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Cloning Human NK Cells 1
1
Cloning Human Natural Killer Cells
Marina Cella and Marco Colonna
1. Introduction
Our understanding of the phenotypical and functional heterogeneity of
human natural killer (NK) cells has greatly advanced over the past few years.
This advancement has been greatly helped by the development of culture con-
ditions for clonal proliferation of NK cells. Analysis of human NK cell clones
has led to the original observation that different NK cell clones recognize distinct
major histocompatibility complex (MHC) class I specificities. This has prompted
the production of monoclonal antibodies directed to NK cell surface antigens
clonally distributed, and, ultimately, the biochemical and molecular definition of
the NK cell surface glycoproteins functioning as MHC class I receptors.
Here we describe a limiting dilution culture protocol that allows establish-
ment of human NK cell clones from peripheral blood leukocytes with high
efficiency.
2. Materials
1. RPMI 1640 (Gibco, cat. no. 31870-025).
2. RPMI + HEPES (25 mM): (Gibco, cat. no. 42401-018).
3. Lymphocyte separation medium (LSM): (ICN Biomedicals, cat. no. 50494/36427).
4. Human recombinant interleukin (IL)-2 Liquemin (25000 UI/5 mL, Roche).
5. RPMI 8866 cells (available from Dr. Bice Perussia, Jefferson Medical College, Kimmel
Cancer Institute, BLSB 750, 233 S 10th Street, Philadelphia, PA 19107, USA).
6. Antibodies: Anti-human CD56 (Pharmingen, cat. no. 31661A, mouse IgG ), anti-
1
human CD3 (OKT3, mouse IgG , ATCC, cat. no. CRL-8001), anti-mouse IgG -
2a 1
phycoerythrin (PE; SBA, cat. no. 1070-09), anti-mouse IgG -fluorescein
2a
isothiocyanate (FITC; SBA, cat. no. 1080-02).
7. Phosphate-buffered saline (PBS).
8. PBS supplemented with 1% fetal calf serum (FCS).
From: Methods in Molecular Biology, vol. 121: Natural Killer Cell Protocols: Cellular and Molecular Methods
Edited by: K. S. Campbell and M. Colonna © Humana Press Inc., Totowa, NJ
1
2 Cella and Colonna
9. Complete medium (CM): RPMI 1640 medium (Gibco, cat. no. 31870-025)
supplemented with 5% human serum (filtered through 0.8-µm filter unit, Nalgene,
cat. no. 380-0080; not heat treated), 500U/mL of human recombinant IL2
(Roche), nonessential amino acids (from 100X stock; Gibco, cat. no. 11140-035),
sodium pyruvate (from 100×stock; Gibco, cat. no. 11360-039), L-glutamine(from
100×stock; Glutamax I, Gibco, cat. no. 35050-038), kanamycin (100 µg/mL final
from 100×; Gibco, cat. no. 15160-047), and 2-mercaptoethanol (5 ×10–5Mfinal
from sterile stock). Do not add HEPES to CM. Filter through 0.22-µm filter unit.
10. Phytohemagglutinin (PHA) (Murex Diagnostics, HA16).
11. Freezing mix : 70% FCS, 10%DMSO, 20% RPMI-HEPES.
12. Cell sorter.
13. Gamma irradiation source.
14. 96-well plates, 24-well plates, 6-well plates, cryotubes. (No particular commer-
cial source is required.)
3. Method
3.1. Preparation of NK Cells
Use sterile technique throughout the following procedures.
1. Collect 5 mL of blood from a blood donor with anticoagulants (heparin or EDTA)
and dilute 1:1 with RPMI + HEPES.
2. In a 15-mL plastic tube gently lay 10 mL of diluted blood on 5 mL of LSM using
a 5-mL wide mouth plastic pipet. Centrifuge for 30 min at 940gat room tempera-
ture with no brake. Red blood cells and granulocytes will sediment in the pellet,
while peripheral blood mononuclear cells (PBMCs) will localize at the interface
between LSM (below) and plasma (above).
3. Collect PBMCs at the interface with a pasteur pipet. Transfer PBMCs to a sepa-
rate tube, wash them in RPMI + HEPES, and collect them by centrifuging for 15 min
at 500g. Discard supernatant. Flick tube gently to resuspend pelleted cells.
4. Wash the pelleted PBMCs 2×with RPMI + HEPES. Collect by centrifuging for
10 min at 300g.
5. Resuspend the pellet of the heparinized blood cells in a 15-mL Falcon tube in 500 µL
of ice-cold PBS–1% FCS containing anti-CD56 antibody (mouse IgG1, 10 µg/mL)
and anti-CD3 antibody (mouse IgG , 10 µg/mL). Incubate 30 min on ice.
2a
6. Fill the tube of antibody-treated cells with 15 mL of ice-cold PBS–1% FCS and
centrifuge at 300g for 10 min.
7. Wash 1× with ice-cold PBS–1% FCS by centrifuging for 10 min at 300g.
8. Resuspend the pellet of antibody-treated cells in 200 µL of PBS–1% FCS
containing goat anti-mouse IgG -PE (1:100 dilution) and goat anti-mouse
1
IgG -FITC (1:50) and incubate on ice for 30 min.
2a
9. Fill the tube of antibody-treated cells with 15 mL of ice-cold PBS and centrifuge
at 300g for 10 min.
10. Wash 1× with ice cold PBS–1% FCS by centrifuging for 10 min at 300g.
11. Resuspend the stained cells in PBS with 1% FCS at a concentration of 3–5 ×106
cells/mL. Sort at least 10,000 of the CD3–CD56+ cells on a cell sorter.
Cloning Human NK Cells 3
12. Dilute 10,000 cells in 10 mL of CM. Perform progressive 10-fold limiting dilu-
tions of these cells into CM until the cells have been diluted to 10 cells/mL
(0.5 cells/50 µL) (Note 1). Prepare 50 mL of this final dilution per 10 × 96
U-bottom plates (5 mL/plate).
3.2. Preparation of Feeder Cells
Prepare these cells in parallel with NK cells.
1 To prepare allogeneic feeder cells, collect 50 mL of blood with anticoagulants
from a different blood donor and dilute 1:3 with RPMI + HEPES.
2. Gently lay 30-mL aliquots of diluted blood on 15-mL LSM in a 50mL plastic
tube and centrifuge for 30 min at 940g at room temperature with no brake.
3. Collect PBMCs at the interface between Ficoll and plasma with a pasteur pipet
and wash in RPMI + HEPES by centrifuging for 15 min at 500g.
4. Wash 2×with RPMI + HEPES and collect cells by centrifuging for 10 min at 300g.
5. In parallel, wash 5 × 106 cultured RPMI 8866 cells twice with RPMI+HEPES
(Note 2).
6. Irradiate 5 × 107 PBMC and 5 × 106 RPMI 8866 cells with 5000 Rads.
7. Wash the irradiated cells once with RPMI+HEPES by centrifuging at 300gfor 10 min.
8. Resuspend the irradiated cells together in CM at concentrations of 1 ×106/mL of
PBMC and 1 × 105/mL of RPMI 8866. Add 2 µg/mL of phytohemagglutinin
(PHA) to these cells. This mixture is referred to as “restimulation mix” in subse-
quent procedures.
3.3. Plating and Growing NK Cell Clones
1. Mix 50 mL of NK cells and 50 mL of irradiated feeder cells in a flask and plate
100µL/well in 96-well round bottom plates. Culture at 37°C in 5% CO .
2
2. Inspect culture clones for cell growth after 10–14 d (Note 3). Tranfer each well
with clearly enlarged pellet when viewed from beneath to a single well of a 24-well
plate and add 150 µL of CM. After about 3 d add an additional 250 µL of CM, and
three days later, split to two wells. About 3 d later, transfer cells into one well of a
6-well plate. Expand as necessary when medium is turning yellow. Cells should be
split when they reach a concentration of 1–2 × 106/mL. Usually, cells can be
expanded up to 3–6 wells of a 6-well plate at 1–2 × 106/mL. Clone size ranges
between ~10–40 million cells after 21–28 d of culture without restimulation.
3. Check NK cell surface phenotype of cloned cells by fluorescence-activated cell
sorter (FACS) after staining with anti-CD3 and anti-CD56.
3.4. Maintenance of NK Cell Clones
1. Every 20–30 d NK cell clones slowly stop dividing. At this point they need to be
restimulated with feeder cells.
2. Usually, we take aliquots of 3 × 105 NK cells/mL and mix them with 1 mL of
restimulation mix prepared as described previously and plate them in one well of
a 24-well plate.
4 Cella and Colonna
3. Each well can be expanded into 3 wells of a 6-well plate containing about 3–6
million cells (Notes 4–8).
3.5. Storage of NK Cell Clones
1. Collect 106–107 cells and centrifuge for 5 min at 1200 rpm.
2. Discard supernatant, resuspend cells in 1 mL of freezing mix, and transfer to a
cryotube.
3. Store cryotubes overnight at –80°C and then transfer the tubes to liquid N .
2
3.6. Plating and Growing NK Cell Bulk Cultures
1. For each 96-well round bottom plate mix 10 mL of the first dilution of NK cells
with 10 mL of restimulation mix.
2. Plate the mix in 96-well round bottom plates at 200 µL/well.
3. Culture bulk NK cells for 5–8 d at 37°C in 5% CO .
2
4. Transfer 12 wells of the 96-well plate into one well of a 6-well plate and maintain
by splitting to a new well of a 6-well plate every 2–4 d when medium begins to
yellow. The cells grow best when kept at a concentration of around 1 ×106/mL
(Note 8).
4. Notes
1. When performing serial dilution of NK cells, one should gently resuspend cells
about 5× with pipettor to thoroughly distribute and dilute cells.
2. RPMI 8866 cells should be used when they are in exponential phase of growth.
3. The frequencies of cells capable of extensive proliferation under these culture
conditions are typically 10–20/plate.
4. We have been able to grow cell clones up to 2 billion cells.
5. Individual clones can be analyzed for expression of killer cell Ig-like receptors
(KIRs) and NKG2/CD94 receptors. The expression of these receptors is stable
over years.
6. Individual clonal cultures can also be analyzed for their lytic activity against
K562 target cells.
7. Transfection of clonal cultures by electroporation is virtually impossible. NK
cell clones can be successfull transfected with vaccinia virus-based constructs.
8. One should routinely monitor bulk cultures for growth of cells expressing CD3
by flow cytofluorimetry to be sure that potentially contaminating T cells are not
overgrowing NK cells.
Acknowledgment
The Basel Institute for Immunology was founded and is supported by
Hoffmann-La Roche, CH-4002 Basel.
NK Cell Clones to Analyze Ly49 5
2
Generation of Short-Term Murine Natural Killer Cell
Clones to Analyze Ly49Gene Expression
Werner Held, Bente Lowin-Kropf, and David H. Raulet
1. Introduction
Natural killer (NK) cells express receptors specific for class I major histo-
compatibility complex (MHC) molecules. In the mouse, the class I specific
receptors identified to date belong to the polymorphic Ly49 receptor family.
Engagement of Ly49 receptors with their respective MHC ligands results in
negative regulation of NK cell effector functions, consistent with a critical role
of these receptors in “missing self” recognition. The Ly49 receptors analyzed
so far are clonally distributed such that multiple distinct Ly49 receptors can be
expressed by individual NK cells (for review see refs. 1–3). The finding that
most NK cells that express the Ly49A receptor do so from a single Ly49A
allele (whereby expression can occur from the maternal or the paternal chro-
mosome) may thus reflect a putative receptor distribution process that restricts
the number of Ly49 receptors expressed in a single NK cell (3–5).
Ly49 receptors are encoded by a small gene family that currently comprises
nine members, denoted Ly49A-I(for review see ref.3). The further and more
detailed analysis of Ly49 receptor expression, however, is hampered owing to:
1. The lack of murine NK cell clones.
2. The limited number of monoclonal antibodies (mAbs) that recognize individual
Ly49 receptors or alleles thereof.
We have thus developed and describe in detail below a procedure that allows
the analysis by reverse transcription and polymerase chain reaction (RT-PCR)
of the expression of Ly49 receptor genes in short-term clonal populations of
mouse NK cells.
From: Methods in Molecular Biology, vol. 121: Natural Killer Cell Protocols: Cellular and Molecular Methods
Edited by: K. S. Campbell and M. Colonna © Humana Press Inc., Totowa, NJ
5
6 Held, Lowin-Kropf, and Raulet
2. Materials
1. Mice: C57BL/6J (B6), > 6 wk old.
2. Recombinant human interleukin-2 (rIL-2).
3. Cell culture medium: Dulbecco’s modified Eagle’s medium (DMEM) containing
L-glutamine and 4.5 g/L glucose (Gibco-BRL, Paisley, UK) supplemented with
HEPES (10 mM), 2-mercaptoethanol (5×10-5M), penicillin (50 µg/mL), strep-
tomycin (50 µg/mL), neomycin (100 µg/mL) (all from Gibco-BRL) and 10% fetal
calf serum (FCS).
4. ACK buffer: 0.16 M NH Cl, 0.1 mM Na EDTA, 0.01 M KHCO .
4 2 3
5. Nylon wool columns: Weigh out 0.6 g of nylon wool (type 200L, combed and
scrubbed) (Robbins Scientific, Sunnyvale, CA). Fluff the nylon wool manually
and package into a 10-mL syringe up to the 6-mL mark (i.e., 0.1 g/mL), wrap into
tin foil, and autoclave. Such a column is good for one spleen (i.e., 108 cells).
6. Monoclonal antibodies (mAbs): anti-CD16/CD32 (2.4G2, anti-FcγII/III recep-
tors) hybridoma supernatant to prevent nonspecific staining (available as
FcBlock™ from Pharmingen, San Diego, CA), phycoerythrin (PE)-labeled anti-
CD3 (145.2C11), fluoroisothiocyanate (FITC)-labeled anti-NK1.1 (PK136). Note
that the NK1.1 antigen is expressed only in a few mouse strains including C57Bl/6
(seeAppendix). The anti-DX5 antibody in conjunction with CD3 can be used to
identify NK cells in all mouse strains. All mAbs are available from Pharmingen
(San Diego, CA).
7. Plasticware: 96-Well U-bottom plates (such as Costar, cat. no. 3799, Cambridge,
MA), tissue culture flasks (such as Falcon, cat. no. 3014, Becton Dickinson,
Franklin Lakes, NJ).
8. Fluorescence activated cell sorter (such as FACStarplus [Becton Dickinson, San
Jose, CA]) equipped with a single cell deposition unit.
9. Total RNA isolation reagent (such as Trizol Reagent [Gibco-BRL]).
10. Oligo-dT (such as primer dT , Roche Molecular Biochemicals, cat. no. 814270,
15
Mannheim, Germany).
11. RNase inhibitor (such as RNAguard, 33 U/µL, Pharmacia, cat. no. 27-0815-01,
Uppsala, Sweden).
12. Reverse transcriptase and buffer (such as AMV RT, 20 U/µL, Roche Molecular
Biochemicals #109 118).
13. Taq polymerase (such as AmpliTaq, 5 U/µL, Perkin Elmer, Emeryville, CA).
14. Thermocycler (such as Uno Thermoblock, Biometra, Tampa, FL).
15. Dideoxynucleotides (such as Roche Molecular Biochemicals).
3. Methods
3.1. Cell Culture and Sorting
Lymphokine-activated Killer cells (LAKs) are prepared following the
method described by Karlhofer et al. (6) with modifications.
1. Warm culture medium to 37°C.
2. Attach a three-way stopcock and a 211/ -gage needle to a sterile nylon wool col-
2
umn. Add prewarmed medium to wet nylon wool. Close stopcock and remove air
NK Cell Clones to Analyze Ly49 7
bubbles by firmly tapping to the sides of the column. Run 10 mL of prewarmed
medium through the column. Close stopcock and cover nylon wool with 1 mL of
medium. Incubate 30 min at 37°C in CO incubator.
2
3. Remove the spleen under sterile conditions. Prepare a single cell suspension by
pressing the spleen through a steel mesh into a sterile Petri dish filled with 10 mL
of medium. Transfer the cell suspension into a tube.
4. Leave for 2 min to sediment large debris.
5. Transfer the supernatant into a new tube and centrifuge for 5 min at 500g.
6. Remove the supernatant and lyse red blood cells by resuspending the cell pellet
in 1 mL of ACK buffer, incubate for 1 min, and add 10 mL of medium.
7. Centrifuge for 5 min at 500g, then wash with 10 mL of medium.
8. Resuspend the cell pellet in 2 mL of prewarmed 37°C medium.
9. Drain equilibrated nylon wool column and apply spleen cell suspension.
10. Stop the flow when the suspension has completely entered the column, and add 1 mL
of prewarmed medium to cover the nylon wool.
11. Incubate for 1 h at 37°C in a CO incubator.
2
12. Elute nylon wool nonadherent cells with 7–10 mL of prewarmed medium
(see Note 1). Centrifuge for 5 min at 500g.
13. Resuspend the cell pellet in 10 mL of medium containing rIL-2 at 250 ng/mL.
Transfer to a small (25-cm2) tissue culture flask and culture in a CO incuba-
2
tor for 3 d.
14. Harvest LAKs. Adherent cells are detached by incubating for a few minutes with
cold PBS containing 1.5 mM EDTA. Pool nonadherent and adherent cells.
15. Count viable cells, centrifuge for 5 min at 500g, and resuspend at 106cells/25µL of
2.4G2 hybridoma supernatant to block Fcγ receptors. Incubate for 20 min on ice.
16. Wash 1× with PBS containing 5% FCS.
17. Incubate the cell suspension with appropriate dilutions of PE-conjugated anti-CD3
plus FITC-labeled NK1.1 mAbs in PBS containing 5% FCS at 106 cells/25 µL.
18. Wash as above and resuspend at 2 × 106 cells/mL for single cell sorting.
19. Sort single CD3–NK1.1+blast cells (the latter is defined by an elevated forward
and side scatter) (seeFig. 1) into wells of a round-bottom 96-well plate, which
contain 200 µL of culture medium plus 250 ng/mL of rIL-2.
20. Wrap plates into tin foil and culture in a CO incubator for 7 d (seeNote 2).
2
3.2. RNA Isolation
The remainder of this procedure requires the usual precautions for work
with RNA. The use of aerosol-resistant tips is recommended to prevent cross-
contamination of the samples to be used later for PCR.
1. Visually inspect wells and mark those containing >10 cells (seeNote 3).
2. From marked wells remove as much supernatant as possible without disturbing
the cells.
3. Isolate total cellular RNA using the acid phenol method developed by
Chomczynski and Sacchi (7). Lyse the cells directly in the well by the addition of
8 Held, Lowin-Kropf, and Raulet
Fig. 1. Lymphokine-activated Killer cells used for NK cell cloning. Foreward (FSC)
and side scatter gate (SSC) of d 3 lymphokine activated cells are shown in (A). Cell
surface expression of CD3 and NK1.1 is assessed in blast cells (R1) (identified based
on an elevated FSC /SSC). To derive short term NK cell clones, a single CD3–NK1.1+
cell is deposited per microwell using a cell sorter equipped with a single cell deposi-
tion unit.
200µL of Trizol reagent to which 10 µg/mL carrier tRNA has been added, mix
well by pipetting up and down, and tranfer the lysate to a 1.5-mL Eppendorf tube.
Incubate for 5 min at room temperature (seeNote 4).
4. Add 40 µL of chloroform, shake by hand for 15 s, and incubate for 2–3 min at
room temperature.
5. Centrifuge in a cooled (4°C) microfuge for 15 min at 12,000g.
6. Recover upper, aqueous phase (approx 60% of the total volume) and transfer to a
new 1.5-mL Eppendorf tube.
7. Precipitate RNA by the addition of 100 µL of isopropanol, mix, and incubate at
room temperature for 10 min.
8. Centrifuge in a cooled (4°C) microfuge for 10 min at 12,000g.
9. Wash the RNA pellet by adding 1 mL of 70% EtOH, mix and centrifuge in a
cooled (4°C) microfuge for 5 min at 7500g.
10. Air-dry RNA pellet for 5–10 min.
3.3. Complementary DNA Preparation
1. Resuspend RNA pellet in a total of 7 µL of H O containing 0.3 µL of oligo-dT
2
(150µM) as a primer.
2. Incubate for 5 min at 72°C.
3. Transfer directly on ice.
4. Add 13 µL of reverse transcriptase mix:
4.0µL 5× concentrated reverse transcriptase buffer
5.0µL 2 mM of each dATP, dCTP, dGTP, and dTTP
2.0µL 0.1 mM DTT
1.1µL H O
2
NK Cell Clones to Analyze Ly49 9
0.6µL RNase inhibitor
0.3µL reverse transcriptase
total volume of 20 µL for cDNA preparation
5. Incubate for 1 h at 42°C, store at –20°C.
3.4. Polymerase Chain Reaction
1. Take 1 µL of the cDNA preparation for PCR.
2. Add 29 µL of PCR mix (seeNote 5):
0.6µL of sense primer (10 mM stock)
0.6µL of antisense primer (10 mM stock)
3µL of 10 x PCR buffer containing 15 mM MgCl
2
3µL of 2 mM of each dATP, dCTP, dGTP, and dTTP
0.15µL of Taq polymerase
total volume of 30 µL for PCR preparation.
3. The PCR is performed using the following conditions:
Preheat PCR machine to 92°C, add samples, and leave at 92°C for 3 min, start
cycles:
92°C for 1 min, 55°C for 1 min, 72°C for 1 min
40 cycles
72°C for 5 min, then hold at 4°C.
4. One microliter of this PCR product (seeNote 6) is used for reamplification using
a set of nested PCR primers (seeFig. 2). Conditions for reamplification are the
same as described previously except that the number of cycles is reduced to 20
(seeNote 7).
3.5. Analysis of the PCR Product
1. One tenth (3 µL) of the second PCR product is run on an agarose gel to identify
positive clones.
2. In the case of Ly49A, the presence of correct amplification product is verified by
restriction enzyme digestions of one tenth (3 µL) of the second PCR product.
Add 2 U of restriction enzyme plus the appropriate digestion buffer and bring
volume to a total of 20 µL. Incubate at the appropriate temperature for 1 h(see
Note 8 and Fig. 2).
3. PCR and/or cleavage products are visualized under UV light following gel
electrophoresis in the presence of ethidium bromide.
4. Notes
1. Nylon wool nonadherent cells are mostly T cells and NK cells with few B cells
(<5% of total). Recovery is usually between 15 and 20 ×106cells per B6 spleen.
2. Ly49 receptor expression is stable at least during the 7 d culture period used for
expansion(5).
3. Approx 20–30% of the wells contain more than 10 cells.
4. The lysate can be stored at this stage at –80°C for at least a month.
5. Ly49-specific PCR primers:
