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