Molecular Cloning and Characterization of MHC Class I– and II–Restricted Tumor Antigens Recognized by T Cells

Rong‐Fu Wang1

1 The Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.10
DOI:  10.1002/0471142735.im2010s84
Online Posting Date:  February, 2009
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Abstract

T cells play a central role in cancer immunosurveillance, autoimmune, and infectious diseases. Identification of MHC class I– and II–restricted T cell peptides is a critical step for the development of effective vaccines against cancer and infectious diseases. This unit describes a cDNA expression system and a genetic targeting expression system for the cloning of genes encoding for MHC class I– and II–restricted antigens recognized by antigen‐specific CD8+ and CD4+ T cells. Curr. Protoc. Immunol. 84:20.10.1‐20.10.29. © 2009 by John Wiley & Sons, Inc.

Keywords: T helper peptides; tumor antigens; cancer vaccines; immunotherapy; CD4+ T cells; cDNA library; targeting expression; MHC class II

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Tumor Cells and Tumor‐Antigen‐Specific T Cells
  • Basic Protocol 2: Cloning of MHC Class I–Restricted Antigens
  • Support Protocol 1: Testing Antibody Blockage of T Cell Recognition
  • Basic Protocol 3: Column Chromatography of Tumor‐Derived Antigen cDNAs
  • Basic Protocol 4: Ligation of Tumor‐Derived cDNA to the Vector and Transformation of Ligated DNA into E. coli by Electroporation
  • Support Protocol 2: Preparation of cDNA Subpool DNAs
  • Basic Protocol 5: Screening a cDNA Library Using Tumor‐Specific T Cells
  • Isolation of Positive Clones and Identification of Epitopes
  • Support Protocol 3: Isolation of a Single cDNA Encoding a Tumor Antigen
  • Basic Protocol 6: Identification of T Cell Epitopes from a cDNA‐Encoding Protein
  • Identification of MHC Class II‐Restricted Antigens
  • Basic Protocol 7: Determination of Restriction Element for T Cell Recognition
  • Basic Protocol 8: Generation of DR‐Matched 293IMDR Cells as “Professional” APC
  • Basic Protocol 9: Construction of an Invariant Chain (Ii) Fusion Library
  • Basic Protocol 10: Screening of Ii‐Fusion Libraries by CD4+ T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Tumor Cells and Tumor‐Antigen‐Specific T Cells

  Materials
  • Tumor specimen
  • Tumor digestion medium (see recipe)
  • RPMI 1640 medium (Invitrogen), serum‐free
  • T cell growth medium with 6000 IU/ml IL‐2 (see recipe)
  • T cell growth medium with 300 IU/ml (see recipe)
  • Tumor growth medium (see recipe)
  • Cryopreservation medium: 90% (v/v) DMSO/10% (v/v) FBS
  • T cell assay medium (see recipe)
  • Allogenic, MHC class I–matched tumor cells (obtained from different patient or from ATCC)
  • GM‐CSF ELISA kit (R&D Systems)
  • IFN‐γ ELISA kit (Endogen, Inc.)
  • Dissecting instruments: sterile scalpels, forceps, and scissors, at least two of each
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Clay Adams Nutator mixer (BD Bioscience)
  • Standard tabletop centrifuge (e.g., IEC Clinical)
  • 96‐well (flat‐bottom), 24‐well, 12‐well, and 6‐well tissue culture plates, sterile
  • Humidified 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3B), separating CD4+ and CD8+ subpopulations of lymphocytes by immunomagnetic separation (unit 7.4), and ELISA (unit 2.1)

Basic Protocol 2: Cloning of MHC Class I–Restricted Antigens

  Materials
  • Tumor cells (see protocol 1)
  • Tumor growth medium (see recipe)
  • Phosphate‐buffered saline ( appendix 2A)
  • Trizol reagent (Invitrogen, cat. no. 15596)
  • Chloroform
  • Isopropanol
  • 75% (v/v) ethanol
  • RNase‐free (DEPC‐treated) H 2O (unit 10.11)
  • PolyATract kit (Promega, cat. no. Z5200) containing:
    • 35 µl biotinylated oligo(dT) probe (50 pmol/µl)
    • 4.2 ml 20× SSC solution (3 × 1.4 ml)
    • 9 ml streptavidin MagneSphere paramagnetic particles (3 × 3 ml)
    • 75 ml nuclease‐free water (3 × 25 ml)
    • 3 mRNA user tubes
    • 1 MagneSphere magnetic separation stand for 12 × 75–mm tubes
  • 3 M sodium acetate ( appendix 2A)
  • SuperScript kit (Invitrogen, cat. no. 18248‐013) containing:
    • NotI primer‐adapter (0.5 µg/µl)
    • 5× first strand buffer [250 mM Tris⋅Cl (pH 8.3), 375 mM KCl, 15 mM MgCl 2]
    • 0.1 M DTT
    • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
    • SuperScript II RT (200 U/µl)
    • 5× second strand buffer [100 mM Tris⋅Cl (pH 6.9), 450 mM KCl,
    • 23 mM MgCl 2, 0.75 mM β‐NAD+, 50 mM (NH 4) 2SO 4]
    • E. coli DNA ligase (10 U/µl)
    • E. coli DNA polymerase I (10 U/µl)
    • E. coli RNase H (2 U/µl)
    • T4 DNA polymerase (5 U/µl)
    • 5× T4 DNA ligase buffer [250 mM Tris⋅Cl (pH 7.6),
    • 50 mM MgCl 2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000]
    • SalI adapters (1 µg/µl)
    • T4 DNA ligase (1 U/µl)
    • REact 3 buffer
    • NotI (15 U/µl)
    • DEPC‐treated water
    • Plasmid pCMV•SPORT6, NotI‐SalI‐cut (50 ng/µl)
    • Plasmid pSPORT 1, NotI‐SalI‐cut (50 ng/µl)
    • Control RNA (0.5 µg/µl)
    • Yeast tRNA (1 µg/µl)
    • cDNA size fractionation columns
  • 0.5 M EDTA
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol prepared with buffered phenol (unit 10.1)
  • 4.0 M ammonium acetate
  • Absolute ethanol, –20°C
  • 70% (v/v) ethanol
  • REact 3 buffer (Invitrogen)
  • 10 U/µl NotI restriction enzyme (New England Biolabs)
  • 175‐cm2 tissue culture flasks
  • 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated centrifuge
  • 16°, 65°, and 70°C water baths
  • Sterile 5‐ml (and larger) RNase‐free tubes
  • MagneSphere magnetic separation stand for 5‐ml tubes (Promega)
  • Additional reagents and equipment for denaturing agarose gel electrophoresis of RNA (unit 10.12), purification and concentration of DNA from aqueous solution (unit 10.1), spectrophotometric quantitation of nucleic acids ( appendix 3L), and column chromatography of cDNAs ( protocol 4)

Support Protocol 1: Testing Antibody Blockage of T Cell Recognition

  Materials
  • Autologous tumor cells ( protocol 1)
  • T cell assay medium (see recipe)
  • Anti‐class I (A, B, and C) antibody (HB95 from ATCC)
  • Anti‐class (DR, DQ, and DP) antibody (HB145 from ATCC)
  • Anti‐DR antibody (HB55 from ATCC)
  • Isotype‐control antibody
  • IFN‐γ ELISA kit (Endogen, Inc.)
  • γ‐irradiator
  • Additional reagents and equipment for counting viable cells by trypan blue exclusion ( appendix 3B) and ELISA (unit 2.1)

Basic Protocol 3: Column Chromatography of Tumor‐Derived Antigen cDNAs

  Materials
  • cDNA ( protocol 2)
  • TEN buffer (see recipe)
  • 100 mg/ml glycogen (VWR, cat. no. JTM 816‐1)
  • 4.0 M ammonium acetate
  • Absolute ethanol, –20°C
  • cDNA size fractionation columns from SuperScript cDNA fractionation kit (provided with SuperScript kit; Invitrogen, cat. no. 18248‐013)

Basic Protocol 4: Ligation of Tumor‐Derived cDNA to the Vector and Transformation of Ligated DNA into E. coli by Electroporation

  Materials
  • 5× T4 ligase buffer [Invitrogen; 250 mM Tris⋅Cl, pH 7.6, 50 mM MgCl 2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG8000]
  • 100 ng/µl eukaryotic expression vector, e.g., pcDNA3.1 from Invitrogen (BstXI/NotI cut; see unit 10.8 for restriction digestion)
  • Size‐fractionated cDNA ( protocol 4)
  • 1 U/µl T4 DNA ligase
  • 1 µg/µl yeast tRNA (provided with SuperScript kit; Invitrogen, cat no. 18248‐013)
  • 7.5 M ammonium acetate
  • Absolute ethanol, –20°C
  • 70% (v/v) ethanol, –20°C
  • ElectroMAX DH10B electrocompetent E. coli cells (Invitrogen)
  • SOC medium (see recipe)
  • LB medium (see recipe)
  • LB plates with 100 µg/ml ampicillin (see recipe)
  • Cryoprotectant solution: 60% (v/v) LB medium (see recipe)/40% (v/v) glycerol
  • Additional reagents and equipment for electroporation of E. coli ( appendix 3N)

Support Protocol 2: Preparation of cDNA Subpool DNAs

  Materials
  • cDNA library ( protocol 5)
  • Primers 1 and 2 (designed based on vector used, also see unit 10.20; optimization is based on successful amplification of insert DNA)
  • QIAprep 96 miniprep kit (Qiagen) including:
    • 96‐well flat‐bottom block
    • QIAprep 96 Turbo plate
    • Buffer P1
    • Buffer P2
    • Buffer N3
    • Buffer PB
    • Blue collection microtube rack with 1.2‐ml microtubes
  • TB (“terrific broth”) medium (see recipe) containing 100 µg/ml ampicillin
  • 10 mM Tris·Cl, pH 8.5 ( appendix 2A)
  • Centrifuge with microtiter plate carrier
  • QIAvac 96 vacuum manifold and vacuum source
  • 96‐well round‐bottom microtiter plates (Corning, cat. no. 3799)
  • Additional reagents and equipment for PCR (unit 10.20), agarose gel electrophoresis (unit 10.4), and determining DNA concentration ( appendix 3L)

Basic Protocol 5: Screening a cDNA Library Using Tumor‐Specific T Cells

  Materials
  • COS‐7 cells (ATCC no. CRL‐1654)
  • Growth medium for COS‐7 cells: DMEM containing 10% FBS
  • Effectene Transfection Reagent Kit (Qiagen) including:
    • Enhancer
    • EC buffer
    • Effectene Reagent
  • Plasmid DNA encoding an appropriate MHC class I molecule (plasmid is constructed in‐house; protocol 3 outlines procedure for determining appropriate MHC class I molecule)
  • Library DNA, purified ( protocol 6)
  • T cell assay medium (see recipe)
  • Antigen‐specific CD8+ T cells ( protocol 1)
  • 96‐well round bottom microtiter plates (Corning, cat. no. 3799)
  • 5‐ml tubes
  • Additional reagents and equipment for cytokine release assays (Chapter 6)

Support Protocol 3: Isolation of a Single cDNA Encoding a Tumor Antigen

  Materials
  • Positive cDNA pools
  • Qiaprep 96 Turbo kit (Qiagen)
  • Additional reagents and equipment for DNA sequencing (Ausubel et al., , Chapter 7)

Basic Protocol 6: Identification of T Cell Epitopes from a cDNA‐Encoding Protein

  Materials
  • Peptides of interest
  • DMSO
  • PBS ( appendix 2A)
  • MHC class I‐matched EBV‐transformed B cells or 293T cells expressing the appropriate MHC class I molecules
  • RPMI 1640 medium containing 2% FBS
  • T cell assay medium (see recipe)
  • Antigen‐specific T cells ( protocol 1)
  • GM‐CSF ELISA kit (R&D Systems)
  • IFN‐γ ELISA kit (Endogen, Inc.)
  • Flat‐bottom 96‐well plate
  • Additional reagents and equipment for ELISA (unit 2.1)

Basic Protocol 7: Determination of Restriction Element for T Cell Recognition

  Materials
  • Allogenic MHC class II–matched tumor cells
  • RPMI 1640 medium containing 500 U/ml interferon γ (IFN‐γ), 10% FBS, 2 mM glutamine, 100 IU penicillin, and 100 µg/ml streptomycin
  • RPMI 1640 medium (serum‐free)
  • T cell assay medium (see recipe)
  • Antigen‐specific CD4+ T cells ( protocol 1)
  • Anti‐DR antibody (ATCC no. HB55)
  • Anti–MHC class I antibody (ATCC no. HB95)
  • Anti–MHC class II antibody (ATCC no. HB145)
  • MHC class II–matched B cells
  • 96‐well plate
  • Tabletop centrifuge (e.g., IEC Clinical)
  • γ‐irradiator
  • Additional reagents and equipment for counting viable cells ( appendix 3B) and determination of cytokines by ELISA (unit 6.4)

Basic Protocol 8: Generation of DR‐Matched 293IMDR Cells as “Professional” APC

  Materials
  • HEK293IMDR cells ( protocol 11)
  • Growth medium for HEK293IMDR cells: DMEM containing 10% FBS and 2 mM glutamine (no antibiotics)
  • OPTI‐MEM or DMEM (Invitrogen)
  • cDNA pools ( protocol 12; see annotation to step 2, below)
  • Lipofectamine 2000 (Invitrogen)
  • OPTI‐MEM I Medium (Invitrogen)
  • RPMI 1640 medium containing 2% type AB human serum and 120 U recombinant interleukin 2 (IL‐2)
  • Antigen‐specific CD4+ T cells
  • 96‐well plates
  • Tabletop centrifuge with microtiter plate carrier
  • Additional reagents and equipment for cytokine release assays (Chapter 6) and counting viable cells ( appendix 3B)
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Figures

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Literature Cited

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