Figure 1.

Expansion of canine NK cells in vitro. (A) Schema of experimental strategy expanding NK cells from PBMC and CD5-depleted starting populations. After processing, respective cell populations were co-cultured with irradiated K562 clone 9 feeder cells and 100 mIU/mL rhIL-2 for 14 days. (B) Representative flow cytometry gating of cells prior to co-culture and following magnetic bead separation for CD5 depletion. Depleted cells showed virtually no CD5 expression in contrast to positively selected CD5+ cells, confirming the efficacy of magnetic separation and phenotype of CD5-depleted cells as a starting population. (C) Cell counts and viability were calculated on days 0, 7, 10, 12, and 14 of the 14-day co-culture using bulk PBMCs (blue) and CD5-depleted cells (red) as starting populations. Mean and SEM for 11 healthy donor dogs are plotted against time. (D) Representative flow cytometry gating of bulk PBMCs before and after 14-day co-culture, corroborating the expansion of NKp46+ NK cells from PBMCs without preceding NK-isolation. NK cells were identified as CD3-NKp46+, reaching a majority at day 14 with minimal CD3+ T cell infiltrate. (E) Representative flow cytometry gating of PBMCs at rest and following 14-day co-culture confirming minimal contamination of CD3+γδTCR+ T cells. NK, natural killer; PBMC, peripheral blood mononuclear cell.