Figure 1.
Surface expression of CD15 and transgene receptors on Clone 9.mbIL21 aAPC, and stability of expression of relevant receptors.
After limiting-dilution cloning, cloned aAPCs were assessed for expression of transgenes, A. Since CD15 expression was variable among the clones and is felt to play a role in NK cell activation, its expression was also assessed. After selection of a clone with expression of all surface proteins, the clone was maintained in culture for 6 months, with periodic re-evaluation of CD137L and mbIL21 expression, B.
Table 1.
Artificial APCs used in this study.
Figure 2.
Schema for NK cell manufacturing with aAPCs.
Artificial antigen-presenting cells (aAPCs) were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells ex vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the aAPCs as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use.
Figure 3.
NK cell expansion and purity after repeated weekly stimulation with aAPCs expressing membrane-bound cytokines.
A, Mean expansion of CD3neg/CD16-or-56pos NK cells from 22 donors expanded for 21 days using aAPCs bearing mbIL15 or mbIL21. NK cells from five donors were also expanded with aAPCs expressing both mbIL15 and mbIL21. B, Mean expansion of NK cells from 4 donors expanded for 42 days using aAPCs bearing mbIL15 or mbIL21. C, Percent of CD3pos T/NKT cells and CD3neg/CD16-or-56pos NK cells in the starting PBMC product (day 0) and at the end of 21 days of expansion on Clone 9.mbIL21 from 20 donors. D, Mean expansion of CD3neg/CD16-or-56pos NK cells on Clone 9.mbIL21 from unfractionated PBMC (n = 19), unfractionated PBMC followed by NK purification at day 14 of expansion (n = 3), or from NK cells purified from PBMC at day 0 prior to expansion (n = 13). Mean +/− SD is shown for each plot. All p values indicated are for t-test of fold expansion at the end of the expansion period.
Figure 4.
Phenotype of NK cells expanded on aAPCs bearing membrane-bound cytokines.
NK cells purified from PBMC were stimulated weekly with either Clone 4 (mbIL15) or Clone 9.mbIL21 for 3 weeks. Expression of NK cell receptors was determined by flow cytometry. A) Representative dot plots of NK cells expanded from the same donor on Clone 4 (mbIL15) or Clone 9.mbIL21. B) Component subpopulations of fresh NK cells or NK cells expanded on Clone 4 (mbIL15) or Clone 9.mbIL21 from 4 donors as determined by flow cytometry. Mean +/− SD is shown. P values are for 2-way repeated-measures ANOVA comparing against mbIL21-expanded NK cells with Bonferroni correction (all significant P values are indicated).
Figure 5.
Gene expression in NK cells stimulated with mbIL15 or mbIL21 as assessed using the nCounter platform.
Paired aliquots of purified NK cells from 4 donors were stimulated for one week with either Clone 4 (mbIL15) or Clone 9.mbIL21. Total RNA was purified and equal quantities hybridized for detection of expression of 96 genes. Gene expression was normalized to LDH (mean 6,076 copies detected), and the remaining data for each gene plotted as mean +/− SEM for each expansion condition. Genes with detection below background (set at ≤10 detected copies) were excluded as not biologically significant (grey box). The ten highest-expressed genes in addition to LDH are labeled, as are genes that are differentially expressed by >2 fold (red) or <0.5 fold (blue) in mbIL21-expanded cells. Differentially expressed genes were replotted (inset) for comparison, and two-tailed t-test applied.
Figure 6.
Change in relative telomere length of purified NK cells after expansion on aAPCs with or without membrane-bound cytokines.
NK cells were purified from normal donor buffy coats. An aliquot was viably frozen for later comparison, and the remaining cells were stimulated with IL-2 and irradiated aAPCs bearing no cytokine (Clone 9), mbIL15 (Clone 4), mbIL21 (Clone 9.mbIL21), or IL-15 fused with a linker to IL15Rα (Clone 27). (A) Telomere length of the NK cells was determined after 7 days by flow cytometry after hybridization with FITC-labeled PNA probe for the TTAGGG telomeric repeat sequence. Mean fluorescence of the NK cells was normalized to the reference cell line CEM-1301, and the telomere length of expanded NK cells was normalized to that of fresh NK cells for each individual donor. A 1-way ANOVA with Dunnett's multiple comparisons test was applied to compare each aAPC to Clone 4. (B) NK cells from four additional donors stimulated weekly for 21 days prior to assessing telomere length, and again normalized to that of fresh cells from each donor. P value shown for two-tailed t-test.
Figure 7.
Direct cytotoxicity and cytokine secretion of fresh NK cells, mbIL15-expanded, and mbIL21-expanded NK cells.
NK cells were purified from four normal donor buffy coats and assessed directly, and then retested after being expanded for 21 days with Clone 9.mbIL21 and Clone 4 (mbIL15) (with CD3 depletion on day 14). NK cells were assessed for cytotoxicity against 721.221 (A) or cytokine secretion in response to K562 (B). NK cells from another four donors were purified and cryopreserved, and expanded with Clone 9.mbIL21 and then cryopreserved. Fresh (circles) and expanded (squares) NK cells were then thawed and activated in parallel for 24 h in 50 IU/mL IL-2, and then tested for cytotoxicity against HLAnull 721.221 targets (C), C*0702-transduced (Group C1) 721.221 targets (D), or B*5801-transduced (Group Bw4) 721.221 targets (E). All data points shown are mean +/− SEM of the means of four donors tested in triplicate.
Figure 8.
Direct and antibody-dependent cellular cytotoxicity of mbIL21 expanded NK cells toward tumor cell lines.
NK cells were expanded for 21 days from four normal donors with CD3 depletion on day 14, and then cryopreserved in aliquots. NK cells were thawed and rested for 24 h in 50 IU/mL IL-2, and then tested for cytotoxicity against the indicated tumor cell lines using the calcein release assay. Assays were performed in triplicate, and results are shown as mean +/− SD for each donor against representative AML (A, B), B-cell (C, D), neuroblastoma (E, F), colon carcinoma (G), and melanoma (H) tumor cell lines. Squares, circles, triangles, and inverted triangles correspond to the expanded NK cells from the same four independent donors in each panel. I–L, expanded NK cells were tested for direct cytotoxicity (circles) or ADCC (squares) against B cell tumors (I, J) using anti-CD20 antibody (rituximab), or neuroblastoma (K) and melanoma (L) using anti-GD2 antibody (14G.2a). Data shown are mean +/− SEM of each donor tested in triplicate (A–H) or of the means of all four donors (I–L).