natural killer cell protocols

Methods in Molecular Biology Methods in Molecular Biology VOLUME 121 HUMANA PRESS HUMANA PRESS Natural Killer Cell Protocols Cellular and Molecular Methods Natural Killer Cell Protocols Cellular and Molecular Methods TM TM Edited by Kerry S. Campbell Marco Colonna Edited by Kerry S. Campbell Marco Colonna Cloning Human NK Cells 1 1 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 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 1 ), anti- human CD3 (OKT3, mouse IgG 2a , ATCC, cat. no. CRL-8001), anti-mouse IgG 1 - phycoerythrin (PE; SBA, cat. no. 1070-09), anti-mouse IgG 2a -fluorescein isothiocyanate (FITC; SBA, cat. no. 1080-02). 7. Phosphate-buffered saline (PBS). 8. PBS supplemented with 1% fetal calf serum (FCS). 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 –5 M final 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 940g at 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 2a , 10 µg/mL). Incubate 30 min on ice. 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 1 -PE (1:100 dilution) and goat anti-mouse IgG 2a -FITC (1:50) and incubate on ice for 30 min. 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 × 10 6 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 × 10 6 cultured RPMI 8866 cells twice with RPMI+HEPES (Note 2). 6. Irradiate 5 × 10 7 PBMC and 5 × 10 6 RPMI 8866 cells with 5000 Rads. 7. Wash the irradiated cells once with RPMI+HEPES by centrifuging at 300g for 10 min. 8. Resuspend the irradiated cells together in CM at concentrations of 1 × 10 6 /mL of PBMC and 1 × 10 5 /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 100 µL/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 × 10 6 /mL. Usually, cells can be expanded up to 3–6 wells of a 6-well plate at 1–2 × 10 6 /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 × 10 5 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 10 6 –10 7 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 × 10 6 /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 5 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 2 Generation of Short-Term Murine Natural Killer Cell Clones to Analyze Ly49 Gene 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. 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 -5 M), 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 4 Cl, 0.1 mM Na 2 EDTA, 0.01 M KHCO 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., 10 8 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 (see Appendix). 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 FACStar plus [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 15 , Roche Molecular Biochemicals, cat. no. 814270, 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 21 1 / 2 -gage needle to a sterile nylon wool col- 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 2 incubator. 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 2 incubator. 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-cm 2 ) tissue culture flask and culture in a CO 2 incuba- 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 10 6 cells/25 µL 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 10 6 cells/25 µL. 18. Wash as above and resuspend at 2 × 10 6 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) (see Fig. 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 2 incubator for 7 d (see Note 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 (see Note 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 200 µL 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 (see Note 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 2 O containing 0.3 µL of oligo-dT (150 µM) as a primer. 2. Incubate for 5 min at 72°C. 3. Transfer directly on ice. 4. Add 13 µL of reverse transcriptase mix: 4.0 µL5× concentrated reverse transcriptase buffer 5.0 µL2 mM of each dATP, dCTP, dGTP, and dTTP 2.0 µL 0.1 mM DTT 1.1 µLH 2 O 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. NK Cell Clones to Analyze Ly49 9 0.6 µL RNase inhibitor 0.3 µL 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 (see Note 5): 0.6 µL of sense primer (10 mM stock) 0.6 µL of antisense primer (10 mM stock) 3 µL of 10 x PCR buffer containing 15 mM MgCl 2 3 µL of 2 mM of each dATP, dCTP, dGTP, and dTTP 0.15 µL 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 (see Note 6) is used for reamplification using a set of nested PCR primers (see Fig. 2). Conditions for reamplification are the same as described previously except that the number of cycles is reduced to 20 (see Note 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 × 10 6 cells 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: [...]... morphology, and lytic activity of IL-2-activated natural killer cells J Immunol 150, 3747–3754.s Development of NK and T Cells 25 4 Techniques for Studying Development of Human Natural Killer Cells and T Cells Hergen Spits, Pieter Res, and Ana-Cristina Jaleco 1 Introduction It is now commonly accepted that natural killer (NK) cells are closely related to T cells Some severe combined immunodeficiency (SCID)... subset of human CD3– CD16– natural killer cells Role in cell activation and regulation of cytolytic function J Exp Med 171, 695–714 4 Kuribayashi, K., Gillis, S., Kern, D E., and Henney, C S (1981) Murine NK cell cultures: effects of interleukin 2 and interferon on cell growth and cytotoxic reactivity J Immunol 126, 2321–2327 5 Dennert, G (1980) Cloned lines of natural killer cells Nature 287, 47–49 6... lymphocytes and natural killer cells Cell 69, 139–150 20 Brooks, C G., Georgiou, A., and Jordan, R K (1993) The majority of immature fetal thymocytes can be induced to proliferate to IL-2 and differentiate into cells indistinguishable from mature natural killer cells J Immunol 151, 6645–6656 21 Ballas, Z K., Rasmussen, W L., Alber, C A., and Sandor, M (1997) Ontogeny of thymic NK1 1+ cells J Immunol... cloning of cells with NK cell characteristics (5–8) However, the finding that many of the lines and clones obtained under these conditions expressed CD8 (7), coupled with the discovery that conventional T cells could acquire not only NK cell markers such as asialo-GM1 and NK1.1 (9,10) but also NK cell funcFrom: Methods in Molecular Biology, vol 121: Natural Killer Cell Protocols: Cellular and Molecular... and Brooks, C G (1998) MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells Eur J Immunol 28, 47–56 Fetal Mouse NK Cells 13 3 Cloning and Culturing of Fetal Mouse Natural Killer Cells Colin G Brooks 1 Introduction The ability to study the properties and functions of individual cells is a major goal of cell biologists Nowhere is this more true than in studies of the... discovery of killer cell immunoglobulin-like inhibitory (KIR) receptors (2,3; and Chapter 1) By contrast, for unknown reasons, it has proven exceedingly difficult to clone murine natural killer (NK) cells In the early 1980s, following the discovery that interleukin-2 (IL-2) was not only a growth factor for T cells but also for NK cells (4), laboratories reported a number of reports of the cloning of cells... been described lacking T and NK cells, but having normal numbers of B and myeloid cells, suggesting a common origin of T and NK cells (1) Furthermore, T and NK cells share a number of phenotypic and functional characteristics, not present in B cells (reviewed in [2,3]) In additional, both in humans (4) and mice (5) cells have been found with T and NK cell, but no B cell, progenitor activities There... Method Some precursor cells that express low levels of CD34 should be isolated using a depletion method to enrich for CD34+ thymocytes 1 The most convenient isolation method is to deplete for cells expressing CD4, CD8, and CD3 Because CD3–CD4–CD8– cells contain red blood cells, mature NK cells, and B cells, it is advised to include antibodies against CD19 (B cells), CD56 (NK cells), and glycophorin... tumor target cells, their recognition capacity was found to be remarkably similar to that of adult NK cells Most importantly, different clones of fetal NK cells displayed a similar broad specificity both to each other and to that of uncloned bulk populations of fetal or adult NK cells (24), suggesting that positive recognition of these target cells by NK cells is either dominated by a single NK cell receptor... in development 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 25 26 Spits, Res, and Jaleco of these cells as mice deficient for the common γ chain, shared by the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, lack NK cells Strong perturbations of NK development were also observed . Molecular Biology VOLUME 121 HUMANA PRESS HUMANA PRESS Natural Killer Cell Protocols Cellular and Molecular Methods Natural Killer Cell Protocols Cellular and Molecular Methods TM TM Edited by Kerry. target cells from lysis by Ly49-deficient fetal NK cells. Eur. J. Immunol. 28, 47–56. Fetal Mouse NK Cells 13 13 From: Methods in Molecular Biology, vol. 121: Natural Killer Cell Protocols: Cellular. NJ 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