Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire

Jeffrey R. Currier1, Mary Ann Robinson2

1 Henry M. Jackson Foundation, Rockville, Maryland, 2 National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.28
DOI:  10.1002/0471142735.im1028s38
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Measuring the diversity of TCRs utilized by specific primary and memory T cell responses is critical to the fundamental understanding of regulation of the immune system. This unit describes the Spectratype/Immunoscope technique which permits an in depth analysis of the TCR repertoire present in a variety of biological samples from mice to humans. Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different CDR3 lengths from specific TCR variable region genes in a competitive manner. The PCR products are then resolved on polyacrylamide sequencing gels to reveal precise sizes in nucleotide base pairs. The unit also includes protocols that have been optimized to process and produce the starting materials required for spectratype analysis.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Spectratype Analysis of Human and Mouse TCR Repertoire Diversity
  • Alternate Protocol 1: Spectratype Analysis Using [32P]dCTP
  • Basic Protocol 2: Fine‐Specificity Spectratype Analysis Using Junctional Region Primers
  • Basic Protocol 3: Rapid High‐Throughput Sequencing of Amplified TCR Gene Products
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Spectratype Analysis of Human and Mouse TCR Repertoire Diversity

  Materials
  • Cell population (e.g. purified T cells) or tissue sample to be analyzed
  • 20 µM solutions of β‐actin (or other housekeeping gene) primers (Actin‐F and Actin‐R; see Table 10.28.1; stored at −70°C)
  • 1.5% (w/v) agarose gel made with 1× TBE buffer containing 0.5 µg/ml ethidium bromide (unit 10.4)
  • Sterile H 2O, DNase free
  • 10× PCR amplification buffer with 15 mM MgCl 2 (e.g., PCR Buffer I, PE Biosystems, or as described in unit 10.20)
  • 1.25 mM 4dNTP mix (e.g., Pharmacia, or as described in unit 10.20)
  • 20 µM solutions of human (Tables 10.28.1 and 10.28.2) or mouse (Tables 10.28.3 and 10.28.4) constant region gene (C‐gene) TCR primers (HTCA3 or HTCB3; store at −70°C)
  • 5 U/µl Taq DNA polymerase modified for hot‐start PCR (e.g., AmpliTaq Gold, PE Biosystems, or Platinum Taq DNA polymerase, Life Technologies) and 5 U/µl unmodified Taq polymerase (e.g., AmpliTaq, PE Biosystems)
  • 20 µM solutions of AV and BV human (Tables 10.28.1 and 10.28.2) or mouse (Tables 10.28.3 and 10.28.4) TCR primer (stored at −70°C)
  • 4 µM solutions of 5′ fluorescently (6‐FAM)‐labeled nested C‐gene primers, HTCA1‐FAM or HTCB1‐FAM, or mouse equivalents (Tables 10.28.1 to 10.28.4; stored at −70°C)
  • 4% (w/v) denaturing polyacrylamide sequencing gel (unit 10.25)
  • Deionized formamide
  • GeneScan lane standard GS‐500 TAMRA (PE Biosystems)
  • EDTA/blue dextran gel loading dye (PE Biosystems)
  • 0.6‐ml or 0.3‐ml sterile polycarbonate PCR amplification tubes, or 96‐well PCR reaction array trays (e.g., Marsh Biomedical)
  • Thermal cycler with heated lid (e.g., MJ Research or PE Biosystems)
  • Automated sequencing machine with GeneScan software installed (e.g., ABI PRISM model 377, PE Applied Biosystems)
  • 36‐lane sharkstooth comb
  • Additional reagents and equipment for purifying human T cells (units 7.3 & 7.4) or mouse T cells (units 3.2, 3.3, & 3.5), preparation of RNA (unit 10.11), reverse transcription of RNA into cDNA (unit 10.27), agarose gel electrophoresis (unit 10.4), and denaturing polyacrylamide sequencing gel (unit 10.25)
CAUTION: When working with human blood, cells, or infectious agents, biosafety practices must be followed (see Chapter introduction).

Alternate Protocol 1: Spectratype Analysis Using [32P]dCTP

  • 20 µM solutions of human (Tables 10.28.1 and 10.28.2) or mouse (Tables 10.28.3 and 10.28.4) constant region gene (C‐gene) TCR primers (HTCA1 or HTCB1; store at −70°C)
  • 0.1 mCi/µl [α‐32P]dCTP (3000 Ci/mmol; NEN Life Science Products or Amersham Pharmacia Biotech)
  • 6% (w/v) polyacrylamide sequencing gel (unit 10.25)
  • recipeGel loading dye (see recipe)
  • Whatman 3MM filter paper
  • PhosphorImager cassette and PhosphorImaging Scanner and ImageQuant software or equivalent (Molecular Dynamics; optional)
  • Additional reagents and equipment for manual gel sequencing by electrophoresis (see unit 10.25, protocol 2) and autoradiography ( appendix 3J)

Basic Protocol 2: Fine‐Specificity Spectratype Analysis Using Junctional Region Primers

  Materials
  • 4 µM solutions of each 5′ fluorescently‐labeled (6‐FAM) BJ‐gene human or mouse reverse‐strand primers (see Table 10.28.5)
  • PCR reaction products (see protocol 1, step )
  • Additional reagents and equipment for spectratype analysis (see protocol 1)

Basic Protocol 3: Rapid High‐Throughput Sequencing of Amplified TCR Gene Products

  Materials
  • Sterile H 2O DNase free
  • 10× ligation buffer (provided with enzyme)
  • 2.5 ng/µl Ampr β‐gal+ T‐A cloning vector (e.g., Invitrogen pCR 2.1 vector or equivalent; see Finney et al., , for generation of T‐A overhang vector)
  • 4 U/µl T4 DNA ligase (e.g., New England Biolabs)
  • PCR product (see protocol 1, step ; freshly prepared)
  • Transformation‐competent Amps β‐galE. coli cells (e.g., DH5α)
  • recipeLB agar plates containing 100 µg/ml ampicillin and treated with Xgal (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Proteinase K buffer (see recipe)
  • 10× PCR amplification buffer with 15 mM MgCl 2 (e.g., PCR Buffer I, PE Biosystems, or as described in unit 10.20)
  • 1.25 mM 4dNTP mix (e.g., Pharmacia, or as described in unit 10.20)
  • 20 µM C‐gene primer (Tables 10.28.1 to 10.28.4)
  • 20 µM V‐gene primer (Tables 10.28.1 to 10.28.4)
  • 1.5% (w/v) agarose gel made with 1× TBE buffer containing 0.5 µg/ml ethidium bromide (unit 10.4)
  • 1 U/µl shrimp alkaline phosphatase (SAP)
  • 10 U/µl E. coli exonuclease I
  • 10× SAP buffer (supplied with enzyme)
  • ABI PRISM Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase FS (PE Biosystems; or equivalent)
  • 75% (v/v) isopropanol
  • 1.5 ml sterile microcentrifuge tubes
  • 14°C and 37°C water baths
  • 96‐well array PCR trays (e.g., Marsh)
  • Themal cycler with heated lid (e.g., MJ Research or PE Applied Biosystems)
  • ABI PRISM model 377 automated sequencing machine (PE Biosystems; or similar automated sequencing machine)
  • Strip caps or 3M Scotch Tape 425‐3 adhesive backed aluminum foil tape
  • Tube‐tray adaptor for tabletop centrifuge (e.g., Beckman GS‐6 centrifuge with microplus carriers)
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., ) and agarose gel electrophoresis (unit 10.4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Blish, C.A., Gallay, B.J., Turk, G.L., et al. 1999. Chronic modulation of the TCR repertoire in the lymphoid periphery. J. Immunol. 162:3131‐3140.
   Bousso, P., Levraud, J.P., Kourilsky, P., and Abastado, J.P. 1999. The composition of a primary T cell response is largely determined by the timing of recruitment of individual T cell clones. J. Exp. Med. 189:1561‐1600.
   Currier, J.R., Deulofeut, H., Barron, K.S., Kehn, P.J., and Robinson, M.A. 1996. Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity. Human Immunol. 48:39‐51.
   Davis, M.M. and Bjorkman, P.J. 1988. T‐cell antigen receptor genes and T‐cell recognition. Nature 334:395‐402.
   Finney, M., Nisson, P.E., and Rashtchian, A. 1995. Molecular cloning of PCR products. In Current Protocols in Molecular Biology (F.M. Ausabel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 15.7.1‐15.7.11. John Wiley & Sons, New York.
   Garboczi, D.N., Ghosh, P., Utz, U., Fan, Q.R., Biddison, W.E., and Wiley, D.C. 1996. Structure of the complex between human T‐cell receptor, viral peptide and HLA‐A2. Nature 384:134‐141.
   Garcia, K.C., Degano, M., Stanfield, R.L., Brunmark, A., Jackson, M.R., Peterson, P.A., Teyton, L., and Wilson, I.A. 1996. An αβ TCR structure at 2.5A and its orientation in the TCR‐MHC complex. Science 274:209‐219.
   Gorochov, G., Neuman, A.U., Kereveur, A., Parizot, C., Li, T., Katlama, C., Karmochine, M., Raguin, G., Autran, B., and Debre, P. 1998. Perturbation of CD4+ and CD8+ T‐cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy. Nature Med. 4:215‐221.
   Gorski, J., Yassai, M., Zhu, X., Kisella, B., Keever, C., and Flomenberg, N. 1994. Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status. J. Immunol. 152:5109‐19.
   Han, M., Harrison, L., Kehn, P., Stevenson, K., Currier, J., and Robinson, M.A. 1999. Invariant or highly conserved TCRα chains are expressed on double‐negative (CD3+ CD4− CD8−) and CD8+ T cells. J. Immunol. 163:301‐311.
   Kronenberg, M., Siu, G., Hood, L.E., and Shastri, N. 1986. The molecular genetics of the T cell receptor and T cell antigen recognition. Annu. Rev. Immunol. 4:529‐91.
   Lin, M.Y. and Welsh, R.M. 1997. Stability and diversity of T cell receptor repertoire usage during lymphocytic choriomeningitis virus infection of mice. J. Exp. Med. 188:1993‐2005.
   Pannetier, C., Cochet, M., Darche, S., Casourage, A., Zoller, M., and Kourilsky, P. 1993a. The sizes of the CDR3 hypervariable regions of the murine T‐cell receptor β chains vary as a function of the recombined germ‐line segments. Proc. Natl. Acad. Sci. USA. 90:4319‐4323.
   Pannetier, C., Delassus, S., Darche, S., Saucier, C., and Kourilsky, P. 1993b. Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation. Nucleic Acids Res. 21:577‐583.
   Rowen, L., Koop, B.F., and Hood, L. 1996. The complete 685‐kilobase DNA sequence of the human beta T cell receptor locus. Science 272:1755‐1762.
   Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. In Current Protocols in Molecular Biology (F.M. Ausabel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.8.1‐1.8.10. John Wiley & Sons, New York.
   Wei, S., Charmley, P., Robinson, M.A., and Concannon, P. 1994. The extent of the human germline T‐cell receptor V beta gene segment repertoire. Immunogenetics 40:27‐36.
   Yassai, M., McFarland, J.G., Newton‐Nash, D., Newman, P.J., Eckels, D.D., and Gorski, J. 1992. T cell receptor and alloimmune thrombocytopenias: a model for autoimmune diseases? Ann. Med. Interne. 143:365‐370.
Key References
   Pannetier, C., Even, J., and Kourilsky, P. 1995. T‐cell repertoire diversity and clonal expansions in normal and clinical samples.. Immunol. Today 16:176‐181.
  Application of spectratype analysis in humans and mice.
   Currier et al., 1996. See above.
  An overview of human TCRB analyses.
   Han et al., 1999. See above.
  An overview of human TCRA analyses.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library