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. 2020 Feb 21;15(2):e0224775. doi: 10.1371/journal.pone.0224775

Fig 2. Canine NK cells are enriched in NKp46 and Granzyme B expression with time-dependent changes in cytotoxic function.

Fig 2

(A) Canine NK cells were isolated from peripheral blood mononuclear cells via CD5 antibody depletion (left), thereby enriching for a CD3-NKp46+ population of cells (right). (B) Ex vivo expansion with an irradiated feeder cell line (human K562 leukemia line transduced with 4-1BBL and membrane bound IL-21) yields a CD3 population of cells that are markedly positive for NKp46+ (left) and Granzyme B+ (right) after 14 days of stimulation. For panels A and B, representative flow cytometry plots from 10 study dogs and 6 healthy beagles are shown. (C) The cytotoxicity of NK cells was assayed at multiple time points (using CTAC cells as targets) and compared to fresh PBMCs (incubated with 100 IU/mL rhIL-2) in 12–16 hour killing assays. Data from one experiment performed in triplicate are shown. Mean values ± SD are shown. This experiment was repeated with 3 different beagle donors. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 via one-way ANOVA with Tukey’s post-test.