Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins

Jerome Thiery1, Michael Walch1, Danielle K. Jensen1, Denis Martinvalet1, Judy Lieberman1

1 Department of Pediatrics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.37
DOI:  10.1002/0471143030.cb0337s47
Online Posting Date:  June, 2010
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Abstract

Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography. Curr. Protoc. Cell Biol. 47:3.37.1‐3.37.29. © 2010 by John Wiley & Sons, Inc.

Keywords: cytotoxic granules; CTL; NK cells; perforin; granzyme; granulysin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Borregaard Method of CTL and NK Cell Cytotoxic Granule Isolation
  • Alternate Protocol 1: Purification of Cytotoxic Granules from Rat RNK‐16 Cells
  • Basic Protocol 2: Purification of Perforin from Cytotoxic Granules
  • Alternate Protocol 2: Purification of Granzyme and Granulysin from Cytotoxic Granules
  • Support Protocol 1: Immunostaining Assay for Perforin and/or Granzyme Expression
  • Support Protocol 2: Flow Cytometry Assay for Perforin and/or Granzyme Expression
  • Support Protocol 3: Measuring Perforin Activity by Hemoglobin Release Assay
  • Support Protocol 4: Measuring Perforin Activity by Propidium Iodide Uptake
  • Support Protocol 5: Measuring GzmB Activity
  • Support Protocol 6: Measuring GzmA Activity
  • Support Protocol 7: Measuring GNLY‐Mediated Antibacterial Activity by Colony‐Forming Unit Assay
  • Support Protocol 8: Measuring GNLY‐Mediated Antibacterial Activity by Turbidimetry
  • Support Protocol 9: Granzyme‐Mediated Chromium Release Apoptosis Assay
  • Support Protocol 10: Granzyme‐Mediated Annexin‐V/Propidium Iodide (PI) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Borregaard Method of CTL and NK Cell Cytotoxic Granule Isolation

  Materials
  • Human blood or buffy coats for LAK cell production or YT‐Indy NK cells (ATCC)
  • Ficoll‐Paque PLUS (GE Healthcare)
  • K10 medium (see recipe)
  • K10 + IL‐2: K10 medium plus 1000 IU/ml human IL‐2 (Chiron)
  • PHA‐P (Sigma)
  • Hanks' balanced salt solution (HBSS) without calcium, magnesium, and phenol red
  • Relaxation buffer (see recipe)
  • Continuous 40% (w/v) adjusted Percoll gradient (see recipe)
  • Solubilization buffer (see recipe)
  • 50‐ml centrifuge tubes
  • Centrifuge
  • 1000‐µl pipettor or serological pipet
  • Cavitation/disruption bomb (Parr Instrument Company)
  • Magnetic stirrer
  • Refrigerated centrifuge with Sorvall SS‐34 rotor (or equivalent), and corresponding 26‐ml rigid polycarbonate tube (Beckman Instruments)
  • 20‐G spinal needle (Popper & Sons)
  • Beckman ultracentrifuge with Sw28Ti rotor (or equivalent) and thick‐wall polycarbonate tubes (cat. no. 355631)
  • Additional reagents and equipment for counting the cells (unit 1.1), flow cytometry ( protocol 6), and fluorescence microscopy ( protocol 5)
NOTE: All procedures, buffers, tubes, and equipment must be maintained at 4°C during the purification unless otherwise noted.NOTE: Make sure to handle human blood products with universal precautions following the rules of your institution.

Alternate Protocol 1: Purification of Cytotoxic Granules from Rat RNK‐16 Cells

  Materials
  • F344 rats (200 to 225 g; Harlan Laboratories)
  • Pristane (2,5,10,14‐tetramethyl‐pentadecane; Sigma)
  • RNK‐16 cells
  • Hanks' balanced salt solution (HBSS) without calcium, magnesium, and phenol red
  • HHH buffer (see recipe)
  • Ice
  • Red blood cell lysis buffer (Sigma)
  • 3‐ml syringe and 23‐G needle
  • Dissecting pad
  • Scissors
  • 10‐ml pipet
  • Additional reagents and equipment for euthanizing the rat (Donovan and Brown, )

Basic Protocol 2: Purification of Perforin from Cytotoxic Granules

  Materials
  • Cytotoxic granules ( protocol 1), frozen
  • Ice
  • Equilibration buffer A1 (see recipe)
  • IMAC resin charged with Cobalt (TALON Superflow Metal Affinity Resin; Clontech)
  • 0.5 M EGTA in H 2O
  • Elution buffer B1 (see recipe)
  • BCA assay (Pierce)
  • Rabbit anti‐PFN (Cell Signaling, cat. no. 3693)
  • 0.45‐µm low‐protein‐binding syringe filter (Millipore)
  • 10‐ml desalting columns (Econo‐Pac 10DG disposable chromatography columns; Biorad)
  • 50‐ml Superloop (Amersham)
  • Fast Protein Liquid Chromatography (FPLC) system (Biorad) and columns (Pharmacia Biotechnology)
  • Ultrafiltration concentrator (Amicon Ultra centrifugal filter device with low binding; Millipore)
  • Nitrocellulose membrane
  • Additional reagents and equipment for hemoglobin release assay ( protocol 6), BCA assay ( appendix 3H), SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and silver staining (unit 6.6)
NOTE: All buffers and suspensions are kept on ice.

Alternate Protocol 2: Purification of Granzyme and Granulysin from Cytotoxic Granules

  Materials
  • Granules from YT‐Indy cells ( protocol 1)
  • Phenyl Sepharose chromatography elution buffers A2 and B2 (see reciperecipes)
  • Liquid nitrogen
  • Heparin chromatography buffers A3 and B3 (see reciperecipes)
  • SP Sepharose (or MonoS) chromatography elution buffers A4 and B4 (see reciperecipes)
  • BCA protein assay kit (Pierce)
  • Fast Protein Liquid Chromatography (FPLC) and columns (GE Healthcare)
  • Centrifuge
  • 0.45‐µm low‐protein‐binding syringe filter (Millipore)
  • Phenyl Sepharose High Performance, SP Sepharose High Performance, 5 ml of each densely packed in a column (GE Healthcare)
  • Ultracentrifugation concentrator
  • HiTrap heparin column, 5 ml (GE Healthcare)
  • MonoS column (GE Healthcare)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), staining with Coomassie brilliant blue (unit 6.6), and immunoblotting (unit 6.2)
NOTE: For the purification of rat granzymes the use of a MonoS column (GE Healthcare) instead of SP Sepharose is highly recommended.

Support Protocol 1: Immunostaining Assay for Perforin and/or Granzyme Expression

  Materials
  • Poly‐L‐lysine (Sigma)
  • Cultures of CTL or NK cells
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐031‐CV)
  • 4% (w/v) PFA in PBS, pH 7.4
  • Permeabilization buffer: 0.2% (v/v) Triton X‐100 in PBS
  • Blocking buffer: 10% (v/v) FBS in PBS
  • Mouse anti‐human PFN, clone Pf80/164 (Mabtech); 1:600 dilution for microscopy
  • Mouse anti‐human GzmB, clone GB11 (Caltag Laboratories); 1:300 dilution for microscopy
  • Mouse anti‐human GzmA, clone CB9 (BD Pharmingen); 1:300 dilution for microscopy
  • Mouse anti‐granulysin (BD Pharmingen)
  • Incubation buffer: PBS/0.05% (v/v) Triton X‐100
  • Fluorochrome‐conjugated secondary antibody (Molecular Probe)
  • Vectashield mounting medium containing DAPI (Vector Laboratories)
  • 18‐mm circle coverslips
  • 96‐well plates
  • Centrifuge, adaptors for plates
  • Fluorescence microscope

Support Protocol 2: Flow Cytometry Assay for Perforin and/or Granzyme Expression

  Materials
  • Jurkat (ATCC), LAK cells, or YT‐Indy cells from protocol 1 (step 10)
  • 4% (w/v) PFA in PBS, pH 7.4
  • FACS buffer (see recipe)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐031‐CV)
  • Saponin (Sigma)
  • Mouse anti‐human PFN, clone δG9 (BD Pharmingen) for flow cytometry
  • Mouse anti‐granulysin (BD Pharmingen) for flow cytometry
  • Mouse anti‐GzmA (clone CB9, BD Pharmingen)
  • Mouse anti‐GzmB (clone GB11, Catlag)
  • Secondary antibody
  • Microcentrifuge tubes or 96‐well V‐bottom plates
  • Centrifuge
  • Flow cytometer

Support Protocol 3: Measuring Perforin Activity by Hemoglobin Release Assay

  Materials
  • Sheep red blood cells (Rockland Immunochemicals)
  • Hanks' balanced salt solution (HBSS), without calcium, magnesium, and phenol red, ice cold
  • HBSS + 4 mM CaCl 2, ice cold
  • FPLC fractions of perforin purification ( protocol 3)
  • Saponin (Sigma)
  • 96‐well round‐bottom microplate
  • 96‐well flat‐bottom microplates
  • Microplate reader

Support Protocol 4: Measuring Perforin Activity by Propidium Iodide Uptake

  Materials
  • Target cells
  • Buffer C (see recipe)
  • FPLC fractions from perforin purification ( protocol 3)
  • Buffer P (see recipe)
  • An5 buffer
  • Propidium iodide (PI; Sigma)
  • 37°C incubator
  • Flow cytometer

Support Protocol 5: Measuring GzmB Activity

  Materials
  • AAD assay buffer (see recipe)
  • FPLC fractions of GzmB purification ( protocol 4)
  • 96‐well flat‐bottom plates
  • Microplate reader

Support Protocol 6: Measuring GzmA Activity

  Materials
  • BLT assay buffer (see recipe)
  • FPLC fractions for GzmA purification ( protocol 4)
  • 96‐well flat‐bottom plate
  • 37°C incubator
  • Microplate reader

Support Protocol 7: Measuring GNLY‐Mediated Antibacterial Activity by Colony‐Forming Unit Assay

  Materials
  • Bacteria: E. coli, Listeria, Salmonella, or whatever lab‐strain is available
  • LB medium and LB agar plates ( appendix 2A)
  • Colony forming and turbidimetry unit assay buffer (see recipe)
  • Fractions for GNLY purification ( protocol 4)
  • 37°C incubator
NOTE: NaCl concentrations higher than 50 mM interfere with GNLY activity.

Support Protocol 8: Measuring GNLY‐Mediated Antibacterial Activity by Turbidimetry

  Materials
  • Bacteria: E. coli, Listeria, Salmonella, any non‐virulent bacterial lab‐strain
  • LB medium ( appendix 2A)
  • Colony forming and turbidimetry unit assay buffer (see recipe)
  • Fractions for GNLY purification ( protocol 4)
  • 96‐well transparent flat‐bottomed plates
  • 37°C incubator
  • Microplate reader (with ability to monitor kinetics at 37°C and discontinuous shaking; Spectra Max 340PC, Molecular Devices)

Support Protocol 9: Granzyme‐Mediated Chromium Release Apoptosis Assay

  Materials
  • Target cells
  • K10 medium: RPMI 1640 + 10% (v/v) FBS
  • 51Cr‐labeled Na 2CrO 4 (NEN BioLabs), 5 µCi/µl
  • Hanks' balanced salt solution (HBSS)
  • Buffer C (see recipe)
  • Purified granzymes and perforin
  • Buffer P (see recipe)
  • 96‐well V‐bottom plate
  • 37°C incubator
  • TopCount scintillation counter and LumaPlate 96 (PerkinElmer)

Support Protocol 10: Granzyme‐Mediated Annexin‐V/Propidium Iodide (PI) Assay

  Materials
  • Target cells
  • Buffer C (see recipe)
  • Buffer P (see recipe)
  • Purified granzymes and perforin
  • An5 buffer (see recipe)
  • Annexin‐V‐APC (Caltag)
  • Propidium iodide (PI)
  • 96‐well V‐bottom plates
  • 37°C incubator
  • Centrifuge with plate‐adaptor
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Figures

Videos

Literature Cited

Literature Cited
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