Unique molecular identifiers ( UMIs) have been introduced during cDNA synthesis to minimize the impact of PCR and sequencing errors.Īmong them, multiplex PCR approaches are the most common and can be used for both gDNA and RNA. There are three common methods for the construction of TCR profiling libraries: multiplex PCR, target enrichment, and 5'RACE cDNA synthesis and nested PCR (Figure 2). Although CDR1 and CDR2 do not directly interact with antibodies, they play a vital role contacting the MHC molecules and thus affecting the sensitivity and affinity of the TCR binding. The CDR3 region has been a preferential target in many TCR profiling studies. PCR-based methods can amplify both α and β chains simultaneously, but they are often separated during sequencing in order to increase the precision and specificity of the outcome. The β chain is the main target of interest because of its uniqueness in single cells and higher combinatorial potential compared to α chains. Γδ T cells are less interesting since they are frequently found at mucosal sites. We mainly focus on cell population-based TCR sequencing in this review. Next-generation sequencing (NGS) has revolutionized TCR repertoire profiling through the ability to massively analyze TCR and antibody repertoires from blood samples. Interaction between an antigen-presenting cell (APC) and a T cell (a), and V(D)J recombination (b) (Rosati et al., 2017). ![]() Hence, it is more important to determine the immune repertoire status under different disease conditions.įigure 1. The sum of all TCRs of a human body is termed the TCR repertoire or TCR profile, which can change greatly with the onset and progression of diseases. The antigen specificity of TCRs is mainly determined by the hypervariable CDR3 of the beta chain, which is formed by recombination of V, D, and J gene segments with the addition of random nucleotides at the gene segment junctions. Each TCR chain contains three hypervariable loops, i.e., complementarity determining regions (CDR1–3). The variable region of α and δ chains is encoded by variable (V) and joining (J) genes, while β and γ chains are encoded by diversity (D) genes. The TCR chains consist of a variable region for antigen recognition and a constant region. TCRs normally consist of highly variable α and β chains expressed by a majority of T cells, or γ and δ chains expressed by a subset of T cells (1–5%). TCRs are responsible for the recognition of the Ag-MHC (major histocompatibility complex) molecules. Human adaptive immune system drives the immune response via B cell-generated immune-globulins and immunoglobulin-like TCRs on T lymphocytes.
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