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Chapter 29 : Evaluation of the T-Cell Receptor Repertoire

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Abstract:

During the process of differentiation in the thymus, T cells undergo rearrangement of their T-cell receptor (TCR) genes and mature into single positive cells of either the CD4+ or the CD8+ phenotype. This chapter updates methods for assessment of the TCR Vβ repertoire in peripheral blood by CDR3 length spectratyping by multiplex PCR and with monoclonal antibodies (MAbs) by flow cytometry. Whole blood is collected by venipuncture into a tube containing an anticoagulant such as heparin, acid-citrate-dextrose, or EDTA. The reverse transcriptase (RT) enzyme uses RNA dependent DNA polymerase activity to synthesize cDNA from an RNA template. The cDNA may be synthesized with oligo(dT) or TCR-specific C-region (Cβ-14) primers. Bands in the sequencing gel are converted into peaks by the ImageQuant software and give information similar to that of the fluorescent PCR but without the CDR3 sizes that are computed by the automated DNA sequencer. A reduction in the number of peaks compared to that of the control range represents restrictions in the repertoire. Southern blotting can be used for assessment of TCR Vβ and Jβ segments. The extracted DNA is treated with a set of restriction enzymes. The products of digestion are separated by agarose gel electrophoresis, blotted onto a nylon membrane, and probed with a set of labeled Jβ probes.

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29

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Figures

Image of FIGURE 1
FIGURE 1

Organization of the a (3 TCR showing the constant, transmembrane, and variable regions of the a and (3 chains.

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
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Image of FIGURE 2
FIGURE 2

Schematic representation of the β-chain transcript of the TCR. For CDR3 length analysis PCR, a nested reverse Cβ-R primer is used in combination with multiplexed forward Vβ primers. The locations of one set of Vβ primers (set A), primer Cβ-R for PCR and reverse primer Cβ-14 for reverse transcription, are indicated (single-headed arrows). -Y-(L/F)-C-A-S- and -F-G-X-G-T-, the amino acid residues that flank the CDR3 region of the β chain of the TCR. The V, CDR3, J, and C regions are also indicated (double-headed arrows).

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
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Image of FIGURE 3
FIGURE 3

Flow chart indicating various steps for the CDR3 length analysis of CD4 and CD8 T cells by the radioactive and fluorescent multiplex PCR methods.

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
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Image of FIGURE 4
FIGURE 4

CDR3 length analysis of the Vβ chain of TCR showing radiolabeled PCR products resolved by polyacrylamide gel electrophoresis. The figure shows an example of dominance of Vβ 7 from CD8 T cells of a healthy subject. The Vβ7 family (rectangle) is shown as an example of the step for further analysis with ImageQuant software.

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
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Image of FIGURE 5
FIGURE 5

CDR3 spectratype generated after the fluorescent PCR products were resolved on an automated DNA sequencer (ABI 310). The axis for each set represents the size of the PCR product, and the axis represents the signal intensity. (A) TCR repertoire of cord blood CD4 T cells showing normal Gaussian patterns for all Vβ families. (B) TCR repertoire of peripheral blood CD8 T cells from an HIV-infected child, showing perturbation (arrows).

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
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Tables

Generic image for table

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table
TABLE 1

Primers used for reverse transcription-PCR

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table
TABLE 2

Primers used for TCR Vβ analysis with fluorescently labeled Vβ primers

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table
TABLE 3

Panel of Vβ-specific MAbs

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29
Generic image for table
TABLE 4

Methods for assessing the TCR repertoire

Citation: Pahwa S. 2006. Evaluation of the T-Cell Receptor Repertoire, p 249-261. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch29

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