T Cell Antigen Receptor Sequencing

The T cell antigen receptor (TCR) is a characteristic marker of T cells, playing a crucial role in the immune system. Each T cell typically expresses a specific TCR, which has a unique structure and sequence that can recognize different antigens, including bacteria, viruses, tumor cells, and other pathogens. Once the TCR binds to the antigen, the T cell initiates an immune response, generating cytotoxic effects, secreting cytokines, and coordinating the activities of other immune cells.

 

There are millions of different T cells in the human body, each with a different TCR. The diversity of T cell antigen receptors primarily comes from the diversity of their genes. In the human genome, there are two main TCR gene families: the α chain and β chain. There is a large number of different combinations and variations in the genes in these two gene families, leading to the diversity of T cell antigen receptors. In recent years, the development of T cell antigen receptor sequencing technology has provided us with important tools for a deeper understanding of the function and diversity of T cells.

 

T Cell Antigen Receptor Sequencing Technologies

1. High-Throughput Sequencing (HTS) Technology

High-throughput sequencing (HTS) technology can simultaneously analyze the gene sequences of thousands of T cell receptors. This technology identifies the diversity and specificity of T cells by measuring the sequences of the rearrangement regions of the TCRβ chain or α chain genes.

 

(1) Sample Preparation: Total RNA or DNA is extracted from blood, tissue, or other samples enriched with immune cells.

(2) Amplification: PCR amplification is performed using specific primers for multiple regions of the TCR genes, increasing the sensitivity and specificity of detection.

(3) High-Throughput Sequencing: The amplified products are used for library construction, and then sequenced on a high-throughput sequencing platform.

(4) Data Analysis: The sequencing data are processed and analyzed using specialized bioinformatics tools to identify different TCR variants.

 

2. Mass Spectrometry Technology

Mass spectrometry technology directly measures the mass and structure of T cell receptor proteins to identify differences among different T cell clones. By analyzing specific proteolytic fragments of TCR proteins, this technology can identify the types of TCR, thereby revealing the diversity and specificity of T cells.

 

(1) Sample preparation: T cells are isolated from blood, tissue, or samples enriched with immune cells. It is necessary to ensure that there are enough T cells in the sample for subsequent analysis.

(2) Protein extraction and purification: Various protein extraction methods are used to extract total protein from T cells, and protein purification techniques are used to remove non-target proteins in complex samples.

(3) Protein digestion: The extracted protein samples are enzymatically treated, typically with trypsin, to cut the TCR protein into smaller peptides for mass spectrometry analysis.

(4) Mass spectrometry analysis: The processed samples are analyzed with a mass spectrometer. The specificity of the TCR sequences is identified by measuring the mass-to-charge ratio (m/z) and fragment spectra of the peptides.

(5) Data analysis: Specialized bioinformatics tools and algorithms are used to process and analyze mass spectrometry data, identifying and quantifying the different TCR variants present in the sample.

 

The development of T cell antigen receptor sequencing technologies provides us with opportunities for a deeper understanding of the immune system. By studying the diversity and function of T cell antigen receptors, we can better understand immune responses, disease pathogenesis, and the development of immunotherapies. Continuous advances in this field will help improve our understanding of the immune system and are expected to bring breakthrough progress in the treatment of various immune-related diseases.