NK cell expansion

From NKcells.info

Contents 1 Introduction 2 Short Protocols 2.1 Cella and Colonna, 2000 2.2 Guven, 2003 2.3 Igarashi, 2004 2.4 Harada, 2004 2.5 Imai, 2005 2.6 Torelli, 2002 & 2005, Palmieri 1995 2.7 Alici 2008 3 additional information 4 References 4.1 2008 4.2 2005 4.3 2004 4.4 2003 4.5 2002 4.6 2001 4.7 2000 4.8 1990s

Introduction

Natural Killer cells can be expanded upon activation either by cytokines (such as IL-2) or by cell line (e.g. K562, HFWT, LCLs) proteins binding to triggering NK cell receptors (such as NKG2D, NKp46). NK cell activation appears to induce NK cell proliferation.

Tsujimoto et al. (JLB 2005 [1]) demonstrated that treatment of murine NK cells with flagellin induces NK cell proliferation but no strong interferon-gamma (IFN-gamma) production, indicating that NK cell proliferation and IFN-gamma production may be regulated differentially, proposing that flagellin is a potent inducer of NK cell proliferation.

Alici et al. (2008) described expansion of NK cells from Multiple Myeloma patients' Peripheral Blood Mononuclear Cells (see short protocols). NK cells were expanded 502 to 2658 fold (average 1625 fold) within 20 days, with an average NK cell content in the final cell product of 65% (22% T cells, 11% CD3+CD56+ NKT like cells). Expanded NK cells had an increased expression of activating receptors (including CD16, NCRs and NKG2D) and should increased cytotoxicity against both K562 and autologous Multiple Myeloma cells.

Short Protocols

Cella and Colonna, 2000

• prepare PBMC from "target donor" and allogeneic donor
• select CD3-CD56+ NK cells from "target donor" (donor for NK cell clones)
• resuspend 1E3 NK cells in 10 mL of medium (RPMI8866 in RPMI 1640, 5%human serum, 500 U/mL rhIL-2, non-essential amino acids, sodium pyruvate, L-glutamine, 100µg/mL kanamycin, 5E-5M 2-mercaptoethanol) per 96-well plate

• irradiate (5000 Rads) 1E7 allogeneic PBMC per 96-well plate
• irradiate 1E6 RPMI 8866 per well in exponential growth phase
• resuspend irradiated feeder cells together at 1E6/ml PBMC and 1E5/ml RPMI8866 in medium
• add 2µg/ml PHA (Phytohemagglutinin)

• mix NK cells and Feeder Cells and plate @ 200µl/well in 96-well round bottom plates
• after 5-8 days transfer 12 wells into one well of a 6-well plate, split every 2-4 days.

Guven, 2003

• prepare PBMC
• culture at 1E6/mL in
  • CellGro SCGM serum-free medium
  • 5% human serum
  • 500U/mL IL-2
  • 10ng/mL OKT-3 (d1 - d5)
• wash out OKT-3 containing medium on d5/d6
• replenish with fresh medium every 2-3 days until d21, avoid contact inhibition of cell growth
• a 200 to 500 fold expansion of NK cells can be expected

Igarashi, 2004

• prepare PBMC
• isolate NK cells by magnetic depletion of non-NK cells
• culture with 100 Gy irradiated feeder cells (1000:1 allogeneic EBV-LCL / NK cell ratio)
  • HLA-C group match/mismatch of feeder cells has no impact on net fold expansion of NK cells but on percentage of CD158a/b positive NK cells
  • CellGro SCGM serum-free medium
  • 10% human AB serum
  • 500 IU/mL IL-2
    • when using feeder cells 5 IU/mL IL-2 resulted in similar NK Expansion compared to 500 IU/mL IL-2
  • PenStrep
• optinally enrich or deplete CD158a+ or CD158b+ NK cells
• in case of CD158a/b selected expand for another 14 days to remove cell bound antibody
• replace half of the medium weekly
• a 10^2 fold expansion of NK cells within 14 days can be expected without feeder cells, a 10^4 fold expansion of NK cells within 14 days can be expected with feeder cells

Harada, 2004

• prepare PBMC
• coculture 1E6 PBMC and 1E5 HFWT (irradiated with 50 Gy of X rays) in 24 well plates in RHAMa medium with 5% (v/v) autologous plasma and 200 U/ml IL-2
  • RHAMa medium is a 3:1:1 mixture of RPMI1640, Ham/F12 and MEMa media supplemented with 0.02 mg/l alpha-tocopherol, 0.002 mg/l sodium selenite, 0.004 mg/l linoleic acid, 0.2 mg/l cholesterol, 500 mg/l human serum albumine, 0.6 mg/l 2-mercaptoethanol, 0.6 mg/l ethanolamine, 5 mg/l recombinant human insulin, 5 mg/l transferrin
• change half of the medium every 2 days

Imai, 2005

• coculture PBMC and variants of K562 cell line in RPMI1640, 10%FCS, 10 U/ml IL-2
  • median 2.5-fold expansion of CD56(+) CD3(-) cells at 1 week of culture, 20-fold expansion when K562-mb15-41BBL were used (K562 transfected with 4-1BBL and membrane-bound IL-15)
  • median >10000-fold expansion of CD56(+) CD3(-) cells at 2-3 weeks of culture, without T lymphocyte expansion (K562-mb15-41BBL)

alternatively:

• culture purified NK cells with 7 µg/ml PHA and 1000 U/ml IL-2
  • 2-5 fold expansion after 1 week, but T cell expansion is much higher, thus T cells overgrow NK cells.

Torelli, 2002 & 2005, Palmieri 1995

• remove adherent cells by culturing PBMC in RPMI 1640 for 2 hours
• coculture 4E5/ml non-adherent PBMC with 1E5/ml 3000 rad irradiated RPMI 8866 for 10-12d
• during the last 24h of culture add 100 U/ml IL-2, 10ng/ml IL-12, 50ng/ml IL-15 or 10 U/ml IL-2 + 10ng/ml IL-12 or 10 U/ml IL-2 + 50ng/ml IL-15.
  • NK cells represented 62-95% of bulk culture, NK cell expansion was about 40-fold

Alici 2008

• (nearly identical to Guven, 2003)
• prepare PBMC
• culture at 0.5E6/mL in
  • CellGro SCGM serum-free medium
  • 5% human serum
  • 500U/mL IL-2
  • 10ng/mL OKT-3
• wash out OKT-3 containing medium on d5
• replenish with fresh medium (without OKT-3) every 2 days until d20, avoid contact inhibition of cell growth
• a 500 to 2500 fold expansion of NK cells was observed (Multiple Myeloma patients)

additional information

• see generation of NK cell clones

References

2008

Alici E, Sutlu T, Bjorkstrand B, Gilljam M, Stellan B, Nahi H, Quezada HC, Gahrton G, Ljunggren HG, Dilber MS.
Autologous anti-tumor activity by NK cells expanded from myeloma patients using GMP-compliant components.
Blood. 2008 Jan 11;. Epub, PMID:18192509

Giancola R, Olioso P, Di Riti M, Capone A, Contento A, Pompetti F, Iacone A.
Evaluation of an automated closed fluid management device for processing expanded cytokine-induced killer cells to use in immunotherapy programs for cancer.
Transfusion. 2008 Jan 15;. Epub, PMID:18208417

2005

Koehl U, Esser R, Zimmermann S, Tonn T, Kotchetkov R, Bartling T, Sorensen J, Gruttner HP, Bader P, Seifried E, Martin H, Lang P, Passweg JR, Klingebiel T, Schwabe D.
Ex vivo Expansion of Highly Purified NK Cells for Immunotherapy after Haploidentical Stem Cell Transplantation in Children.
Klin Padiatr. 2005 Nov;217(6):345-350. PMID: 16307421 NKref0501

Chihaya Imai, Shotaro Iwamoto, and Dario Campana
Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells
Blood 2005;106 376-383 NKref0452

Torelli GF, Guarini A, Maggio R, Alfieri C, Vitale A, Foa R.
Expansion of natural killer cells with lytic activity against autologous blasts from adult and pediatric acute lymphoid leukemia patients in complete hematologic remission.
Haematologica. 2005 Jun;90(6):785-92. PMID: 15951291 NKref0451

Tsujimoto H, Uchida T, Efron PA, Scumpia PO, Verma A, Matsumoto T, Tschoeke SK, Ungaro RF, Ono S, Seki S, Clare-Salzler MJ, Baker HV, Mochizuki H, Ramphal R, Moldawer LL.
Flagellin enhances NK cell proliferation and activation directly and through dendritic cell-NK cell interactions.
J Leukoc Biol. 2005 Jul 20; Epub PMID: 16033815 free full text

2004

Klingemann HG, Martinson J.
Ex vivo expansion of natural killer cells for clinical applications.
Cytotherapy. 2004;6(1):15-22. PMID: 14985163 NKref0349

Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells
Takehito Igarashi, Jason Wynberg, Ramprasad Srinivasan, Brian Becknell, J. Phillip McCoy, Jr, Yoshiyuki Takahashi, Dante A. Suffredini, W. Marston Linehan, Michael A. Caligiuri, and Richard W. Childs
Blood 2004;104 170-177 NKref0319

Peng BG, Liang LJ, He Q, Huang JF, Lu MD.
Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma.
World J Gastroenterol. 2004 Jul 15;10(14):2119-23. PMID: 15237448 (HFWT reference, NKref0335)

Harada H, Saijo K, Ishiwata I, Ohno T.
A GFP-transfected HFWT cell line, GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay.
Hum Cell. 2004 Mar;17(1):43-8. PMID: 15369136 NKref0373

Ferlazzo G, Pack M, Thomas D, Paludan C, Schmid D, Strowig T, Bougras G, Muller WA, Moretta L, Munz C.
Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs.
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16606-11. Epub 2004 Nov 09. PMID: 15536127
(Role of membrane bound IL-15)

2003

Guven H, Gilljam M, Chambers BJ, Ljunggren HG, Christensson B, Kimby E, Dilber MS.
Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy.
Leukemia. 2003 Oct;17(10):1973-80.
PMID: 14513047 NKref0232

Oikawa T, Kawai K, Ishiwata I, Ohno T, Akaza H.
Induction of potent antitumour natural-killer cells from peripheral blood of patients with advanced prostate cancer.
BJU Int. 2003 Dec;92(9):1009-15. PMID: 14632866 (HFWT reference)

2002

Harada H, Saijo K, Watanabe S, Tsuboi K, Nose T, Ishiwata I, Ohno T.
Selective expansion of human natural killer cells from peripheral blood mononuclear cells by the cell line, HFWT.
Jpn J Cancer Res. 2002 Mar;93(3):313-9. PMID: 11927014 (HFWT reference, NKref0334)

Torelli GF, Guarini A, Palmieri G, Breccia M, Vitale A, Santoni A, Foa R.
Expansion of cytotoxic effectors with lytic activity against autologous blasts from acute myeloid leukaemia patients in complete haematological remission.
Br J Haematol. 2002 Feb;116(2):299-307. PMID: 11841430 NKref0453

2001

Condiotti R, Zakai YB, Barak V, Nagler A.
Ex vivo expansion of CD56+ cytotoxic cells from human umbilical cord blood.
Exp Hematol. 2001 Jan;29(1):104-13. PMID: 11164111

2000

Cella, Marina; Colonna, Marco
Cloning Human Natural Killer Cells
in Natural Killer Cell Protocols, Cellular and Molecular Methods, February 2000, ISBN: 1-59259-044-6 (includes bulk culture for NK cell expansion)

Goodier MR, Londei M.
Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10.
J Immunol. 2000 Jul 1;165(1):139-47., PMID:10861046

1990s

Warren HS, Kinnear BF, Skipsey LJ.
Human natural killer (NK) cells: requirements for cell proliferation and expansion of phenotypically novel subpopulations.
Immunol Cell Biol. 1993 Apr;71 ( Pt 2):87-97. PMID: 8486400

Robertson MJ, Manley TJ, Donahue C, Levine H, Ritz J.
Costimulatory signals are required for optimal proliferation of human natural killer cells.
J Immunol. 1993 Mar 1;150(5):1705-14. PMID: 7679691

Warren HS, Kinnear BF, Phillips JH, Lanier LL.
Production of IL-5 by human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and IL-12.
J Immunol. 1995 May 15;154(10):5144-52. PMID: 7730620 NKref0384

Palmieri G, Serra A, De Maria R, Gismondi A, Milella M, Piccoli M, Frati L, Santoni A.
Cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors enhances natural killer cell cytotoxic activity.
J Immunol. 1995 Dec 1;155(11):5314-22. PMID: 7594545

Miller JS, Klingsporn S, Lund J, Perry EH, Verfaillie C, McGlave P.
Large scale ex vivo expansion and activation of human natural killer cells for autologous therapy.
Bone Marrow Transplant. 1994 Oct;14(4):555-62., PMID:7532064

Miller JS, Oelkers S, Verfaillie C, McGlave P.
Role of monocytes in the expansion of human activated natural killer cells.
Blood. 1992 Nov 1;80(9):2221-9., PMID:1421393

Rabinowich H, Sedlmayr P, Herberman RB, Whiteside TL.
Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.
Cell Immunol. 1991 Jul;135(2):454-70., PMID:1709827