Unbiased Molecular Analysis of T Cell Receptor Expression Using Template‐Switch Anchored RT‐PCR

Máire F. Quigley1, Jorge R. Almeida1, David A. Price2, Daniel C. Douek1

1 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 2 Department of Infection, Immunity, and Biochemistry, Cardiff University School of Medicine, Heath Park, Cardiff, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.33
DOI:  10.1002/0471142735.im1033s94
Online Posting Date:  August, 2011
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Abstract

A detailed knowledge of the principles that guide clonal selection within the memory and effector T cell pools is essential to further our understanding of the factors that influence effective T cell‐mediated immunity and has direct implications for the rational design of vaccines and immunotherapies. This unit provides methods for the unbiased quantification and characterization of all expressed T cell receptor (TCR) gene products within any defined T cell population. The approach is based on a template‐switch anchored reverse transcription–polymerase chain reaction (RT‐PCR) and is optimized for the analysis of antigen‐specific T cells isolated directly ex vivo. Curr. Protoc. Immunol. 94:10.33.1‐10.33.16. © 2011 by John Wiley & Sons, Inc.

Keywords: T cell receptor; polymerase chain reaction; antigen‐specific T cell

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Antigen‐Specific T Cell Populations for Analysis of TCR Gene Expression
  • Basic Protocol 2: Extraction of mRNA from Isolated T Cells
  • Basic Protocol 3: Synthesis of Anchor Sequence‐Containing cDNA
  • Basic Protocol 4: cDNA Clean‐Up Using Nucleospin Extract II Columns
  • Basic Protocol 5: PCR Amplification of Rearranged TCR Products
  • Support Protocol 1: Remedy for Unsuccessful PCR Amplification
  • Basic Protocol 6: Gel Extraction and Purification of Amplicons
  • Basic Protocol 7: Amplicon Ligation into TA Cloning Vector
  • Basic Protocol 8: Vector Transformation into Competent E. coli
  • Basic Protocol 9: Colony PCR and Sequencing
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Antigen‐Specific T Cell Populations for Analysis of TCR Gene Expression

  Materials
  • Single cell suspension of live T cells defined with an appropriate staining procedure
  • RNAlater (Applied Biosystems)
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • Vortex, optional
  • Microcentrifuge
  • Flow‐cytometric cell sorter

Basic Protocol 2: Extraction of mRNA from Isolated T Cells

  Materials
  • Oligotex Direct mRNA Mini kit (Qiagen) containing:
    • Buffer OL1 (lysis buffer)
    • Buffer ODB (dilution buffer)
    • Buffer OW1 (wash buffer 1)
    • Buffer OW2 (wash buffer 2)
    • Buffer OEB (elution buffer; incubate at 70°C)
    • Oligotex suspension (incubate at 37°C)
  • 2‐mercaptoethanol (2‐ME; Sigma‐Aldrich)
  • Frozen vial of sorted cells in RNAlater (see protocol 1)
  • Two heating blocks or thermomixers (37°C, 70°C)
  • Vortex
  • Microcentrifuge
  • QIAshredders with spin columns (Qiagen)
  • 2‐ml collection tubes
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • Spin columns
  • −80°C freezer

Basic Protocol 3: Synthesis of Anchor Sequence‐Containing cDNA

  Materials
  • SMARTer PCR cDNA Synthesis kit (Clontech) containing:
    • SMART II anchor oligo (12 µM): 5′‐AAGCAGTGGTATCAACGCAGAGTACGCGGG‐3′ containing three G ribonucleotides (not dG) at the end
  • 5′ CDS oligo(dT) primer (12 µM): 5′‐(T) 25VN‐3′ (V = A, C, G; N = A, C, G, T)
  • PolyA+ mRNA (see protocol 2)
  • 5× RT buffer: 250 mM Tris⋅Cl (pH 8.3), 375 mM KCl and 30 mM MgCl 2
  • Dithiothreitol (DTT, 20 mM; Invitrogen)
  • RNaseOUT (RNase inhibitor; Invitrogen)
  • dNTP mix (10 mM; Invitrogen)
  • Superscript II RNase H Reverse Transcriptase (RT; 200 U/µl; Invitrogen)
  • Tricine buffer: 10 mM Tricine‐KOH (pH 8.5), 1 mM EDTA
  • Two heating blocks (42°C, 70°C)
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • −20°C freezer
  • Microcentrifuge
  • Pipets
NOTE: The SMARTer PCR cDNA Synthesis Kit (Clontech) contains several components required for cDNA synthesis, including a proprietary modified SMART oligo, which can be successfully used with this method. However, the 5′ CDS oligo(dT) should be synthesized as described in the materials list. The 5′ PCR primer II A from the SMARTer PCR cDNA Synthesis Kit (Clontech) can be used as an alternative to the Universal Primer Mix in protocol 5.

Basic Protocol 4: cDNA Clean‐Up Using Nucleospin Extract II Columns

  Materials
  • cDNA (see protocol 3)
  • NucleoSpin Extract II kit (Clontech) containing:
    • Buffer NT
    • Buffer NT3 (requires addition of 24 ml ethanol)
    • Buffer NE
    • NucleoSpin Extract II Column with clear collection tube
  • DNase/RNase‐free water, molecular biology grade (Sigma)
  • ≥99.5% ethanol
  • Microcentrifuge
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)

Basic Protocol 5: PCR Amplification of Rearranged TCR Products

  Materials
  • cDNA (stored at −80°C; see protocol 4)
  • Advantage2 PCR Kit (Clontech) containing:
    • 10× PCR buffer
    • AdvanTaq2 (a mixture of two enzymes, polymerase and proofreading)
    • PCR‐grade water
    • dNTP mix (10 mM)
  • 10× UPM (Universal Primer Mix); the 10× UPM can be prepared by mixing the following primers at the indicated concentrations:
    • Long (0.4 µM): 5′‐CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
    • Short (2 µM): 5′‐CTAATACGACTCACTATAGGGC
  • MBC2 primer [25 µM; custom‐synthesized commercially (e.g., Invitrogen)]
  • 100‐bp DNA ladder
  • 1% agarose gel made with 1× TAE buffer (unit 10.4)
  • SYBR Gold (Invitrogen)
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • 0.2‐ml PCR tubes
  • Microcentrifuge
  • Thermal cycler
  • Gel box/thick combs/power source
  • UV illuminator or blue light transilluminator
  • Disposable scalpel or disposable genecatcher (The Gel Company)
  • Additional reagents and equipment for running the product on a 1% agarose gel (unit 10.4)
NOTE: The Advantage 2 PCR Kit contains all necessary reagents except the TCR‐specific primer and the 10× UPM.

Support Protocol 1: Remedy for Unsuccessful PCR Amplification

  Materials
  • mRNA (see protocol 2)
  • DNase/RNase‐free water, molecular biology grade (Sigma)
  • Amicon Ultra 0.5‐ml centrifugal filters (Millipore)
  • Collection tubes
  • Microcentrifuge

Basic Protocol 6: Gel Extraction and Purification of Amplicons

  Materials
  • Gel‐bound PCR product (see protocol 5)
  • NucleoSpin Extract II kit (Clontech) containing:
    • Buffer NT
    • Buffer NT3 (requires addition of 24 ml ethanol)
    • Buffer NE
    • NucleoSpin Extract II Column with clear collection tube
  • ≥99.5% ethanol
  • Scale
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • Thermomixer (50°C)
  • Microcentrifuge
  • Vortex

Basic Protocol 7: Amplicon Ligation into TA Cloning Vector

  Materials
  • Gel‐extracted PCR product (see protocol 7)
  • pGEM‐T Easy Vector System I kit (Promega) containing:
    • pGEM‐T easy vector
    • T4 DNA ligase
    • 2× ligation buffer #1
    • Control insert
  • DNase/RNase‐free water, molecular biology grade (Sigma)
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • Vortex
  • Microcentrifuge
  • 4°C incubator

Basic Protocol 8: Vector Transformation into Competent E. coli

  Materials
  • Ligation product (see protocol 8)
  • Transformation‐competent, ampicillin‐sensitive E. coli cells (e.g., Max Efficiency DH5α Competent Cells; Invitrogen)
  • Ice
  • SOC medium (Invitrogen)
  • Beaker containing ethanol
  • LB agar plates containing 100 µg/ml ampicillin, 50 µg/ml isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), and 50 µg/ml X‐galactosidase (X‐gal)
  • 1.5‐ml polypropylene O‐ring microcentrifuge tubes (Sarstedt)
  • Heating block (42°C)
  • Thermomixer (37°C)
  • Bunsen burner
  • Plate spinner
  • Glass spreader
  • 37°C incubator

Basic Protocol 9: Colony PCR and Sequencing

  Materials
  • Transformation plates (see protocol 9)
  • Platinum Taq DNA Polymerase High Fidelity (HiFi; Invitrogen) with 10× HiFi buffer and MgSO 4 (50 mM)
  • dNTP mix (10 mM; Invitrogen)
  • M13F and M13R primers, 5 µM each (M13F: 5′‐TTTTCCCAGTCACGAC‐3′; M13R: 5′‐CAGGAAACAGCTATGAC‐3′)
  • DNase/RNase‐free water, molecular biology grade (Sigma)
  • LB agar plates containing 100 µg/ml ampicillin, 50 µg/ml isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), and 50 µg/ml X‐galactosidase (X‐gal)
  • Ice
  • 15‐ml polypropylene tubes (Falcon)
  • Skirted 96‐well PCR plate (AB1000, Applied Biosystems)
  • Sterile plastic disposable reservoir enabling multichannel pipet access (Costar, Corning)
  • Multichannel pipets
  • Sterile toothpicks
  • 37°C incubator
  • Snap caps (MicroAmp 8‐strip cap; Applied Biosystems)
  • Cap installing tool (Applied Biosystems)
  • Vortex
  • Benchtop centrifuge
  • Thermal cycler
  • Aluminum foil seals (Seal & Sample; Beckman Coulter)
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Figures

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Literature Cited

Literature Cited
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