T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

Abu Dhabi Stem Cells Center Abu Dhabi United Arab Emirates

Center for Molecular and Cellular Biology Skolkovo Institute of Science and Technology Moscow Russia

Central European Institute of Technology Masaryk University Brno Czechia

Department of Biomolecular Sciences and Department of Molecular Neuroscience Weizmann Institute of Science Rehovot Israel

Department of Molecular Technologies Institute of Translational Medicine Pirogov Russian National Research Medical University Moscow Russia

Dmitry Rogachev National Medical and Research Center of Pediatric Hematology Oncology and Immunology Moscow Russia

Genomics of Adaptive Immunity Department Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Moscow Russia

Zobrazit více v PubMed

Lefranc M-P, Lefranc G. The T cell Receptor factsBook. 1st edition. London: Academic Press; (2001).

Lefranc M-P. Immunoglobulin and T cell receptor genes: IMGT® and the birth and rise of immunoinformatics. Front Immunol (2014) 5:22. doi: 10.3389/fimmu.2014.00022 PubMed DOI PMC

Schatz DG, Ji Y. Recombination centres and the orchestration of V(D)J recombination. Nat Rev Immunol (2011) 11:251–63. doi: 10.1038/nri2941 PubMed DOI

Giudicelli V, Chaume D, Lefranc M-P. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res (2005) 33:D256–61. doi: 10.1093/nar/gki010 PubMed DOI PMC

Schatz DG, Swanson PC. V(D)J recombination: mechanisms of initiation. Annu Rev Genet (2011) 45:167–202. doi: 10.1146/annurev-genet-110410-132552 PubMed DOI

Ciubotaru M, Surleac MD, Metskas LA, Koo P, Rhoades E, Petrescu AJ, et al. . The architecture of the 12RSS in V(D)J recombination signal and synaptic complexes. Nucleic Acids Res (2015) 43:917–31. doi: 10.1093/nar/gku1348 PubMed DOI PMC

Eastman QM, Leu TM, Schatz DG. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature (1996) 380:85–8. doi: 10.1038/380085a0 PubMed DOI

Hempel WM, Stanhope-Baker P, Mathieu N, Huang F, Schlissel MS, Ferrier P. Enhancer control of V(D)J recombination at the TCRbeta locus: differential effects on DNA cleavage and joining. Genes Dev (1998) 12:2305–17. doi: 10.1101/gad.12.15.2305 PubMed DOI PMC

Curry JD, Schlissel MS. RAG2’s non-core domain contributes to the ordered regulation of V(D)J recombination. Nucleic Acids Res (2008) 36:5750–62. doi: 10.1093/nar/gkn553 PubMed DOI PMC

Chien YH, Iwashima M, Wettstein DA, Kaplan KB, Elliott JF, Born W, et al. . T-cell receptor delta gene rearrangements in early thymocytes. Nature (1987) 330:722–7. doi: 10.1038/330722a0 PubMed DOI

Biondi A, di CPF, Rossi V, Casorati G, Matullo G, Giudici G, et al. . High prevalence of T-cell receptor Vδ2-(D)-Dδ3 or Dδ1/2-Dδ3 rearrangements in B-precursor acute lymphoblastic leukemias. Blood (1990) 75:1834–40. doi: 10.1182/blood.V75.9.1834.1834 PubMed DOI

Punt J, Stranford S, Jones P, Owen JA. Kuby Immunology, Eighth edition. W.H. Freeman and Company; (2019) pp. 241–2.

Safonova Y, Pevzner PA. V(DD)J recombination is an important and evolutionarily conserved mechanism for generating antibodies with unusually long CDR3s. Genome Res (2020) 30:1547–58. doi: 10.1101/gr.259598.119 PubMed DOI PMC

Liu P, Liu D, Yang X, Gao J, Chen Y, Xiao X, et al. . Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains. Protein Cell (2014) 5:603–15. doi: 10.1007/s13238-014-0060-1 PubMed DOI PMC

Pogorelyy MV, Elhanati Y, Marcou Q, Sycheva AL, Komech EA, Nazarov VI, et al. . Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires. PloS Comput Biol (2017) 13:e1005572. doi: 10.1371/journal.pcbi.1005572 PubMed DOI PMC

Benedict CL, Gilfillan S, Thai T-H, Kearney JF. Terminal deoxynucleotidyl transferase and repertoire development. Immunol Rev (2000) 175:150–7. doi: 10.1111/j.1600-065X.2000.imr017518.x PubMed DOI

Sycheva AL, Komech EA, Pogorelyy MV, Minervina AA, Urazbakhtin SZ, Salnikova MA, et al. . Inactivated tick-borne encephalitis vaccine elicits several overlapping waves of T cell response. Front Immunol (2022) 13:970285. doi: 10.3389/fimmu.2022.970285 PubMed DOI PMC

Dupic T, Marcou Q, Walczak AM, Mora T. Genesis of the αβ T-cell receptor. PloS Comput Biol (2019) 15:e1006874. doi: 10.1371/journal.pcbi.1006874 PubMed DOI PMC

Marcou Q, Mora T, Walczak AM. High-throughput immune repertoire analysis with IGoR. Nat Commun (2018) 9:561. doi: 10.1038/s41467-018-02832-w PubMed DOI PMC

Pogorelyy MV, Minervina AA, Chudakov DM, Mamedov IZ, Lebedev YB, Mora T, et al. . Method for identification of condition-associated public antigen receptor sequences. Elife (2018) 7:e33050. doi: 10.7554/eLife.33050 PubMed DOI PMC

Pogorelyy MV, Minervina AA, Shugay M, Chudakov DM, Lebedev YB, Mora T, et al. . Detecting T cell receptors involved in immune responses from single repertoire snapshots. PloS Biol (2019) 17:e3000314. doi: 10.1371/journal.pbio.3000314 PubMed DOI PMC

Hou X, Zeng P, Zhang X, Chen J, Liang Y, Yang J, et al. . Shorter TCR β-chains are highly enriched during thymic selection and antigen-driven selection. Front Immunol (2019) 10:299. doi: 10.3389/fimmu.2019.00299 PubMed DOI PMC

de Greef PC, de Boer RJ. TCRβ rearrangements without a D segment are common, abundant, and public. Proc Natl Acad Sci (2021) 118:e2104367118. doi: 10.1073/pnas.2104367118 PubMed DOI PMC

Nazarov VI, Minervina AA, Komkov AY, Pogorelyy MV, Maschan MA, Olshanskaya YV, et al. . Reliability of immune receptor rearrangements as genetic markers for minimal residual disease monitoring. Bone Marrow Transplant (2016) 51:1408–10. doi: 10.1038/bmt.2016.148 PubMed DOI

Brüggemann M, Kotrová M, Knecht H, Bartram J, Boudjogrha M, Bystry V, et al. . Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study. Leukemia (2019) 33:2241–53. doi: 10.1038/s41375-019-0496-7 PubMed DOI PMC

Tirtakusuma R, Szoltysek K, Milne P, Grinev V, Ptasinska A, Chin PS, et al. . Epigenetic regulator genes direct lineage switching in MLL/AF4 leukaemia. Blood (2022) 140(17):1875–90. doi: 10.1182/blood.2021015036 PubMed DOI PMC

Semchenkova A, Mikhailova E, Komkov A, Gaskova M, Abasov R, Matveev E, et al. . Lineage conversion in pediatric B-cell precursor acute leukemia under blinatumomab therapy. Int J Mol Sci (2022) 23:4019. doi: 10.3390/ijms23074019 PubMed DOI PMC

Komkov A, Miroshnichenkova A, Nugmanov G, Popov A, Pogorelyy M, Zapletalova E, et al. . High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid Malignancies. Br J Haematol (2020) 188:723–31. doi: 10.1111/bjh.16230 PubMed DOI

Bolotin DA, Poslavsky S, Mitrophanov I, Shugay M, Mamedov IZ, Putintseva EV, et al. . MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods (2015) 12:380–1. doi: 10.1038/nmeth.3364 PubMed DOI

Shugay M, Bagaev DV, Turchaninova MA, Bolotin DA, Britanova OV, Putintseva EV, et al. . VDJtools: unifying post-analysis of T cell receptor repertoires. PloS Comput Biol (2015) 11:e1004503. doi: 10.1371/journal.pcbi.1004503 PubMed DOI PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol (2013) 30:772–80. doi: 10.1093/molbev/mst010 PubMed DOI PMC

Crooks GE, Hon G, Chandonia J-M, Brenner SE. WebLogo: A sequence logo generator. Genome Res (2004) 14:1188–90. doi: 10.1101/gr.849004 PubMed DOI PMC

Smirnova AO, Miroshnichenkova AM, Olshanskaya YV, Maschan MA, Lebedev YB, Chudakov DM, et al. . The use of non-functional clonotypes as a natural calibrator for quantitative bias correction in adaptive immune receptor repertoire profiling. Elife (2023) 12:e69157. doi: 10.7554/eLife.69157 PubMed DOI PMC