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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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Functional activity of ex vivo-generated CD56+ NK cells using Method II with GBGM.(a) Specific cytotoxicity of a CD56+ NK cell product (98% purity) against the myeloid leukemia cell lines K562 and KG1a. Specific lysis was determined after 4 and 24 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 2∶1. Data are displayed as mean ± SD of triplicate wells. (b) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells. Results are depicted as mean ± SD of triplicate wells. (c) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (d) Specific cytotoxicity of another CD56+ NK cell product (95% purity) against the myeloid leukemia cell lines K562, Lama, Kasumi and KG1a, and the melanoma cell lines BLM and FM3. Specific lysis was determined after 18 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 1∶1. Data are displayed as mean ± SD of triplicate samples. (e) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells after overnight stimulation with different targets. Data are depicted as mean ± SD of triplicate samples. (f) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (g) Specific cytotoxicity of a third CD56+ NK cell product (97% purity) against primary AML cells from 5 different patients. Specific lysis was determined after 24, 48 and 72 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 3∶1. Data are displayed as mean ± SD of triplicate samples.
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pone-0009221-g006: Functional activity of ex vivo-generated CD56+ NK cells using Method II with GBGM.(a) Specific cytotoxicity of a CD56+ NK cell product (98% purity) against the myeloid leukemia cell lines K562 and KG1a. Specific lysis was determined after 4 and 24 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 2∶1. Data are displayed as mean ± SD of triplicate wells. (b) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells. Results are depicted as mean ± SD of triplicate wells. (c) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (d) Specific cytotoxicity of another CD56+ NK cell product (95% purity) against the myeloid leukemia cell lines K562, Lama, Kasumi and KG1a, and the melanoma cell lines BLM and FM3. Specific lysis was determined after 18 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 1∶1. Data are displayed as mean ± SD of triplicate samples. (e) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells after overnight stimulation with different targets. Data are depicted as mean ± SD of triplicate samples. (f) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (g) Specific cytotoxicity of a third CD56+ NK cell product (97% purity) against primary AML cells from 5 different patients. Specific lysis was determined after 24, 48 and 72 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 3∶1. Data are displayed as mean ± SD of triplicate samples.

Mentions: To determine the cytotoxic potential of the ex vivo-generated NK cell products, we performed flow cytometry-based cytotoxicity assays using various myeloid leukemia cell lines (K562, Lama, Kasumi and KG1a), primary AML cells and two melanoma cell lines (BLM and FM3). Ex vivo-generated NK cells in GBGM® mediated efficient lysis of K562 cells (∼40% and ∼90% after 4 and 24 hrs, respectively) at a very low E∶T ratio of 2∶1 (Figure 6a). Furthermore, profound lysis could be observed against MHC class I-expressing KG1a cells (∼20% and ∼40% after 4 and 24 hrs, respectively). Interestingly, NK cells cultured in GBGM® showed higher cytotoxic and degranulating activity as compared to NK cells cultured in H3000 medium from the same UCB donor (Figure S5a–c). Furthermore, we found that GBGM-derived NK cells showed higher expression of the activating receptors NKG2D, NKp30, NKp44 and NKp46 (Figure S5d).


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)

Functional activity of ex vivo-generated CD56+ NK cells using Method II with GBGM.(a) Specific cytotoxicity of a CD56+ NK cell product (98% purity) against the myeloid leukemia cell lines K562 and KG1a. Specific lysis was determined after 4 and 24 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 2∶1. Data are displayed as mean ± SD of triplicate wells. (b) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells. Results are depicted as mean ± SD of triplicate wells. (c) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (d) Specific cytotoxicity of another CD56+ NK cell product (95% purity) against the myeloid leukemia cell lines K562, Lama, Kasumi and KG1a, and the melanoma cell lines BLM and FM3. Specific lysis was determined after 18 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 1∶1. Data are displayed as mean ± SD of triplicate samples. (e) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells after overnight stimulation with different targets. Data are depicted as mean ± SD of triplicate samples. (f) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (g) Specific cytotoxicity of a third CD56+ NK cell product (97% purity) against primary AML cells from 5 different patients. Specific lysis was determined after 24, 48 and 72 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 3∶1. Data are displayed as mean ± SD of triplicate samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2821405&req=5

pone-0009221-g006: Functional activity of ex vivo-generated CD56+ NK cells using Method II with GBGM.(a) Specific cytotoxicity of a CD56+ NK cell product (98% purity) against the myeloid leukemia cell lines K562 and KG1a. Specific lysis was determined after 4 and 24 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 2∶1. Data are displayed as mean ± SD of triplicate wells. (b) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells. Results are depicted as mean ± SD of triplicate wells. (c) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (d) Specific cytotoxicity of another CD56+ NK cell product (95% purity) against the myeloid leukemia cell lines K562, Lama, Kasumi and KG1a, and the melanoma cell lines BLM and FM3. Specific lysis was determined after 18 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 1∶1. Data are displayed as mean ± SD of triplicate samples. (e) Degranulation of CD56+ NK cells was determined by FCM as the percentage of CD107a+ cells after overnight stimulation with different targets. Data are depicted as mean ± SD of triplicate samples. (f) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (g) Specific cytotoxicity of a third CD56+ NK cell product (97% purity) against primary AML cells from 5 different patients. Specific lysis was determined after 24, 48 and 72 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 3∶1. Data are displayed as mean ± SD of triplicate samples.
Mentions: To determine the cytotoxic potential of the ex vivo-generated NK cell products, we performed flow cytometry-based cytotoxicity assays using various myeloid leukemia cell lines (K562, Lama, Kasumi and KG1a), primary AML cells and two melanoma cell lines (BLM and FM3). Ex vivo-generated NK cells in GBGM® mediated efficient lysis of K562 cells (∼40% and ∼90% after 4 and 24 hrs, respectively) at a very low E∶T ratio of 2∶1 (Figure 6a). Furthermore, profound lysis could be observed against MHC class I-expressing KG1a cells (∼20% and ∼40% after 4 and 24 hrs, respectively). Interestingly, NK cells cultured in GBGM® showed higher cytotoxic and degranulating activity as compared to NK cells cultured in H3000 medium from the same UCB donor (Figure S5a–c). Furthermore, we found that GBGM-derived NK cells showed higher expression of the activating receptors NKG2D, NKp30, NKp44 and NKp46 (Figure S5d).

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