Brief introduction to TCR sequencing and analysis: immune repertoire sequencing (Rep-seq) Part One

1. Introduction

T cell-mediated antigen recognition depends on the interaction of the T cell receptor (TCR) with the antigen major histocompatibility complex (MHC) molecule. TCR is a highly diverse heterodimer composed of the alpha and beta chains (αβ TCR) expressed by most T cells, or from T cells (1-5%) in peripheral blood or T-cell expressed γδ chain (γδ TCR) in mucosal sites.

Similar to immunoglobulins expressed by B cells, membrane-bound immunoglobulins are commonly referred to as B cell receptors (BCRs), which consist of variable and constant regions recognized by the antigen. The variable regions of the TCR alpha and delta chains are encoded by a plurality of variable (V) and ligation (J) genes, while the TCR beta and gamma chains are encoded by a diversity (D) gene. During VDJ recombination, one random allele of each gene fragment is recombined with other gene segments to form a functional variable region.

A functional TCR strand transcript is produced by recombining the variable region with a constant gene segment. This process results in a strong combination (depending on which gene region will recombine) and linkage diversity (how many nucleotides will be added/deleted), resulting in a large and highly variable TCR profiling, which ultimately identifies a large number of TCR profiling antigens. Diversity is achieved by pairing alpha and beta or gamma and delta chains to form a functional TCR.

Each TCR chain contains three hypervariable loops, termed complementarity determining regions CDR1-3. CDR1 and CDR2 are encoded by the V gene and are critical for interaction of TCR with MHC complexes. However, CDR3 is encoded by the junction region between V and J or D and J, and thus the CDR3 changes to a greater extent. Since CDR3 is a TCR region that is in direct contact with the antigen, CDR3 plays an important role in the interaction of TCR with the peptide-MHC complex.

Therefore, CDR3 is a common cloning region of T cells. Unless T cells are derived from the same form, it is generally unlikely that T cells express the same CDR3 sequence.

The TCR sum of all T cells became TCR profiling (TCR spectrum). As the disease continues to worsen, TCR profiling will change a lot. Researchers are increasingly concerned about the state of the immune spectrum under different disease conditions, such as cancer, autoimmunity, inflammation, and infectious diseases.

TCR profiling of T cells in patients with cirrhosis suggests that autologous stem cell transplantation can have a dramatic impact on the patient's immune system. In cancer patients, cytotoxic T cells can kill tumor-specific antigens after they have been identified. Some studies have attempted to identify specific T cell clone types involved in the process by analyzing tumor infiltrating lymphocyte profiles.

At present, the biggest problem in immunohistochemistry sequencing is that it is too diverse. In theory, VDJ rearrangement can produce 1015~1020 different clone types, and in fact about 1013 are different. This means that VDJ rearrangement is seemingly random, but actually regular and subject to various conditions.

In addition, the TCR of T cells in the normal population was previously considered to be basically similar. In fact, recent studies have shown that many TCRs are rare. This requires a fast, efficient and accurate method to study the personal immune library.

In the past few decades, different technologies have been developed to study immune libraries. For example, monoclonal antibodies can be used to analyze specific V genome subtypes by fluorescence microscopy or flow cytometry, while PCR techniques can analyze immune group information at relatively low resolution. Until the advent of high-throughput sequencing technology, researchers were able to perform parallel analysis of TCR sequences in large quantities for the first time. But there is still no gold standard because each method has its advantages and disadvantages.

2. Select the appropriate experimental material for TCR sequencing

The choice of materials before the TCR project is crucial. Whether to choose DNA or RNA has its own application scenarios, each with its own advantages and disadvantages. The DNA level will be more stable, and the TCR region can be cloned and sequenced using a single cell as a template. However, the shortcomings of the DNA level are also obvious, and it is impossible to obtain information on the transcription level of the gene. In addition, the sequencing results are inaccurate due to the presence of introns and other reasons. When selecting RNA samples, TCR sequencing will face more serious challenges. For example, T cells will contain multiple TCR transcripts, which will encounter relatively large problems at the single cell level. In addition, the actual concentration of DNA and RNA is also worth considering.

In general, due to the complexity of the target and the batch effects that may affect downstream data analysis, the processing of all samples should be as uniform as possible, for example using the same concentration and attempting to provide supersaturation for each sample.

3. TCR sequencing

Although there are many TCR sequencing methods for single cell levels, here we mainly discuss sequencing methods for cell populations. Because cell populations are more commonly used to study TCR diversity and to study different profiling in larger groups. However, this method has the disadvantage that it can only provide the frequency information of a single TCR chain, but it cannot be paired with the cells one by one.

Therefore, the method of database construction of single cells becomes more and more important. Only this method can identify two TCR chain pairs (αβ, γδ) very accurately at the cellular level, so that the analysis reaches a higher level of complexity. At the same time, it can also reflect the function of the living body more accurately.

However, the method of building a single cell is very expensive, and this method covers a limited number of cells compared to the method of building a large number of immune cells. In addition, the single cell construction method requires fresh samples for the separation and sorting of living cells, which makes the application of this method extremely limited and has certain limitations. It is also unrealistic for researchers not to get fresh disease samples for each experiment.

4. Choosing target sequences: chain and CDR regions

Although a large number of reagent companies or sequencer manufacturers currently offer kits that include sequencing services for various chains, even a small number of companies claim to be able to measure almost all TCR chains. Since the αβ T cell population accounts for the vast majority of the total T cell population, the α chain and β chain are still the most common target regions.

In previous studies, the beta chain was the primary target region of the study because the beta chain (the major component of the D gene in VDJ) has a higher combined potential than the alpha chain. A single cell can only express a unique beta chain, and the same cell may express two alpha chains, which greatly increases the complexity.

There are few papers about γδ T cell receptor, as the proportion of γδ T cells is not high in the total T cell population. Compared to αβ TCR, γδ TCR has a lower level of diversity overall and a certain abundance bias in its analysis region. Because γδ T cells have a higher frequency at the mucosa. Therefore, the current mainstream research subjects are still the whole blood / peripheral blood.

Multiplex PCR-based methods can simultaneously amplify alpha and beta chains, but this approach typically causes some errors in both the library preparation and sequencing. In any case, PCR amplification has been shown to improve the accuracy and specificity of the results.

CDR3 is the preferred target region for many TCR sequencing because the TCR of CDR3 is capable of interacting directly with peptides and antigens.

There are currently not many studies on CDR1 and CDR2, as CDR1 and CDR2 do not interact directly with antigen. However, CDR1 and CDR2 play an important role in assisting interaction with MHC, which ultimately affects the sensitivity and affinity of TCR binding. Therefore, understanding the sequence information including CDR1, CDR2 and CDR3 can help researchers to fully understand and speculate the TCR structure and its binding characteristics.

However, at present, not all methods can detect CDR1 and CDR2. This conditional limitation can only be achieved by multiple pairs of primer designs, which is called multiplex PCR. In fact, multiple pairs of specific primers can be designed at different positions of the V gene, which can be used to eliminate the possibility of detecting non-CDR3 regions.

To be continued in Part Two…

Scientists from Creative Biolabs have accomplished over hundreds of sequencing projects and accumulated sufficient knowledge and understanding of the immune system. The company is confident in providing the best service of Rep-seq at the single-cell level, and it also provides the most comprehensive germline genes and alleles identification service with the most accurate sequencing results for our global customers.

Creative Biolabs offers the unparalleled mass sequencing service to analyze the immune repertoires for customers all over the world. Depending on its cutting-edged SuPrecision™ platform which mainly based on next-generation sequencing (NGS) technique, its scientists have accumulated extensive experience in identifying the whole four types of T-cell receptor (TCR) chains (α, β, γ, and δ) and the vast majority of B-cell receptor (BCR) V(D)J sequences contained in a single sample.