Evaluating Human T Cell Receptor Gene Expression by PCR

Emanuela Gussoni1, Michael A. Panzara2, Lawrence Steinman3

1 Children's Hospital, Boston, Massachusetts, 2 Massachusetts General Hospital, Boston, Massachusetts, 3 Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.26
DOI:  10.1002/0471142735.im1026s22
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of PCR to characterize and quantify rearranged transcripts from specific T cell receptor variable gene families in human tissue and peripheral blood lymphocytes. The strategy outlined in this unit has been extensively used on different sources of human tissue including brain, spinal cord, and skeletal muscle. A protocol is provided to clone and sequence PCR‐amplified cDNA transcripts to study the junctional diversity of the expressed genes. A support protocol describes a method for reverse transcribing total RNA to make the cDNA required by the other protocols.

     
 
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Table of Contents

  • Basic Protocol 1: PCR Analysis of T Cell Receptor Gene Expression
  • Alternate Protocol 1: Quantitation of TCR Variable Sequence Transcripts by PCR
  • Support Protocol 1: Sequence Analysis of Rearranged V‐D‐J Regions
  • Support Protocol 2: Synthesis of cDNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR Analysis of T Cell Receptor Gene Expression

  Materials
  • 50 µM solutions of each TCR primer (Table 10.26.1; store at −20°C)
  • Sterile water
  • 10× amplification buffer with 15 mM MgCl 2 (unit 10.20)
  • 0.01 µg/µl cDNA (see protocol 4)
  • 2.5 U/µl Taq DNA polymerase (e.g., AmpliTaq, Perkin‐Elmer Cetus)
  • Mineral oil
  • 1.5% (w/v) NuSieve GTG agarose (FMC Bioproducts)/0.5% (w/v) agarose in 1× TBE electrophoresis buffer (unit 10.4)
  • Denaturing solution: 0.5 M NaOH/1.5 M NaCl (store at room temperature)
  • Neutralizing solution: 0.5 M Tris⋅Cl (pH 7.0)/1.5 M NaCl (store at room temperature)
  • 20× SSPE (Table 97.80.4711)
  • 1% (w/v) SDS
  • recipeHybridization solution (see recipe)
  • Horseradish peroxidase (HRPO)–labeled oligonucleotide probes (Operon Technologies)
  • 1× SSPE/1% (w/v) SDS
  • 0.1× SSPE/1% (w/v) SDS
  • PBS ( appendix 2A)
  • Enhanced chemiluminescence (ECL) Gene Detection System (Amersham)
  • 0.5‐ml polycarbonate microcentrifuge tubes, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Thermal cycler
  • Nylon membrane (Gene Screen Plus, New England Nuclear)
  • UV cross‐linker
  • Heat‐sealable hybridization bags
  • X‐ray film
  • Additional reagents and equipment for PCR (unit 10.20), agarose gel electrophoresis and ethidium bromide staining (unit 10.4), Southern blotting by downward capillary transfer (unit 10.6), and UV cross‐linking (unit 10.6)
  • CAUTION: Biosafety procedures must be followed when handling human blood or tissue sample.

Alternate Protocol 1: Quantitation of TCR Variable Sequence Transcripts by PCR

  • 50 µM TCR Cβ 3′, Cα 5′, and Cα 3′ primers for reference template (Table 10.26.1)
  • Densitometer

Support Protocol 1: Sequence Analysis of Rearranged V‐D‐J Regions

  • CloneAmp cloning system (GIBCO/BRL) containing:
    • pAMP1 vector
    • 25 ng/µl 1 U/µl uracil DNA glycosylase
    • 10 × PCR amplification buffer
    • DH5α transformation‐competent E. coli
  • 50 µM TCR sense primer (V primer) with the adapter 5′‐CUACUACUACUA‐3′ attached to the 5′ end
  • 50 µM TCR antisense primer (C primer) with the adapter 5′‐CAUCAUCAUCAU‐3′ attached to the 5′ end
  • recipeLB plates containing 50 µg/ml ampicillin (see recipe)
  • recipeTB medium (see recipe)
  • 0.7% (w/v) agarose gel
  • Sequenase version 2.0 T7 DNA polymerase kit (U.S. Biochemical) containing:
  •   5× annealing buffer: 200 mM Tris⋅Cl (pH 7.5)/100 mM MgCl 2/250 mM NaCl
  •   0.1 M dithiothreitol (DTT)
  •   Labeling nucleotide mix: 1.5 µM each dGTP, dCTP, and dTTP
  •    recipeTermination nucleotide mixtures (also see recipe)
  •    recipeStop solution (also see recipe)
  •   Enzyme dilution buffer: 10 mM Tris⋅Cl (pH 7.5)/5 mM DTT/0.05%   (w/v) BSA
  •   200 U or 1000 U Sequenase version 2.0 T7 DNA polymerase
  • Centricon‐100 microconcentrator spin column with conical cap (Perkin‐Elmer Cetus)
  • Beckman J2‐21M centrifuge and JA‐20.1 rotor
  • 37°C bacterial incubator with shaker
  • Additional reagents and equipment for alkaline lysis preparation of plasmid DNA (unit 10.3)

Support Protocol 2: Synthesis of cDNA

  Materials
  • Fresh or frozen samples of tissue or peripheral blood lymphocytes
  • 10× amplification buffer with 15 mM MgCl 2 (unit 10.20)
  • 2.5 mM 4dNTP mix (unit 10.20)
  • 50 A 260 U random hexamers (Pharmacia Biotech) resuspended in 1 ml DEPC‐treated water
  • 10 U/µl human placental RNase inhibitor (GIBCO/BRL)
  • 200 U/µl Superscript MuLVH‐reverse transcriptase (GIBCO/BRL)
  • DEPC‐treated sterile water (unit 10.11)
  • Additional reagents and equipment for preparing total RNA by the guanidium method (unit 10.11)
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Figures

Videos

Literature Cited

Literature Cited
   Bragado, R., Lauzurica, P., Lopez, D., and Lopez‐De Castro, J.A. 1990. T cell receptor Vβ usage in a human alloreactive response. J. Exp. Med 171:1189‐1204.
   Bugawan, T.L., Horn, G.T., Long, C.M., Mickelson, E., Hansen, J.A., Ferrara, G.B., Angelini, G., and Erlich, H.A. 1988. Analysis of HLA‐DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP‐α and DP‐β loci. J. Immunol. 141:4024‐4030.
   Bugawan, T.L., Begovich, A.B., and Erlich, H.A. 1990. Rapid HLA‐DPβ‐typing using enzymatically amplified DNA and nonradioactive sequence‐specific oligonucleotide probes. Immunogenetics 32:231‐241.
   Bugawan, T.L. and Erlich, H.A. 1991. Rapid typing of HLA‐DQB1 DNA polymorphism using nonradioactive oligonucleotide probes and amplified DNA. Immunogenetics 33:163‐170.
   Choi, Y., Kotzin, B., Herron, L., Callahan, J., Marrack, P., and Kappler, J. 1989. Interaction of Staphylococcus aureus toxin “superantigens” with human T cells. Proc. Natl. Acad. Sci. U.S.A. 86:8941‐8945.
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   Erlich, H. A., Gelfand, D., and Sninsky, J. J. 1991. Recent advances in the polymerase chain reaction. Science 252:1643‐1651.
   Gussoni, E., Pavlath, G.K., Miller, R.G., Panzara, M.A., Powell, M., Blau, H.M., and Steinman, L. 1994. Specific TCR gene rearrangements at the site of muscle degeneration in Duchenne muscular dystrophy. J. Immunol. 153:4798‐4805.
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   Oksenberg, J.R., Stuart, S., Begovich, A.B., Bell, R.B., Erlich, H.A., Steinman, L., and Bernard, C.C. 1990. Limited heterogeneity of rearranged T cell receptor Vα transcripts in brains of multiple sclerosis patients. Nature 345:344‐346.
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   Panzara, M.A., Oksenberg, J.R., Begovich, A., Erlich, H., Murray, R., Appel, S.H., and Steinman, L. 1991. Restricted T cell receptor usage in spinal cord and brain of patients with amyotrophic lateral sclerosis. J. Neuroimmunol. Suppl. 1:79.
   Panzara, M.A., Gussoni, E., Steinman, L., and Oksenberg, J.R. 1992. Analysis of the T cell receptor repertoire using the polymerase chain reaction and specific oligonucleotide primers. BioTechniques 12:728‐735.
   Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1986. Analysis of enzymatically amplified β‐globin and HLA‐DQ α DNA with allele‐specific oligonucleotide probes. Nature 324:163‐166.
   Sottini, A., Imberti, L., Gorla, R., Cattaneo, R., and Primi, D. 1991. Restricted expression of T cell receptor Vβ but not Vα genes in rheumatoid arthritis. Eur. J. Immunol. 21:461‐466.
   Tomai, M.A., Aelion, J.A., Dockter, M.E., Majumdar, G., Spinella, D.G., and Kotb, M. 1991. T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. J. Exp. Med. 174:285‐288.
   Wucherpfennig, K., Ota, K., Endo, K., Seidman, J.G., Rosenzweig, A., Weiner, H.L., and Hafler, D.A. 1990. Shared human T cell receptor Vβ usage to immunodominant regions of myelin basic protein. Science 248:1016‐1019.
   Yssel, H., Shanafelt, M.C., Soderberg, C., Schneider, P.V., Anzola, J., and Peltz, G. 1991. Borrelia burgdorferi activates a T helper type 1 like T cell subset in Lyme arthritis. J. Exp. Med. 174:593‐601.
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