“Adherent” versus Other Isolation Strategies for Expanding Purified, Potent, and Activated Human NK Cells for Cancer Immunotherapy

Table 2

Change in the MFI of CD16 on CD3⁻CD56⁺ cells of PBMCs and “adherent” enriched and expanded cells. Analysis of CD16 expression on CD3⁻CD56⁺ cells of PBMCs and “adherent” enriched and expanded cells. PBMC populations and two-week expanded cells were stained with Pacific Blue conjugated anti-CD3 (Clone HIT3a, Biolegend), Phycoerythrin-Cyanine 7 (PE/Cy7) conjugated anti-CD56 (Clone MEM-188, Biolegend), and FITC conjugated anti-CD16 (Clone 3G8, Biolegend) antibodies and fixed in 2% paraformaldehyde. For all flow cytometry experiments, appropriate IgG isotype controls were used to assess nonspecific staining. Cells were analyzed using a BD LSRII flow cytometer (BD Biosciences) and the data was analyzed using FlowJo Flow Cytometry Analysis Software (TreeStar Inc.).

Donor	Percent NK cells in PBMCs	Percent NK cell purity on day 14	CD16 MFI of NK cells in PBMCs	CD16 MFI of day 14 NK cells	Change in CD16^+ NK cell MFI after enrichment and expansion for 14 days
Cancer patient donor PBMCs
SB	30.5	87.9	22,746	11,512	49.4% lower
CH	5.4	64.2	13,600	24,478	80.0% higher
TP	7.9	85.5	10,198	10,833	6.2% higher
RS	8.8	81.7	15,150	10,545	30.4% lower
JB	14.3	88.4	10,644	8,191	23.0% lower
WN	25.4	95.3	11,369	19,061	67.7% higher
GW	6.4	90.6	14,215	3,221	77.3% lower
MT	14.3	87.0	9,624	12,104	25.8% higher
LS	4.0	98.0	10,698	10,833	1.2% higher
Healthy donor PBMCs
2	13.1	94.2	25,808	36,272	40.5% higher
JL	17.2	84.8	14,687	19,563	33.2% higher
4	5.7	69.8	13,828	22,281	61.1% higher
AS	17.0	95.2	24,726	22,696	8.2% lower
SS	15.7	97.9	14,761	21,258	44.0% higher
^*SS	13.9	98.8	14,613	20,062	37.3% higher
21	19.8	94.9	11,484	14,322	24.7% higher
^*21	19.8	92.0	11,484	10,732	6.5% lower

Large-scale expansion.