The methods and advances of adaptive immune receptors repertoire sequencing

Abstract

The adaptive immune response is a powerful tool, capable of recognizing, binding to, and neutralizing a vast number of internal and external threats via T or B lymphatic receptors with widespread sets of antigen specificities. The emergence of high-throughput sequencing technology and bioinformatics provides opportunities for research in the fields of life sciences and medicine. The analysis and annotation for immune repertoire data can reveal biologically meaningful information, including immune prediction, target antigens, and effective evaluation. Continuous improvements of the immunological repertoire sequencing methods and analysis tools will help to minimize the experimental and calculation errors and realize the immunological information to meet the clinical requirements. That said, the clinical application of adaptive immune repertoire sequencing requires appropriate experimental methods and standard analytical tools. At the population cell level, we can acquire the overview of cell groups, but the information about a single cell is not obtained accurately. The information that is ignored may be crucial for understanding the heterogeneity of each cell, gene expression and drug response. The combination of high-throughput sequencing and single-cell technology allows us to obtain single-cell information with low-cost and high-throughput. In this review, we summarized the current methods and progress in this area.

Keywords: T or B cell receptors; adaptive immune repertoire sequencing; bioinformatics; high-throughput sequencing.

Figure 1

The structure of antigen-specific lymphocyte receptors and the generation of diversity. (A, B) The structure of BCR and TCR. The heavy and light chains of antibodies are shown, and they are connected by disulfide bonds (bold blue line); TCR that is across the cell membrane is a heterodimer comprised of αβ chains or γδ chains. The upper part is the variable (V) region composed of V(D)J gene in the figure. The V region is composed of CDR regions and FR domains. CDR1, CDR2, and CDR3 are shown in different colors (yellow, blue, and green, respectively). The lower part (white area) is a constant region that is conservative. The structure of lymphocyte receptors shown here contributes to explaining how immune response occurs. (C) The mechanism of lymphocyte receptors repertoire diversity. During the development of lymphocytes, BCR heavy chains or TCR β(δ) chains suffer from the rearrangement of VDJ genes, while the IgL chain or α(γ) chain lack D gene in the rearrangement. Afterward, the rearranged V-DJ or V-J sequences are linked to the C gene fragments. Finally, two independent chains are assembled into unique receptor proteins. Germline gene V(D)JC undergoes rearrangement and insertion and deletion of nucleotides, resulting in the diversity of the receptor library .

Figure 2

The process of immune repertoire sequencing and analysis. The detailed steps of immune repertoire analysis are shown in the figure. The extraction and amplification of samples are the key techniques in library preparation. The starting materials that are either gDNA or mRNA have both advantages and disadvantages as templates. The choice of templates mainly depends on research purposes. gDNA was amplified by multiple pairs of primers, but there were large introns in the DNA, which led to amplification errors. The amplification for mRNA is operated by using 5'RACE which avoids PCR amplification bias, but the operation is more complicated. A large amount of data obtained from high-throughput sequencing of PCR products are used for health assessment, disease diagnosis, monitoring and prognosis.

Figure 3

Treemap of TCR and BCR repertoires in PBMCs of healthy individuals and COVID-19 patients. A. Treemap of 1 healthy individual and 2 patients, respectively. B. Treemap of total TCR Beta chains from patients 4, patient 5, and healthy control. The larger the clones in the picture, the worse the diversity. The diversity of healthy people is better than that of patients. Adapted with permission from Niu, copyright 2020 . Pt4: patient 4, Pt5: patient 5.

Figure 4

Schematic diagram of single B cell sequencing. The magnetic beads with barcodes and UMIs are used to capture individual cells. Magnetic beads and individual cells are coated in oil. Then the cells are lysed and reverse transcription into cDNA. The barcode-encoded cDNA is then reversed transcription into a library and sequenced. The bioinformatic pipeline is used to analyze the heavy and light chain sequences from a single cell. Adapted with permission from Goldstein, copyright 2019 .

Figure 5

Cancer immunotherapy. The function of immune checkpoint and treatment are shown in figures A, B, and C. Co-stimulatory and co-suppressive receptors which include CD28, PD-1, and CTLA-4 are expressed on the surface of T cells. T cells activate and initiate an immune response when it accepts a stimulus signal; otherwise, T cells inactivate when the suppression signal is received, the T cell immune response is suppressed. Therefore, the target of the inhibitor is to block a combination of the ligands on tumor cells and its receptors, thereby activating T cells response to cancer cells. Nevertheless, the therapeutic effect of checkpoint inhibitors varies from person to person. Adoptive cell therapy is demonstrated in Figure 5D. The figure shows the cell source, isolation, optimization, and reinfusion process of adoptive cell therapy. TILs from patients, T cells in peripheral blood of patients, and peripheral T cells of healthy people are cultured in vitro, optimized, expanded, and then reinfused into the human body to increase accurate antigen recognition and enhance the lethality, making it possible to improve the treatment of tumors. APC: antigen presenting cell; DC: dendritic cell.