day 7 was significantly greater after co-culture with recombinant human cytokines IL-12 (10 ng/mL), IL-15 (10 ng/mL), and IL-18 (10 ng/mL) compared to rhIL-2 alone (5000 IU/mL). E. Using ALAKS expanded with rhIL-12/15/18 from a healthy 7-year old Rat Terrier, we performed a 12–16 h killing assay at the indicated effector:target ratios with OSCA-32. Dose-dependent cytotoxicity was again observed. **** P < 0.0001 via one-way ANOVA with Tukey’s post-test. (TIFF 104 kb
