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PMID: 18432693 Published · ppublish English Journal Article Review

Spectratype/immunoscope analysis of the expressed TCR repertoire.

Current protocols in immunology ·Vol. Chapter 10 ·2001-05-00 ·Pages 10.28.1-10.28.24

Currier JR, Robinson MA

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.

MeSH Terms
Animals Complementarity Determining Regions/genetics,immunology Gene Rearrangement, T-Lymphocyte/genetics,immunology Humans Mice Polymerase Chain Reaction/methods T-Lymphocytes/cytology,immunology
Chemicals
Complementarity Determining Regions
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Currier Jeffrey R
Henry M. Jackson Foundation, Rockville, Maryland.
Robinson Mary Ann
National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland.
References (42)
42 references, click to expand
  1. 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.
  2. 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.
  3. 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.
  4. Davis, M.M. and Bjorkman, P.J. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395-402.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. 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.
  14. 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.
  15. 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.
  16. 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.
  17. 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.
  18. 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.
  19. 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.
  20. Currier et al., 1996. See above.
  21. Han et al., 1999. See above.
  22. 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.
  23. 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.
  24. 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.
  25. Davis, M.M. and Bjorkman, P.J. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395-402.
  26. 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.
  27. 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.
  28. 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.
  29. 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.
  30. 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.
  31. 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.
  32. 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.
  33. 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.
  34. 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.
  35. 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.
  36. 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.
  37. 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.
  38. 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.
  39. 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.
  40. 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.
  41. Currier et al., 1996. See above.
  42. Han et al., 1999. See above.
Article Info
Journal
Current protocols in immunology
Abbr.
Curr Protoc Immunol
ISSN
1934-368X
DOI
10.1002/0471142735.im1028s38
Published
2001-05-00
Pages
10.28.1-10.28.24
Language
English
Region
United States
NLM ID
9101651
Subset
IM
External Links
PubMed source DOI: 10.1002/0471142735.im1028s38
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