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High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

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Responsiveness of ex vivo-generated KIR+ and NKG2A+ NK cells to MHC class I-deficient target cells.Ex vivo-generated NK cells using Method II with GBGM were incubated alone, or 18 hours with MHC class I-negative K562 or MHC class I-expressing KG1a cells at an E∶T ratio of 1∶1. Cells were then stained for CD56, CD3, KIR or NKG2A, and the degranulation antigen CD107a. (a) Degranulation of total CD56+CD3− NK cells and KIR2DL2/DL3+ NK cell subset expanded for 5 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the KIR2DL2/DL3+ NK cells. (b) Degranulation of total CD56+CD3− NK cells and NKG2A+ NK cell subset expanded for 6 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the NKG2A+ NK cells.
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pone-0009221-g005: Responsiveness of ex vivo-generated KIR+ and NKG2A+ NK cells to MHC class I-deficient target cells.Ex vivo-generated NK cells using Method II with GBGM were incubated alone, or 18 hours with MHC class I-negative K562 or MHC class I-expressing KG1a cells at an E∶T ratio of 1∶1. Cells were then stained for CD56, CD3, KIR or NKG2A, and the degranulation antigen CD107a. (a) Degranulation of total CD56+CD3− NK cells and KIR2DL2/DL3+ NK cell subset expanded for 5 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the KIR2DL2/DL3+ NK cells. (b) Degranulation of total CD56+CD3− NK cells and NKG2A+ NK cell subset expanded for 6 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the NKG2A+ NK cells.

Mentions: Phenotypic analysis showed that our ex vivo-generated NK cell products contained up to 10% CD56+KIR+ cells and a high proportion (40–60%) CD56+NKG2A+ cells. To determine cytolytic activity of these subsets and investigate if this activity is regulated by the expressed inhibitory receptors, we performed CD107a-based degranulation assays using HLA-negative K562 cells and KG1a cells expressing relatively high levels of HLA-ABC and -E (see Table 1). For these experiments, we used two ex vivo-generated NK cell products that contained approximately 15–30% CD56+CD107a+ cells upon co-culture with K562 (Figure 5 and Figure S3). In contrast, KG1a cells hardly stimulated degranulation of these NK cell products, indicating that cytolytic activity is inhibited by HLA expression. Similarly, around 35% of the CD56+KIR2DL2/DL3+ subset degranulated upon triggering by K562, while only a small percentage (<5%) expressed CD107a following co-culture with KG1a (Figure 5a and Figure S3). In agreement with these results, degranulation by the CD56+KIR2DL2/DL3+ subset could be specifically inhibited by K562 cells transfected with the HLA-C group 1 allele HLA-Cw3, while transfection of the HLA-C group 2 allele HLA-Cw4 allele had not effect (Figure S4). Finally, we observed that also the dominant CD56+NKG2A+ subset was able to efficiently degranulate in response to K562 (28% CD107a+ cells), while degranulation towards KG1a was low probably due to relative high expression of HLA-E molecules (Figure 5b and Table 1). These data demonstrate that the KIR+ and NKG2A+ subsets within the UCB-derived NK cell products are fully responsive mediating strong degranulating activity, which is regulated by the expression of MHC class I molecules on the engaged target cells.


High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Responsiveness of ex vivo-generated KIR+ and NKG2A+ NK cells to MHC class I-deficient target cells.Ex vivo-generated NK cells using Method II with GBGM were incubated alone, or 18 hours with MHC class I-negative K562 or MHC class I-expressing KG1a cells at an E∶T ratio of 1∶1. Cells were then stained for CD56, CD3, KIR or NKG2A, and the degranulation antigen CD107a. (a) Degranulation of total CD56+CD3− NK cells and KIR2DL2/DL3+ NK cell subset expanded for 5 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the KIR2DL2/DL3+ NK cells. (b) Degranulation of total CD56+CD3− NK cells and NKG2A+ NK cell subset expanded for 6 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the NKG2A+ NK cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821405&req=5

pone-0009221-g005: Responsiveness of ex vivo-generated KIR+ and NKG2A+ NK cells to MHC class I-deficient target cells.Ex vivo-generated NK cells using Method II with GBGM were incubated alone, or 18 hours with MHC class I-negative K562 or MHC class I-expressing KG1a cells at an E∶T ratio of 1∶1. Cells were then stained for CD56, CD3, KIR or NKG2A, and the degranulation antigen CD107a. (a) Degranulation of total CD56+CD3− NK cells and KIR2DL2/DL3+ NK cell subset expanded for 5 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the KIR2DL2/DL3+ NK cells. (b) Degranulation of total CD56+CD3− NK cells and NKG2A+ NK cell subset expanded for 6 weeks from CD34+ UCB cells. Density plots are gated on CD56+CD3− NK cells and the histogram plots show the CD107a degranulation of the NKG2A+ NK cells.
Mentions: Phenotypic analysis showed that our ex vivo-generated NK cell products contained up to 10% CD56+KIR+ cells and a high proportion (40–60%) CD56+NKG2A+ cells. To determine cytolytic activity of these subsets and investigate if this activity is regulated by the expressed inhibitory receptors, we performed CD107a-based degranulation assays using HLA-negative K562 cells and KG1a cells expressing relatively high levels of HLA-ABC and -E (see Table 1). For these experiments, we used two ex vivo-generated NK cell products that contained approximately 15–30% CD56+CD107a+ cells upon co-culture with K562 (Figure 5 and Figure S3). In contrast, KG1a cells hardly stimulated degranulation of these NK cell products, indicating that cytolytic activity is inhibited by HLA expression. Similarly, around 35% of the CD56+KIR2DL2/DL3+ subset degranulated upon triggering by K562, while only a small percentage (<5%) expressed CD107a following co-culture with KG1a (Figure 5a and Figure S3). In agreement with these results, degranulation by the CD56+KIR2DL2/DL3+ subset could be specifically inhibited by K562 cells transfected with the HLA-C group 1 allele HLA-Cw3, while transfection of the HLA-C group 2 allele HLA-Cw4 allele had not effect (Figure S4). Finally, we observed that also the dominant CD56+NKG2A+ subset was able to efficiently degranulate in response to K562 (28% CD107a+ cells), while degranulation towards KG1a was low probably due to relative high expression of HLA-E molecules (Figure 5b and Table 1). These data demonstrate that the KIR+ and NKG2A+ subsets within the UCB-derived NK cell products are fully responsive mediating strong degranulating activity, which is regulated by the expression of MHC class I molecules on the engaged target cells.

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

Show MeSH
Related in: MedlinePlus