Characterization and Functional Analysis of Mouse Semi‐invariant Natural T Cells

Amrendra Kumar1, Jelena S. Bezbradica2, Aleksandar K. Stanic3, Sebastian Joyce1

1 Vanderbilt University Medical Center, Nashville, Tennessee, 2 University of Oxford, Oxford, 3 University of Wisconsin‐Madison, Madison
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.13
DOI:  10.1002/cpim.22
Online Posting Date:  April, 2017
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Abstract

Semi‐invariant natural killer T (iNKT) cells are CD1d‐restricted innate‐like lymphocytes that recognize lipid agonists. Activated iNKT cells have immunoregulatory properties. Human and mouse iNKT cell functions elicited by different glycolipid agonists are highly conserved, making the mouse an excellent animal model for understanding iNKT cell biology in vivo. This unit describes basic methods for the characterization and quantification (see Basic Protocol 1) and functional analysis of mouse iNKT cells in vivo or in vitro. This unit also contains protocols that describe enrichment and purification of iNKT cells, generation of CD1d tetramer, and lipid antigen loading onto cell‐bound and soluble CD1d for activation of NKT cell hybridomas. © 2017 by John Wiley & Sons, Inc.

Keywords: CD1d; CD1d tetramers; cells; cytokines; innate immunity; immune regulation; lipid agonists; murine iNKT; NKT cells; self ligands; T cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification, Quantification, and Phenotyping of iNKT Cells by Flow Cytometry
  • Identification, Quantification, and Phenotyping of iNKT Cell Subsets by Flow Cytometry
  • Basic Protocol 2: Identification, Quantification, and Phenotyping of iNKT Cell Subsets by Flow Cytometry Using Cell Surface Markers
  • Alternate Protocol 1: Identification, Quantification, and Phenotyping of iNKT Cell Subsets by Flow Cytometry Using Transcription Factor Expression Profiling
  • Basic Protocol 3: iNKT Cell Functional Responses to Activation In Vivo
  • Alternate Protocol 2: iNKT Cell Functional Responses to Activation In Vitro
  • Basic Protocol 4: Use of NUR77gfp Reporter Mice as a Tool to Investigate iNKT Cell Stimulation by αGalCer
  • Alternate Protocol 3: Use of NUR77gfp Reporter Mice as a Tool to Investigate iNKT Cell Activation In Vivo During Bacterial Infection
  • Basic Protocol 5: Maintenance and Use of Vα14+ NKT Cell Hybridomas in Functional Studies
  • Basic Protocol 6: Antibody‐Mediated Inhibition of Vα14+ NKT Cell Hybridoma Activation to Ascertain CD1d Restriction and the Nature of the iNKT Cell Agonist
  • Basic Protocol 7: Enrichment of iNKT Cells from Mouse Lymphoid Organs Using Magnetic Cell Separation (Positive Selection)
  • Alternate Protocol 4: Enrichment of iNKT Cells From Mouse Lymphoid Organs Using Magnetic Cell Separation (Negative Selection)
  • Alternate Protocol 5: Enrichment of iNKT Cells From Mouse Lymphoid Organs by Flow Sorting (FACS)
  • Support Protocol 1: Production of CD1d‐β2m Monomers and the Preparation of CD1d‐αGalCer Tetramers
  • Support Protocol 2: Lipid Loading on CD1d at the Cell Surface of Antigen‐Presenting Cells
  • Support Protocol 3: Lipid Loading on Plate‐Bound Soluble CD1d
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Identification, Quantification, and Phenotyping of iNKT Cells by Flow Cytometry

  Materials
  • Mouse lymphoid organs (unit 1.9; Reeves & Reeves, )
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% FBS, cold
  • Dasatinib (optional; see recipe)
  • AccuCheck counting beads (optional; Thermo Fisher Scientific, cat. no. PCB100)
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Purified anti‐mouse CD16/CD32 mAb (2.4G2, Fc block, BD Biosciences)
  • Conjugated anti‐mouse CD3ϵ mAb or conjugated anti‐mouse TCRβ mAb (BD Biosciences)
  • Conjugated anti‐mouse CD8α mAb (BD Biosciences)
  • Conjugated anti‐mouse B220 mAb (BD Biosciences)
  • PE‐ APC‐ or Brilliant Violet 421 (BV421)‐conjugated CD1d tetramer (see protocol 13; CD1d tetramer can be obtained from the NIAID tetramer facility)
  • 2% (w/v) paraformaldehyde (see recipe)
  • Conjugated mAbs to other cell surface proteins of interest (as available, BD Biosciences, Molecular Probes, eBiosciences, BioLegend, and Tonbo Biosciences)
  • 15‐ml polypropylene tubes
  • 5‐ml polystyrene FACS tubes or 96‐well round‐bottom polystyrene microtiter plates
  • Sorvall RT7 centrifuge (or equivalent)
  • Four‐, six‐, or more‐color flow cytometer (FACSCalibur, FACSCanto, or LSRII)
  • Analysis software for FACS data (e.g., FlowJo, Treestar; Chapter 5)
  • Additional reagents and equipment for isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), counting cells ( appendix 3A; Strober, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), and FACS analysis (Chapter 5)

Basic Protocol 2: Identification, Quantification, and Phenotyping of iNKT Cell Subsets by Flow Cytometry Using Cell Surface Markers

  Materials
  • Mouse lymphoid organs (unit 1.9; Reeves & Reeves, )
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% FBS, cold
  • Dasatinib (optional; see recipe)
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Purified anti‐mouse CD16/CD32 mAb (Fc block, BD Biosciences)
  • Fluorochrome‐conjugated antibody panels for surface marker‐based subset characterization (see Table 14.13.4 for author‐recommended antibodies/clones and fluorochrome conjugates)
  • BV421‐conjugated CD1d tetramer (see protocol 13; or CD1d tetramer can be obtained from the NIAID tetramer facility)
  • 2% (w/v) paraformaldehyde (see recipe)
Table 4.3.4   MaterialsRecommended mAbs for iNKT Cell Subset Analysis based on Surface Markers

Antigen Fluorochrome Clone
CD103 FITC 2E7
CCR6 PE 29‐2L17
CD24 Percp‐Cy5.5 M1/69
CD27 APC LG.7F9
CD1d tetramer BV421 NA
CD44 V500 IM7
NK1.1 PE‐Cy7 PK136
CD4 BV605 RM4‐5
TCRB APC‐eF780 H57‐597
CD8α Alexa Fluor 700 53‐6.7

  • 15‐ml polypropylene tubes
  • 5‐ml polystyrene FACS tubes or 96‐well round or V‐bottom polystyrene microtiter plates
  • Sorvall RT7 centrifuge (or equivalent)
  • 7‐ to 8 (or more) color flow cytometer (FACSCanto, LSRII, or LSR Fortessa)
  • Additional reagents and equipment for isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), counting cells ( appendix 3A; Strober, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), and FACS analysis (Chapter 5)

Alternate Protocol 1: Identification, Quantification, and Phenotyping of iNKT Cell Subsets by Flow Cytometry Using Transcription Factor Expression Profiling

  Additional Materials (also see protocol 2)
  • Fluorochrome‐conjugated antibody panel for surface marker and nuclear transcription factor (Table 14.13.5) staining
  • Fixation/Permeabilization concentrate (Affimetrix/eBiosciences)
  • Fixation/Permeabilization diluent (Affimetrix/eBiosciences)
  • Permeabilization buffer (Affimetrix/eBiosciences)
Table 4.3.5   Additional Materials (also see protocol 2)Recommended mAbs for iNKT Cell Subset Analysis Based on Transcription Factor Expression Profile

Antigen Fluorochrome Clone
Surface staining
CD24 FITC M1/69
CD1d tetramer BV421 NA
CD3E PE‐Cy7 145‐2C11
CD8a APC‐Cy7 53‐6.7
Nuclear staining
PLZF PE 9E12
RORγt Percp‐Cy5.5 Q31‐378
T‐bet APC 4B10

Basic Protocol 3: iNKT Cell Functional Responses to Activation In Vivo

  Materials
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in U.K., Amy S. Howell of the University of Connecticut at Storrs, and Paul B. Savage of Brigham Young University are generous sources of NKT cell agonists)
  • Vehicle (see recipe)
  • Mice, age matched (∼6 to 10 weeks old)
  • Complete RPMI‐1640 ( appendix 2A)/10% (w/v) FBS, ice cold
  • Brefeldin A (Sigma)
  • FACS buffer (see recipe)
  • Cytofix/Cytoperm fixation/permeabilization buffer for intracellular flow cytometry (BD Biosciences)
  • Perm/Wash staining buffer for intracellular flow cytometry (BD Biosciences)
  • Conjugated anti‐IL‐2 mAb, anti‐IL‐4 mAb, or anti‐IFN‐γ mAb (BD Biosciences)
  • 2% (w/v) paraformaldehyde (see recipe)
  • ELISA blocking buffer: PBS/10% (w/v) FBS
  • 80°C water bath sonicator
  • 1‐ml syringes fitted with 25‐ or 30‐G needles
  • 15‐ml polypropylene tubes
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for anesthetizing animals (unit 1.4; Donovan & Brown, ), injection of animals (unit 1.6; Donovan & Brown, ), euthanizing animals (unit 1.8; Donovan & Brown, ), harvesting mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), blood collection from mouse (unit 1.7; Donovan & Brown, ), preparation of erythrocyte‐free single‐cell suspension (unit 3.1; Kruisbeek, ), counting cells ( appendix 3A; Strober, ), enzyme‐linked immunosorbent assays (ELISA; unit 2.1; Hornbeck, ), and flow cytometry (see protocol 1 and Chapter 5)

Alternate Protocol 2: iNKT Cell Functional Responses to Activation In Vitro

  Materials
  • Mouse lymphoid organs (unit 1.9; Reeves & Reeves, )
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% (w/v) FBS, ice cold and 37°C
  • 5‐(and ‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes)
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in U.K., Amy S. Howell of University of Connecticut at Storrs, and Paul B. Savage of Brigham Young University are generous sources of NKT cell agonists)
  • Vehicle (see recipe)
  • Fixable viability dye eFluor 450 (eBiosciences)
  • 15‐ml polypropylene tubes
  • 96‐well round‐bottom polystyrene microtiter plates with lids
  • 37°C, 5% CO 2 humidified cell culture incubator
  • 80°C water bath sonicator
  • Additional reagents and equipment for harvesting mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparation of erythrocyte‐free single‐cell suspensions (unit 3.1; Kruisbeek, ); ), counting cells ( appendix 3A; Strober, ), CFSE labeling (unit 4.9; Parish, Glidden, & Quah, ), anesthetizing animals (unit 1.4; Donovan & Brown, ), euthanizing animals (unit 1.8; Donovan & Brown, ), enzyme‐linked immunosorbent assays (ELISA; unit 2.1; Hornbeck, ), and flow cytometry (see protocol 1 and Chapter 5)

Basic Protocol 4: Use of NUR77gfp Reporter Mice as a Tool to Investigate iNKT Cell Stimulation by αGalCer

  Materials
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in U.K., Amy S. Howell of University of Connecticut at Storrs ,and Paul B. Savage of Brigham Young University are generous sources of NKT cell agonists)
  • Vehicle (see recipe)
  • C57BL/6 and Nur77gfp reporter mice (C57BL/6‐Tg (Nr4a1‐EGFP/Cre)820Khog/J (Jackson Laboratory), age‐matched (∼6 to 10 weeks old)
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% FBS, cold
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Purified anti‐mouse CD16/CD32 mAb (Fc block, BD Biosciences)
  • Conjugated anti‐mouse CD3ϵ mAb or conjugated anti‐mouse TCRβ mAb (BD Biosciences)
  • Conjugated anti‐mouse CD8α mAb (BD Biosciences)
  • Conjugated anti‐mouse B220 mAb (BD Biosciences)
  • Optional: conjugated CD69 mAb (BD Biosciences)
  • PE‐ APC‐ or BV421‐conjugated CD1d tetramer (see protocol 13; CD1d tetramer can be obtained from the NIAID tetramer facility)
  • 2% (w/v) paraformaldehyde (see recipe)
  • 15‐ml polypropylene tubes
  • 80ºC water bath sonicator
  • 5‐ml polystyrene FACS tubes or 96‐well round or V‐bottom polystyrene microtiter plates
  • Sorvall RT7 centrifuge (or equivalent)
  • Six‐seven color flow cytometer (FACSCanto, LSRII or LSR Fortessa)
  • Additional reagents and equipment for injection of animals (unit 1.6; Donovan & Brown, ), euthanizing animals (unit 1.8; Donovan & Brown, ), isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), counting cells ( appendix 3A; Strober, ), and FACS analysis (Chapter 5)

Alternate Protocol 3: Use of NUR77gfp Reporter Mice as a Tool to Investigate iNKT Cell Activation In Vivo During Bacterial Infection

  Materials
  • F. tularensis live vaccine strain [LVS; unit 14.25 (Elkins, Cowley, & Conlan, ) and unit 19.14 (Conlan, Chen, Bosio, Cowley, & Elkins, )]
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • C57BL/6 and Nur77gfp reporter mice (C57BL/6‐Tg (Nr4a1‐EGFP/Cre)820Khog/J (Jackson Laboratory), age matched (∼6 to 10 weeks old)
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% FBS, cold
  • Collagenase (from Clostridium histolyticum; Sigma, cat. no. C2139‐1G)
  • ACK lysis buffer (Fisher Scientific)
  • AccuCheck counting beads (optional; Thermo Fisher Scientific, cat. no. PCB100)
  • For FACS analysis:
    • Flow cytometry buffer (FACS buffer; see recipe)
    • Purified anti‐mouse CD16/CD32 mAb (Fc block, BD Biosciences)
    • Conjugated anti‐mouse CD3ϵ mAb or conjugated anti‐mouse TCRβ mAb (BD Biosciences)
    • Conjugated anti‐mouse CD8α mAb (BD Biosciences)
    • Conjugated anti‐mouse B220 mAb (BD Biosciences)
    • Optional conjugated CD69 mAb (BD Biosciences)
    • PE‐ APC‐ or BV421‐conjugated CD1d tetramer (see protocol 13; CD1d tetramer can be obtained from the NIAID tetramer facility)
  • 15‐ml polypropylene tubes
  • Dissecting instruments: scalpel and scissors
  • 70‐μm cell strainer
  • Plunger from 5‐ or 10‐ml syringe
  • 5‐ml polystyrene FACS tubes or 96‐well round or V‐bottom polystyrene microtiter plates
  • Sorvall RT7 centrifuge (or equivalent)
  • Six‐seven color flow cytometer (FACSCanto, LSRII or LSR Fortessa)
  • Additional reagents and equipment for anesthetizing animals (unit 1.4; Donovan & Brown, ), injection of animals (unit 1.6; Donovan & Brown, ), euthanizing animals (unit 1.8; Donovan & Brown, ), isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), counting cells ( appendix 3A; Strober, ), and FACS analysis (see Chapter 5, protocol 1, and protocol 6)

Basic Protocol 5: Maintenance and Use of Vα14+ NKT Cell Hybridomas in Functional Studies

  Materials
  • Vα14+ NKT cell hybridomas (Bendelac, ; Gui, Li, Wen, Hardy, & Hayakawa, ); available from either Dr. Albert Bendelac of the University of Chicago or Dr. Kyoko Hayakawa of Fox Chase Cancer Center, Temple Health; our laboratory can provide validated hybridomas to those who have obtained permission for their use from either Dr. Bendelac or Dr. Hayakawa
  • Complete OPTI‐MEM medium (see recipe)
  • CD1d+ stimulator cell lines (made in individual laboratories) or primary cells [e.g., thymocytes, unit 1.9 (Reeves & Reeves, ), or DC, unit 3.7 (Inaba et al., 2009)]
  • Lipid‐loaded soluble mouse CD1d (see protocol 15)
  • Purified anti‐CD3ϵ mAb (BD Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • ELISA blocking buffer: PBS ( appendix 2A)/10% (w/v) FBS
  • 100‐mm tissue culture plates
  • 96‐well flat‐bottom polystyrene microtiter plates with lids
  • 37°C, 5% CO 2 humidified cell culture incubator
  • Sorvall RT7 centrifuge (or equivalent)
  • Additional reagents and equipment for enzyme‐linked immunosorbent assays (ELISA; unit 2.1; Hornbeck, )
NOTE: All cell culture incubations should be performed under sterile conditions in a 37°C, 5% CO 2 humidified incubator unless otherwise specified.

Basic Protocol 6: Antibody‐Mediated Inhibition of Vα14+ NKT Cell Hybridoma Activation to Ascertain CD1d Restriction and the Nature of the iNKT Cell Agonist

  Materials
  • Vα14+ NKT hybridomas ((Bendelac, ; Gui, Li, Wen, Hardy, & Hayakawa, ); available from either Dr. Albert Bendelac of the University of Chicago or Dr. Kyoko Hayakawa of Fox Chase Cancer Center, Temple Health; our laboratory can provide validated hybridomas to those who have obtained permission for their use from either Dr. Bendelac or Dr. Hayakawa)
  • Complete MEM α medium (see recipe)
  • Murine immature DC‐like cell line JAWSII (ATCC) or CD1d+ stimulator cell lines (made in individual laboratories)
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in U.K., Amy S. Howell of the University of Connecticut at Storrs, and Paul B. Savage of Brigham Young University are generous sources of NKT cell agonists)
  • Vehicle
  • Phosphate‐buffered saline (PBS; appendix 2A) L363 monoclonal antibody (Biolegend, cat. no. 140504)
  • Isotype control, purified Mouse IgG2a, κ
  • Complete OPTI‐MEM medium (see recipe)
  • Anti‐mouse CD1d (Clone 1B1) and isotype control purified rat IgG2b, κ (optional)
  • Purified anti‐CD3ϵ mAb (BD Biosciences)
  • 100‐mm tissue culture plates
  • 96‐well flat‐bottomed polystyrene microtiter plates with lids
  • Sorvall RT7 centrifuge (or equivalent)
  • 37°C, 5% CO 2 humidified cell culture incubator
  • Additional reagents and equipment for maintaining Vα14+ NKT cell hybridomas ( protocol 8), enzyme‐linked immunosorbent assays (ELISA; unit 2.1; Hornbeck, )
NOTE: All cell culture incubations should be performed under sterile conditions in a 37°C, 5% CO 2 humidified incubator, unless otherwise specified.

Basic Protocol 7: Enrichment of iNKT Cells from Mouse Lymphoid Organs Using Magnetic Cell Separation (Positive Selection)

  Materials
  • Mouse lymphoid organs (unit 1.9; Reeves & Reeves, )
  • Complete RPMI‐1640/10% FBS, cold
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Purified anti‐mouse CD16/CD32 mAb (2.4G2, Fc block, BD Biosciences)
  • PE‐ or APC‐conjugated CD1d tetramer (see protocol 13; CD1d tetramer can be obtained from the NIAID tetramer facility)
  • MACS running buffer (see recipe)
  • MACS rinsing buffer (see recipe)
  • Anti‐PE or anti‐APC microbeads (Miltenyi Biotech)
  • 15‐ml polypropylene tubes5‐ml capped polystyrene MACS tubes
  • Sorvall RT7 centrifuge (or equivalent)
  • AutoMACS sorter (Miltenyi Biotec)
  • 40‐μm filter
  • Additional reagents and equipment for isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), counting cells ( appendix 3A; Strober, ), immunomagnetic separation (unit 3.5; Thornton, ), and FACS analysis (Chapter 5)

Alternate Protocol 4: Enrichment of iNKT Cells From Mouse Lymphoid Organs Using Magnetic Cell Separation (Negative Selection)

  Materials
  • Mouse lymphoid organs (unit 1.9)
  • Complete RPMI‐1640/10% FBS, cold
  • MACS running buffer (see recipe)
  • Anti‐mouse B220‐coated magnetic beads (Miltenyi Biotec)
  • Anti‐mouse CD8α‐coated magnetic beads (Miltenyi Biotec)
  • Anti‐mouse CD11c‐coated magnetic beads (Miltenyi Biotec)
  • Anti‐mouse CD11b‐coated magnetic beads (Miltenyi Biotec)
  • MACS rinsing buffer (see recipe)15‐ml polypropylene tubes
  • 5‐ml capped polystyrene MACS tubes
  • Sorvall RT7 centrifuge (or equivalent)40‐μm filter
  • AutoMACS sorter (Miltenyi Biotec)
  • Additional reagents and equipment for isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), ), counting cells ( appendix 3A; Strober, ), immunomagnetic separation (unit 3.5; Thornton, ), and FACS analysis (Chapter 5)

Alternate Protocol 5: Enrichment of iNKT Cells From Mouse Lymphoid Organs by Flow Sorting (FACS)

  Materials
  • Mouse lymphoid organs (unit 1.9; Reeves & Reeves, )
  • Complete RPMI‐1640/10% FBS, cold
  • Flow cytometry buffer (FACS buffer; see recipe)
  • Purified anti‐mouse CD16/CD32 mAb (2.4G2, Fc block, BD Biosciences)
  • PE‐ or APC‐ or BV421‐conjugated CD1d tetramer (see protocol 13; CD1d tetramer can be obtained from the NIAID tetramer facility)
  • For total iNKT cells or sorting iNKT at different stages of development: antibody panel as in protocol 1 (Table 14.13.3)
  • For iNKT cell subset sorting: antibody panel as in protocol 2 (Table 14.13.4)
  • AccuCheck counting beads (optional; Thermo Fisher Scientific, cat. no. PCB100)
  • 15‐ml polypropylene tubes
  • 70‐μm cell strainer
  • 5‐ml polystyrene FACS tubes
  • 7‐ to 8 (or more) color flow cytometer (FACSCanto, LSRII, or LSR Fortessa)
  • Sorvall RT7 centrifuge (or equivalent)
  • Additional reagents and equipment for isolating mouse lymphoid organs (unit 1.9; Reeves & Reeves, ), preparing single‐cell suspension (unit 3.1; Kruisbeek, ), and FACS analysis (Chapter 5)

Support Protocol 1: Production of CD1d‐β2m Monomers and the Preparation of CD1d‐αGalCer Tetramers

  Materials
  • High‐Five (Invitrogen) cells transfected with pIZT‐mouse β2m and pIB‐sCD1d1BSP His‐6 (Stanic et al., )
  • Complete Express Five serum‐free medium (complete Express Five SFM; see recipe)
  • Blasticidin S HCl (Invitrogen)
  • Zeocin selection reagent (Invitrogen)
  • 0.05% (w/v) sodium azide
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.0
  • HisTrap kit (Amersham Biosciences) containing:
    • 0.1 M NiSO 4
    • Buffer A
    • Phosphate buffer, pH 7.4
    • 2 M imidazole, pH 7.4
    • HisTrap columns
    • 5‐ml syringe
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • Micro BCA protein assay kit (Pierce)
  • BirA enzyme kit (Avidity) containing:
    • 10× Biomix‐A: 0.5 M bicine buffer, pH 8.3
    • 10× Biomix‐B: 100 mM ATP, 100 mM magnesium acetate, 500 μM D‐biotin
    • BirA enzyme
    • Additional D‐biotin
  • 10 μM αGalCer (or other glycolipids of choice per glycolipid‐specific protocol)
  • 1 mg/ml streptavidin‐APC, streptavidin‐PE, (Molecular Probes), or streptavidin‐BV421 (BioLegend)
  • 150‐ml tissue culture flasks
  • Sorvall RT7 centrifuge (or equivalent)
  • 1‐liter sterile, polystyrene bottles
  • Centramate tangential‐flow concentration system with a 30,000 MWCO Omega membrane (Pall Filtron)
  • Slide‐A‐Lyzer dialysis cassettes (3000 MWCO; Pierce)
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4; Gallagher, ) and staining of proteins in gels (unit 8.9; Sasse & Gallagher, )

Support Protocol 2: Lipid Loading on CD1d at the Cell Surface of Antigen‐Presenting Cells

  Materials
  • MACS‐purified mouse CD1d+ antigen‐presenting cells [e.g., DC, B cells, macrophages: units 3.5 (Thornton, 14.13) & 3.7 (Inaba et al. (2009)] or CD1d+ cell lines
  • Complete RPMI‐1640 ( appendix 2A) supplemented with 10% FBS, room temperature and 37°C
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in UK, Amy S. Howell of the University of Connecticut at Storrs and Paul B. Savage of Brigham Young University in Provo are generous sources of NKT cell agonists)
  • Vehicle (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete OPTI‐MEM medium (see recipe)
  • Low‐binding 96‐well flat‐bottom polystyrene microtiter plates with lids or 5‐ml capped FACS tubes
  • 80°C water bath sonicator
  • 1‐ml syringes fitted with 25‐ or 30‐G needles
  • 15‐ml polypropylene tubes
  • Additional reagents and equipment for injection of animals (unit 1.6; Donovan & Brown, ) and cell sorting [units 3.5 (Thornton, 14.13) & 3.7 (Inaba et al., 2009)]

Support Protocol 3: Lipid Loading on Plate‐Bound Soluble CD1d

  Materials
  • Soluble CD1d monomers (Joyce et al., and see protocol 13)
  • Phosphate‐buffered saline (PBS; appendix 2A), room temperature and 37°C
  • 5 μg/ml BSA in PBS
  • αGalCer (Funakoshi, cat. no. KRN7000) or other glycolipids (as available from the NIAID tetramer facility; Professors Gurdyal Besra of the University of Birmingham in the U.K., Amy S. Howell of the University of Connecticut at Storrs, and Paul B. Savage of Brigham Young University in Provo are generous sources of NKT cell agonists)
  • Vehicle (see recipe)
  • 96‐well ELISA microtiter plates with lid
  • 80°C water bath sonicator
  • Sorvall RT7 rotor (or equivalent)
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Figures

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Literature Cited

Literature Cited
  Akbari, O., Stock, P., Meyer, E., Kronenberg, M., Sidobre, S., Nakayama, T., …, Umetsu D. T. (2003). Essential role of NKT cells producing IL‐4 and IL‐13 in the development of allergen‐induced airway hyperreactivity. Nature Medicine, 9, 582–588. doi: 10.1038/nm851
  Albacker, L. A., Chaudhary, V., Chang, Y. J., Kim, H. Y., Chuang, Y. T., Pichavant, M., … Umetsu, D. T. (2013). Invariant natural killer T cells recognize a fungal glycosphingolipid that can induce airway hyperreactivity. Nature Medicine, 19, 1297–1304. doi: 10.1038/nm.3321
  Aspeslagh, S., Li, Y., Yu, E. D., Pauwels, N., Trappeniers, M., Girardi, E., … Elewaut, D. (2011). Galactose‐modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis. The EMBO Journal, 30, 2294–2305. doi: 10.1038/emboj.2011.145
  Aspeslagh, S., Nemcovic, M., Pauwels, N., Venken, K., Wang, J., Van Calenbergh, S., … Elewaut, D. (2013). Enhanced TCR footprint by a novel glycolipid increases NKT‐dependent tumor protection. Journal of Immunology, 191, 2916–2925. doi: 10.4049/jimmunol.1203134
  Bendelac, A. (1995). Positive selection of mouse NK1+ T cells by CD1‐expressing cortical thymocytes. The Journal of Experimental Medicine, 182, 2091–2096. doi: 10.1084/jem.182.6.2091
  Bendelac, A., Hunziker, R. D., & Lantz, O. (1996). Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. The Journal of Experimental Medicine, 184, 1285–1293. doi: 10.1084/jem.184.4.1285
  Bendelac, A., Killeen, N., Littman, D. R., & Schwartz, R. H. (1994). A subset of CD4+ thymocytes selected by MHC class I molecules. Science (New York, N.Y.), 263, 1774–1778. doi: 10.1126/science.7907820
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