Fig. 1
CD5 Depletion Enriches for a CD5dimNKp46+ Population of Dog NK Cells which Expand in Culture. a – b. Forward scatter/ side scatter was used to identify the putative lymphocyte population in dog PBMCs pre- and post-depletion of CD5bright cells by negative selection using magnetic particles. Post-depletion, the proportion of CD3-NKp46+ cells is markedly increased, and these NKp46+ cells show bimodal expression of CD8 and absent expression of CD4. c. The lymphocyte population was analyzed for the median fluorescence intensity of indicated cell surface markers pre- and post-CD5 depletion. Post-depletion, there is significantly decreased expression of T cell surface markers as shown. d – e. At day14 after CD5-depletion and co-culture with irradiated K562 human feeder cells transduced with 4-1BBL (CD137L) and membrane-bound rh-IL21, dog NK cells demonstrate ongoing expansion of a NKp46+ population which comprise an increasing percentage of the cell product. f. Among 13 diverse health and tumor-bearing donors, the average NK fold expansion 14 days after isolation and co-culture was 19-fold with wide variability (SD 22.7-fold). g. Post-depletion, the average number of CD5dim cells was 4.1 ± 1.9 × 106 cells, and the average yield at day 14 was 75.2 ± 24.1 × 106 cells, with greatest expansion occurring between day 7 and 14. h – i. qPCR results confirm significant down-regulation of T cell surface markers (CD3 and CD5) during co-culture from depletion to day 14 and significant up-regulation of NK cell surface marker NCR1. In each panel, a representative experiment of a minimum of 3 replicates is shown. * P < 0.05, ** P < 0.01, and *** P < 0.001 via paired t-test with Bonferroni correction where indicated