The immune system, as the body's core defense against the invasion of foreign pathogens and the removal of abnormal cells, depends on a huge immune group library composed of T cell receptor (TCR) and B cell receptor (BCR). This "molecular library", which is composed of billions or even trillions of unique receptor sequences, starts a specific immune response by accurately identifying antigenic epitopes, and its composition and dynamic changes directly reflect the functional state of the immune system. However, traditional immunology research methods are limited by resolution and throughput, and it is difficult to fully analyze the diversity, cloning dynamics, and functional correlation of the immune library, which greatly restricts the in-depth understanding of the immune response mechanism.

In this context, Immune repertoire sequencing technology came into being. As a cutting-edge tool for integrating molecular biology and high-throughput sequencing, immune repertoire sequencing has achieved a systematic analysis of the Immune repertoire sequencing library by targeting TCR/BCR variable region sequences, providing a new perspective for revealing the immune response law in infection, tumor, autoimmune diseases, and other scenarios. A deep understanding of the technical basis of immune repertoire sequencing is not only the premise of carrying out related research but also the key to promoting its transformation from basic scientific research to clinical practice.

This article explores the foundations of immune repertoire sequencing, covering the biological significance of immune repertoire, immune repertoire diversity, principles, key sequencing platforms, and discusses its role in advancing immune research and clinical translation.

Biological Significance of Immune Repertoire

The immunologic library is the sum of all functional TCR and BCR gene sequences in the immune system, and its biological significance runs through the whole process of initiation, regulation, and effect of immune response, which is the core basis for understanding the function of the immune system.

Mediate Specific Immune Response

TCR and BCR in the immune library show a high degree of sequence diversity, which comes from the random rearrangement of V (variable region), D (diversity region, only TCR partial chain and BCR heavy chain) and J (junction region) gene fragments and non-template insertion/deletion events. The receptor molecular library generated by it can accurately identify antigen epitopes, covering the conservative domains of foreign pathogens (such as bacteria and viruses) and new antigens of endogenous abnormal cells (such as tumor cells), and activate T cell or B cell clones through signal transduction to drive cellular immunity or humoral immune response, which constitutes the core molecular basis of the body's immune defense.

Reflect Immune System Dynamic State

The composition and diversity of the immune group library are regulated by many factors, showing significant dynamic changes. In the physiological dimension, the age-related decline of thymus and bone marrow function can lead to the decrease of initial T/B cell output, resulting in the decrease of Cookrone type diversity in immune group.

Under pathological conditions, antigen stimulation caused by pathogen invasion or tumorigenesis will induce the selective proliferation and differentiation of antigen-specific T/B cell clones, and change the clone distribution pattern of immune group library. This dynamic response makes the Immune repertoire sequencing library an important molecular marker for quantitative evaluation of immune system function, and provides a basis for health monitoring and disease early warning.

GenAIRR modular architecture to simulate Ig sequences (Konstantinovsky et al., 2024)

Support Disease Diagnosis and Treatment

In the field of precision medicine, Immune repertoire sequencing analysis has become an important tool for disease diagnosis and treatment. The characteristics of the immune bank of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment are closely related to the efficacy of immunotherapy. TCR clones with high clonal expansion and tumor antigen specificity often predict a good therapeutic response.

In autoimmune diseases, abnormally activated T/B cell clones mediate pathological immune response through antigen cross-reaction or the epitope spreading mechanism. Analyzing the characteristics of its immune library is helpful to reveal the pathogenesis of the disease and provide molecular targets for the development of targeted immunomodulation therapy strategies.

What is Immune Repertoire Diversity

The diversity of the immune library refers to the richness of TCR/BCR gene sequences in the immune system, which is the core index to measure the ability of the immune system to recognize antigens, mainly reflected in the heterogeneity of gene sequence and the complexity of clone composition.

Source of Diversity

The structural basis of TCR and BCR genes determines the high complexity of the immune library. Its coding gene consists of a variable region (V), diversity region (D, only in BCR and TCRβ chain), and junction region (J). Through the V (D) J recombination mechanism, that is, the random combination of different V, D and J gene fragments, combined with the random insertion/deletion of nucleotides (N-region insertion) in the recombination process, and the unique somatic high frequency mutation (SHM) mechanism of BCR, the immune system can produce unique receptor sequences with orders of billions to trillions. These genetic rearrangement and modification events at the molecular level constitute the material basis of the diversity of immune groups.

The Core Dimension of Diversity

The diversity of the Immune repertoire sequencing library can be analyzed from two core dimensions:

• One is the diversity of clonal abundance, which represents the quantitative distribution characteristics of different clonal types in the population and reflects the clonal selection preference of immune system response
• The second is sequence diversity, which involves the differences of gene sequences of different clones, including key parameters such as the frequency of V/J gene fragments and the sequence variation of 3 (CDR3).

These two-dimensional diversity characteristics jointly determine the functional state and antigen recognition spectrum of the immune library.

The diversity of T-cell receptor (TCR) αβ is a result of genetic recombination and diversification mechanisms occurring at the α and β TCR chain loci (Aversa et al., 2020)

Biological Value of Diversity

The high diversity of the immune tissue bank is the key factor in maintaining the functional integrity of the immune system. Abundant clonal types and sequence variations endow the immune system with the ability to recognize a wide range of antigenic epitopes, effectively reducing the blind spots for identifying pathogens or abnormal cells.

On the other hand, when the diversity of the immune group library is significantly reduced due to immune deficiency, aging, or disease state, the breadth and specificity of antigen recognition of the immune system will be damaged, which will lead to the decline of immune monitoring function and increase the risk of infectious diseases and tumors.

Principle of Immune Repertoire Sequencing

Immune repertoire sequencing library sequencing captures the variable region sequence of the TCR/BCR gene by a high-throughput sequencing technique, and then analyzes the composition, diversity, and cloning dynamics of the Immune repertoire sequencing library. Its core principle revolves around specific capture of target sequence and Qualcomm quantity analysis of sequence information.

Specific Capture of the Target Sequence

The variable regions of TCR and BCR, especially the CDR3 region encoding antigen-binding sites, constitute the core of the diversity of immune libraries. Because each lymphocyte clone has a unique CDR3 sequence, in the process of IR-seq, the design of specific primers becomes a key technical link to achieve accurate capture.

In order to ensure the complete coverage of the diversity of the immune library, the primer design should follow the principles of species specificity and chain specificity. Taking model organisms such as humans and mice as examples, for the variable regions of TCR α/β chain and BCR heavy chain/light chain, researchers need to systematically integrate the gene annotation information in authoritative databases such as IMGT and design primer combinations that can target all known V and J gene fragments. This double primer design strategy can not only effectively capture low-frequency cloned sequences but also significantly reduce sequencing costs and data analysis complexity.

Construction of Sequencing Library

The captured variable region DNA/RNA (usually extracted from peripheral blood, tissues, or single cells) should be transformed into a sequencing library, and the process should strictly follow the experimental norms of molecular biology.

• In the nucleic acid extraction stage, the tissue sample needs mechanical grinding or enzymolysis to release the intracellular nucleic acid. RNA samples need to be evaluated for completeness after extraction (for example, using Agilent Bioanalyzer to detect RIN value).
• In the process of reverse transcription, random primers or Oligo (dT) combined with SuperScript reverse transcriptase were used to reverse transcribe RNA into cDNA.
• The number of cycles (usually no more than 25 cycles) should be strictly controlled in the PCR amplification step to reduce the influence of PCR preference on sequence abundance.
• In the linker link, Illumina platform often uses TruSeq linker system, which connects the double-stranded DNA fragments containing sequencing primer binding site, sample Barcode, and P5/P7 sequencing linker to both ends of the target sequence through T4 DNA ligase, to ensure that each sequence has the linker information required by the sequencing platform for subsequent sequencing.
• For the construction of a single-cell library, it is necessary to realize the correlation between the cell barcode and transcript through gel bead technology.

The characteristic types and frequency distributions of T cell clones in patients with atherosclerosis (AS) (Lin et al., 2017)

High-throughput Sequencing and Data Analysis

• After obtaining a large number of short-reading or long-reading sequences through sequencing platforms (such as Illumina and PacBio), the data are processed by bioinformatics tools.
• In the quality control of original data, FastQC is used to evaluate the quality of sequencing data, and low-quality reading segments (such as Phred quality value < 20) and linker sequences (such as using the Cutadapt software) are removed. In the process of V (D) J gene fragment allocation, tools such as IMGT/HiHV-Quest or MiXCR are often used to conduct sequence alignment based on the database of the IMGT to accurately identify V, D, and J gene fragments and recombination breakpoints.
• The recognition of the CDR3 region depends on the accurate location of the V (D) J recombination boundary, and its amino acid sequence length and composition are the key indices to evaluate the specificity of the immune response. The definition of Clonotype requires setting a sequence similarity threshold (usually ≥ 97% nucleotide sequence consistency) and classifying clones with the same or highly similar sequence as the same clonotype.
• Finally, the complexity and dynamic changes of the immune library were quantitatively evaluated by core indicators such as diversity index (such as Shannon index and Simpson index) and clone abundance distribution (such as Gini coefficient). In addition, algorithms based on machine learning (such as deep learning models) are being developed to predict antigen specificity and disease-related clonal characteristics.

Genome coverage plots for 15x depth randomly downsampled sequence coverage from the sequencing platforms tested (Quail et al., 2012)

Key Sequencing Platforms for IR-Seq

The differences in technical characteristics (such as reading length, throughput, and accuracy) of different sequencing platforms determine their application scenarios in IR-seq. At present, mainstream platforms can be divided into three categories: short reading and long Qualcomm capacity platforms, long reading and long platform, and single-cell integration platforms.

A. Short-Read HTS Platforms

a) Illumina (such as NovaSeq, MiSeq), as the representative, with its core advantages of Qualcomm, high accuracy, and low cost, can generate millions to billions of reading segments at a time, which is suitable for large-scale analysis of the clonal composition and diversity of Immune repertoire sequencing libraries.

b) However, due to the short reading length (usually 50-300 bp), it is difficult to cover the complete V (D) J recombinant sequence (especially the full length of CDR1-CDR3), so it is necessary to obtain the full-length information indirectly through sequence splicing.

B. Long-Read Sequencing Platforms

a) Represented by PacBio(SMRT sequencing) and Oxford Nanopore (ONT sequencing), the reading length can reach several kilobytes to tens of kilobytes, which can directly capture the complete TCR/BCR variable region full-length sequence, accurately identify the combination of V, D and J gene fragments and the CDR3 region sequence without splicing, and is especially suitable for analyzing somatic high frequency mutation (SHM). However, the platform has relatively low throughput and high single-base cost, and is more suitable for in-depth analysis of a small sample size (such as rare TIL clone analysis in tumor tissues).

C. Single-cell integrated sequencing platform

a) Represented by 10x genomics and BD Rhapsody, it can capture TCR/BCR sequence, cell transcriptome, and protein expression information at the single cell level, and realize the correlation analysis of "clone-cell phenotype-functional state". For example, through this platform, the transcriptome characteristics of a tumor-specific T cell clone (such as whether PD-1 is highly expressed) can be clarified, which provides a direct basis for the screening of immunotherapy targets. However, the cost of this platform is high, and the number of cells that can be analyzed in a single experiment is limited (usually thousands to tens of thousands of cells).

Variable segment spectratype and Variable-Joining segment usage chord diagram (Bagaev et al., 2016)

Conclusion

To sum up, the foundation of Immune repertoire sequencing is based on the deep understanding of the molecular characteristics of TCR and BCR, and the coordinated development of sequencing technology and bioinformatics. TCR-seq focuses on the dynamics of V (D) J recombination and cloning of TCRs, which provides key data for analyzing cellular immune response. BCR-seq helps the study of humoral immune mechanisms by capturing the sequence diversity of BCRs and somatic high-frequency mutations. Although they have different target receptors and slightly different technical emphases, they together constitute the core tool to reveal the function of the immune system.

At present, from the standardization of sample preparation to the optimization of the data analysis algorithm, the basic system of immune library sequencing is still improving. The advancement of this basic research not only enables us to understand more clearly the formation and dynamic changes of the immune library, but also lays a solid foundation for the clinical transformation of TCR-seq and BCR-seq, and will continue to provide core support for the mechanism exploration and precise intervention of immune-related diseases in the future.