Figure 1.
Unselected CB MNCs were cultured for 7 days in a GP500 bioreactor with IL-2 (100 IU/mL) and aAPCs at 2∶1 aAPC:MNC ratio. Cells were immunomagnetically CD3-depleted on Day 7 and re-cultured in same conditions for an additional 7 days. On day 7 cells were again CD3-depleted and subject to phenotypic and functional studies.
Figure 2.
Co-culture of CB MNCs with IL-2 and aAPCs yields significantly greater expansion of NK cells than culture with IL-2 alone.
A. Mean fold growth of CD56+/CD3− NK cells from 8 fresh and 6 frozen cord blood expansions with aAPCs and IL-2 versus 3 expansions with IL-2 alone (14 day culture). B. Time course of NK cell growth over 14 day culture between all 3 conditions. By day 7, the fresh CB aAPC-containing culture demonstrated greater NK cell growth than culture with IL-2 alone (p<0.05). The frozen CB showed a similar trend at day 7, which did not reach statistical significance (p = 0.06). C. All three culture conditions yielded comparable, low percentages of CD3+ cells:. 0.44%, 0.74% and 0.66% CD3+ cells from the culture with IL-2 alone, fresh CB MNCs with aAPC feeders or frozen CB MNCs with aAPC feeders respectively (p>0.5 for all comparisons). Mean +/− SD is shown for each figure. P<0.05 where indicated (*).
Figure 3.
Phenotype of CB-NK cells cultured with aAPCs.
A. Over the 14-day expansion, CB-NK cells cultured with aAPC feeder cells demonstrated a progressively pure, CD56+/CD3− population, (representative dot plots of 17 expansions). B. aAPC-expanded CB-NK cells maintained Eomesoderminhi and T-bethi phenotype after expansion. Representative histograms from 3 different CB-NK expansions; cells are gated on the live CD56+ population. C. CB MNCs from the same CB unit were expanded with aAPCs +IL-2 or IL-2 alone (n = 3 separate CB units). Representative dot plots of NK cell surface receptor expression on day 14 are shown. D. By median fluorescence intensity (MFI), aAPC-expanded CB-NK demonstrated a decreased surface expression of the NCRs NKp30, NKp46 and NKp44. However there was a similar expression between the conditions of the KIR antigens, inhibitory receptor NKG2A, co-receptor CD94 and activating receptor NKG2C) (n = 3 paired expansions, mean +/− SD is shown, p≤0.05 where indicated).
Figure 4.
aAPC-expanded CB-NK cells form immunological synapses with and are cytotoxic against myeloma targets.
A. CMAC-labeled tumor targets (blue) were incubated at a 1∶1 ratio with aAPC-expanded CB-NK cells for 15 minutes. Conjugates were then fixed, permeabilized and stained for NK effector cell F-actin with rhodamine-phalloidin (red). Confocal and brightfield images were acquired; representative images from each slide are shown. aAPC-expanded CB-NK cells form immune synapses with the classic NK target K562 as well as a variety of MM cell lines. B. aAPC-expanded CB-NK cells were co-incubated in triplicate for 4 hours with 51Cr-labeled target cells at ratios as shown. Supernatants were then harvested and analyzed the next day for 51Cr content. % Cytotoxicity = (sample value-spontaneous lysis)/(max-lysis-spontaneous lysis) x 100%. CB-NK cells demonstrate dose-dependent cytotoxicity against K562 (classic NK cell target) and MM cells lines RPMI 8266, ARP-1 and U266 (representative of n>3 assays for each cell line). C. aAPC-Expanded CB-NK cells displayed equal or more cytotoxicity against K562 cells versus CB-NK cells expanded with IL-2 alone (representative from n = 4 assays).
Figure 5.
aAPC-expanded CB-NK cells delay development of myeloma in a NSG murine model.
1×106 GFP firefly luciferase-transduced ARP-1 cells (Clone 24) were given IV on day -1. In the CB-NK treated group, 10×106 ex vivo, aAPC-expanded CB NK cells were given retro-orbitally on days 0, 12 and 19 with IL-2, 2000 IU (IP) three times per week. Serial BLI and kappa ELISA measurements were acquired until day 18. Results represent mean values of n = 5 mice in each group until day 18, by which time 1 mouse in the ARP-1 alone group had died. A. Serial BLI images demonstrate impaired myeloma development in mice receiving CB-NK cells. B. Signal intensity (p/s) was significantly greater in mice receiving Clone 24 ARP-1 cells alone versus those receiving both Clone 24 ARP-1 cells and CB-NK cells. Region of interest (ROI) is indicated by rectangles superimposed on each mouse from Figure 5A, p≤0.05 at days 8–22. C. Serum kappa levels (ng/mL) were significantly higher in mice treated with Clone 24 ARP-1 cells versus those treated with Clone 24 ARP-1 cells and CB-NK cells, p≤0.01 at each time point. D. By Kalpan-Meier method, there was a significant difference in survival of the mice, (p = 0.003) in favor of the NK-treated group. The mice who received Clone 24 ARP-1 cells alone had a median survival of 31 days versus 38 days for the mice who received Clone 24 ARP-1 cells and CB-NK cells.