Tcr development and evaluation

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T-Cell receptor development and evaluation in the laboratory.

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Tcr development and evaluation

  1. 1. T cell receptor development and evaluation Anna Merca VIV, SMI Spring 2007
  2. 2. Outline <ul><li>Overview of T cell development </li></ul><ul><li>Development of αβ and γδ T cells </li></ul><ul><li>Potential Germline Repertoire (non-rearranged) </li></ul><ul><li>Rearragement of V-D-J domains in the TRA, TRB, TRG and TRD loci </li></ul><ul><li>TCR complex </li></ul><ul><li>TCR Evaluation: </li></ul><ul><ul><li>CDR3 length analysis </li></ul></ul><ul><ul><li>Clonotypic TCR tracking </li></ul></ul><ul><ul><li>Microarray for beta chain repertoire </li></ul></ul>
  3. 3. Overview of T cell development
  4. 4. Development of αβ and γδ T cells
  5. 5. Potential germline repertoire Number of functional genes for each domain
  6. 6. TRA Rearrangement
  7. 7. TRB Rearrangement
  8. 8. TRG rearrangement
  9. 9. TRD rearrangement
  10. 10. TCR complex Abbas & Lichtman 2005
  11. 11. TCR complex Abbas & Lichtman 2005
  12. 12. CDR regions Stewart-Jones et al 2003 Nature Immunology Diagram highlighting the CDR loops of the T cell clone JM22 in complex with MP(58−66)−HLA-A2. The CDR loops are colored as follows: V α   CDR1, green; V α   CDR2, red; V α   CDR3, blue; V β   CDR1, magenta; V β   CDR2, orange; V β   CDR3, cyan. * MP(58-66) Influensa virus matrix protein
  13. 13. CDR3 length analysis Figure 1 . Description of the immunoscope technology. Lymphocytes derived from the blood are stimulated in vitro in the presence of the antigen. 3 days later RNA is extracted and cDNA is synthesized. 24 different PCR amplifications are performed (one for each V  /C  combination). 12 run off reaction s are performed for each V  n-C  PCR product, each one using a fluorescent reverse primer specific for one J  . Finally each run-off’s products are separated on the basis of length. Ria et al 2001 Current Molecular Medicine
  14. 14. Clonotypic TCR analysis of HIV-specific CD8+ T cells <ul><li>Peptide stimulation of PBMCs </li></ul><ul><li>Cell surface cytokine capture </li></ul><ul><li>Flow cytometric cell sorting (5000 cells for each peptide stimulation) </li></ul><ul><li>Anchored RT-PCR </li></ul><ul><li>Transfection of E. Coli with PCR products </li></ul><ul><li>Quantitative PCR with clonotype-specific primers and probes. </li></ul><ul><li>Sequencing of CDR3 region </li></ul>
  15. 15. T-array protocol for Beta Chain Repertoire Bonarius et al 2006
  16. 16. Slide representation for T-array Bonarius et al 2006
  17. 17. Results for Spectratyping and T-array of Jurkat T cell clone mixed with peripheral blood CD4+ T cells Bonarius et al 2006
  18. 18. Sensitivity of various methods fo repertoire analysis Bonarius et al 2006

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