The size of an individual αβ TCR repertoire of mature lymphocytes has still not been precisely determined even in humans, with estimates differing by an order of magnitude 5, 6 from 2.5 × 10 7 to 1 × 10 8. The functional TCR repertoire is shaped during maturation in the thymus, where processes of positive and negative selection assure MHC restriction and central tolerance of T cells, respectively (reviewed in Klein et al.) 4. As a result, each new T cell is provided with a potentially unique receptor, generating an extremely diverse TCR repertoire 2. CDR3 encompasses the V(D)J junction, where insertion and/or deletion of random nucleotides between the rearranged segments (N-diversity regions) increases diversity over that introduced by recombination between V, D, and J genes. The most variable fragment of a TCR – the Complementarity Determining Region 3 (CDR3) – is responsible for recognition of the peptide–MHC complex. In the murine genome, there are approximately 100 V and 60 J segments of α chain, and 35 V, 12 J, and 2 D segments of the β chain however, up to one third of them are pseudogenes or otherwise non-functional 3. During somatic recombination, V (variable) and J (joining) segments form the variable domain of the α chain, whereas the equivalent domain of the β chain is composed of the recombination of three segments: V, J, and D (diversity). The variability of TCRs, necessary to ascertain specificity and robustness for interaction with different antigens, is generated similarly to that of immunoglobulins–that is, by a random, somatic recombination of different, germline DNA segments (Fig. The main function of αβ TCRs is recognition of foreign peptides in the context of the major histocompatibility complex (MHC) 2. In general, majority of circulating T cells bear receptors formed by α and β chains (however this varies between tissues and species, with frequencies ranging from 90–99% in human, mice or rat to 60–85% in some ruminants, pigs or chickens) 1. The central role of T cells in the immune response, both humoral and cytotoxic, is mediated by membrane, heterodimeric T-cell receptors (TCRs). comparative immunology, ecology and evolutionary biology. We hope that by providing an easy-to-follow molecular protocol and on-line bioinformatics tools that do not require reference sequences (AmpliTCR and AmpliCDR3), we will encourage HTS immune repertoire profiling in other non-model vertebrates, thus opening new research avenues in e.g. A conservative, lower bound estimation of the TCRβ repertoire was similar to that found for mice (1.7–2.3 × 10 5 clonotypes). Further, we implemented unique molecular identifiers for quantitative analysis of CDR3 repertoire with stringent error correction. Overall orthology to murine genes was preserved, with 11 J and 37 V genes found in voles (although 3 V genes lacked a close orthologue). ![]() We used RNA from spleens, 5′RACE and HTS to describe V and J segments of TCRβ, qualitatively characterize preferential V–J segment usage and CDR3 length distribution. We present the first in-depth characterization of the T-cell receptor (TCR) repertoire in a non-model mammal (bank vole, Myodes glareolus), widely used in ecological and evolutionary research. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity.
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