The TCR repertoire can change greatly with the onset and progression of diseases, which is why scientists are becoming more and more interested in determining the immune repertoire status under different disease conditions, such as cancer, autoimmune, inflammatory and infectious diseases. The sum of all TCRs by the T cells of one individual is termed the TCR repertoire or TCR profile. For this reason, CDR3 is often used as the region of interest to determine T cell clonotypes, as it is highly unlikely that two T cells will express the same CDR3 nucleotide sequence, unless they have derived from the same clonally expanded T cell. It plays an essential role in the interaction of the TCR with the peptide-MHC complex, as it is the region of the TCR in direct contact with the peptide antigen. CDR3, however, is encoded by the junctional region between the V and J or D and J genes and is therefore highly variable. CDR1 and 2 are encoded by V genes and are required for interaction of the TCR with the MHC complex. Įach TCR chain contains three hypervariable loops in its structure, termed complementarity determining regions (CDR1–3). Additional diversity is achieved by the pairing of α and β or γ and δ chains to form a functional TCR. This process leads to strong combinatorial (depending on which gene regions will recombine) and junctional diversity (which and how many nucleotides will be added/deleted), resulting in a large and highly variable TCR repertoire, which will ensure the identification of a plethora of antigens. Additionally, random nucleotides are added and/or deleted at the junction sites between the gene segments. Recombination of the variable region with a constant gene segment results in a functional TCR chain transcript. During VDJ recombination, one random allele of each gene segment is recombined with the others to form a functional variable region (Fig. The variable region of TCRα and δ chains is encoded by a number of variable (V) and joining (J) genes, while TCRβ and γ chains are additionally encoded by diversity (D) genes. Similar to immunoglobulins expressed by B cells – membrane bound immunoglobulins are often referred to as B-cell receptors (BCRs) – the TCR chains consist of a variable region, important for antigen recognition, and a constant region. TCRs are highly diverse heterodimers, consisting of a combination of α and β chains (αβ TCR) expressed by the majority of T cells, or γδ chains (γδ TCR) expressed by T cells in peripheral blood (1–5%) and T cells found at mucosal sites. T cell mediated antigen recognition depends on the interaction of the T-cell receptor (TCR) with the antigen-major histocompatibility complex (MHC) molecules (Fig. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Here, we report on the latest methodological advancements in the field by describing and comparing the available tools from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis.įinally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire. The extreme diversity of the TCR repertoire represents a major analytical challenge this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response.
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