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T-Cell receptor development and evaluation in the laboratory.
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T cell receptor development and evaluation
Anna MercaVIV, SMI
Spring 2007
Outline• Overview of T cell development• Development of αβ and γδ T cells• Potential Germline Repertoire (non-
rearranged)• Rearragement of V-D-J domains in the TRA,
TRB, TRG and TRD loci• TCR complex• TCR Evaluation:
– CDR3 length analysis– Clonotypic TCR tracking– Microarray for beta chain repertoire
Overview of T cell development
Development of αβ
and γδ T cells
Potential germline repertoire
Number of functional genes for each domain
TRA Rearrangement
TRB Rearrangement
TRG rearrangement
TRD rearrangement
TCR complex
Abbas & Lichtman 2005
TCR complex
Abbas & Lichtman 2005
CDR regionsDiagram 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; Vβ CDR1, magenta; Vβ CDR2, orange; V β CDR3, cyan.
* MP(58-66) Influensa virus matrix protein
Stewart-Jones et al 2003 Nature Immunology
CDR3 length analysisFigure 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 Vβ/Cβ combination).
12 run off reactions are performed for each Vβn-Cβ PCR product, each one using a fluorescent reverse primer specific for one Jβ.
Finally each run-off’s products are separated on the basis of length.
Ria et al 2001 Current Molecular Medicine
Clonotypic TCR analysis of HIV-specific CD8+ T cells
1. Peptide stimulation of PBMCs2. Cell surface cytokine capture3. Flow cytometric cell sorting (5000 cells
for each peptide stimulation)4. Anchored RT-PCR5. Transfection of E. Coli with PCR products6. Quantitative PCR with clonotype-specific
primers and probes.7. Sequencing of CDR3 region
T-array protocol for Beta Chain Repertoire
Bonarius et al 2006
Slide representation for T-array
Bonarius et al 2006
Results for Spectratyping and T-array of Jurkat T cell clone mixed with peripheral blood CD4+ T cells
Bonarius et al 2006
Sensitivity of various methods fo repertoire analysis
Bonarius et al 2006