tcr development and evaluation
DESCRIPTION
T-Cell receptor development and evaluation in the laboratory.TRANSCRIPT
![Page 1: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/1.jpg)
T cell receptor development and evaluation
Anna MercaVIV, SMI
Spring 2007
![Page 2: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/2.jpg)
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
![Page 3: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/3.jpg)
Overview of T cell development
![Page 4: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/4.jpg)
Development of αβ
and γδ T cells
![Page 5: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/5.jpg)
Potential germline repertoire
Number of functional genes for each domain
![Page 6: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/6.jpg)
TRA Rearrangement
![Page 7: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/7.jpg)
TRB Rearrangement
![Page 8: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/8.jpg)
TRG rearrangement
![Page 9: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/9.jpg)
TRD rearrangement
![Page 10: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/10.jpg)
TCR complex
Abbas & Lichtman 2005
![Page 11: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/11.jpg)
TCR complex
Abbas & Lichtman 2005
![Page 12: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/12.jpg)
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
![Page 13: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/13.jpg)
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
![Page 14: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/14.jpg)
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
![Page 15: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/15.jpg)
T-array protocol for Beta Chain Repertoire
Bonarius et al 2006
![Page 16: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/16.jpg)
Slide representation for T-array
Bonarius et al 2006
![Page 17: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/17.jpg)
Results for Spectratyping and T-array of Jurkat T cell clone mixed with peripheral blood CD4+ T cells
Bonarius et al 2006
![Page 18: Tcr development and evaluation](https://reader034.vdocuments.net/reader034/viewer/2022052622/5596fbbf1a28abd45e8b45ca/html5/thumbnails/18.jpg)
Sensitivity of various methods fo repertoire analysis
Bonarius et al 2006