{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T17:52:46Z","timestamp":1790790766050,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T00:00:00Z","timestamp":1759276800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Banc de Sang i Teixits institution","award":["I.2019.041"],"award-info":[{"award-number":["I.2019.041"]}]},{"name":"Banc de Sang i Teixits institution","award":["I.2023.025"],"award-info":[{"award-number":["I.2023.025"]}]},{"name":"an Investigator Initiated Studies grant from CSL Behring"}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["IJMS"],"abstract":"<jats:p>T cell receptor (TCR) profiling using next-generation sequencing (NGS) enables high-throughput, in-depth analysis of repertoire diversity, offering numerous clinical applications. We developed a DNA-based strategy to analyse the TCR\u03b2-chain using NGS and established reference values for T cell repertoire characteristics in 74 healthy donors, including 44 adults, 20 paediatrics, and 10 cord blood units (CBUs). Additionally, four paediatric patients with combined immunodeficiency (CID) or severe CID (SCID) due to deleterious mutations in recombination activating genes (RAG) were analysed. The developed strategy demonstrated high specificity, reproducibility, and sensitivity, and all functional variable and joining genes were detected with minimal PCR bias. All donors had a Gaussian-like distribution of complementary-determining region 3 length, with lower presence of non-templated nucleotides and higher proportion of non-functional clonotypes in CBUs. Both CBUs and paediatrics showed greater convergence and TCR\u03b2 diversity was significantly lower in adults and donors with cytomegalovirus-positive serostatus. Finally, an analysis of paediatric patients with RAG-SCID\/CID showed significantly shorter CDR3 region length and lower repertoire diversity compared to healthy paediatrics. In summary, we developed a reliable and feasible TCR\u03b2 sequencing strategy for application in the clinical setting, and established reference values that could assist in the diagnosis and monitoring of pathological conditions affecting the T cell repertoire.<\/jats:p>","DOI":"10.3390\/ijms26199590","type":"journal-article","created":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T11:06:02Z","timestamp":1759316762000},"page":"9590","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A High-Throughput Sequencing Strategy for Clinical Repertoire Profiling of T Cell Receptor Beta Chain: Development and Reference Values Across Healthy Adults, Paediatrics, and Cord Blood Units"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9183-1333","authenticated-orcid":false,"given":"Emma","family":"Enrich","sequence":"first","affiliation":[{"name":"Immunogenetics and Histocompatibility Laboratory, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"},{"name":"Transfusional Medicine Group, Vall d\u2019Hebron Research Institute, Universitat Aut\u00f2noma de Barcelona (VHIR-UAB), 08035 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0630-5222","authenticated-orcid":false,"given":"Mireia","family":"Ant\u00f3n-Iborra","sequence":"additional","affiliation":[{"name":"Immunogenetics and Histocompatibility Laboratory, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"},{"name":"Immunology Department, Hospital Cl\u00ednic, Centre de Diagn\u00f2stic Biom\u00e8dic (CDB), 08036 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Hobeich","sequence":"additional","affiliation":[{"name":"Transfusional Medicine Group, Vall d\u2019Hebron Research Institute, Universitat Aut\u00f2noma de Barcelona (VHIR-UAB), 08035 Barcelona, Catalonia, Spain"},{"name":"Congenital Coagulopathy Laboratory, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0117-0007","authenticated-orcid":false,"given":"Rut","family":"Mora-Buch","sequence":"additional","affiliation":[{"name":"Transfusional Medicine Group, Vall d\u2019Hebron Research Institute, Universitat Aut\u00f2noma de Barcelona (VHIR-UAB), 08035 Barcelona, Catalonia, Spain"},{"name":"Advanced & Cell Therapy Services, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2884-8228","authenticated-orcid":false,"given":"Ana Gabriela","family":"Lara-de-Le\u00f3n","sequence":"additional","affiliation":[{"name":"Advanced & Cell Therapy Services, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"},{"name":"Immunogenetics in the Autoinflammatory Response Group, Cl\u00ednic Foundation for Biomedical Research-August Pi i Sunyer Biomedical Research Institute (FCRB-IDIBAPS), 08036 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alba","family":"Parra-Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Infection and Immunity in Pediatric Patients\u2014Vall d\u2019Hebron Research Institute, Vall d\u2019Hebron Barcelona Hospital Campus, 08035 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bel\u00e9n","family":"S\u00e1nchez","sequence":"additional","affiliation":[{"name":"Infection and Immunity in Pediatric Patients\u2014Vall d\u2019Hebron Research Institute, Vall d\u2019Hebron Barcelona Hospital Campus, 08035 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8089-4945","authenticated-orcid":false,"given":"Francisco","family":"Vidal","sequence":"additional","affiliation":[{"name":"Transfusional Medicine Group, Vall d\u2019Hebron Research Institute, Universitat Aut\u00f2noma de Barcelona (VHIR-UAB), 08035 Barcelona, Catalonia, Spain"},{"name":"Congenital Coagulopathy Laboratory, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Community of Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0346-5570","authenticated-orcid":false,"given":"Pere","family":"Soler-Palacin","sequence":"additional","affiliation":[{"name":"Infection and Immunity in Pediatric Patients\u2014Vall d\u2019Hebron Research Institute, Vall d\u2019Hebron Barcelona Hospital Campus, 08035 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesc","family":"Rudilla","sequence":"additional","affiliation":[{"name":"Immunogenetics and Histocompatibility Laboratory, Banc de Sang i Teixits (Blood and Tissue Bank, BST), 08005 Barcelona, Catalonia, Spain"},{"name":"Transfusional Medicine Group, Vall d\u2019Hebron Research Institute, Universitat Aut\u00f2noma de Barcelona (VHIR-UAB), 08035 Barcelona, Catalonia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mazzotti, L., Gaimari, A., Bravaccini, S., Maltoni, R., Cerchione, C., Juan, M., Azucena-Gonzalez, E., Pasetto, A., Nascimento, D., and Ancarani, V. (2022). T-Cell Receptor Repertoire Sequencing and Its Applications: Focus on Infectious Diseases and Cancer. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23158590"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"994","DOI":"10.1158\/1078-0432.CCR-22-2469","article-title":"T-Cell Receptor Repertoire Sequencing in the Era of Cancer Immunotherapy","volume":"29","author":"Frank","year":"2023","journal-title":"Clin. Cancer Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1007\/s00109-014-1145-2","article-title":"Molecules in medicine mini review: The \u03b1\u03b2 T cell receptor","volume":"92","author":"Clambey","year":"2014","journal-title":"J. Mol. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"883","DOI":"10.1038\/nri1977","article-title":"Structural determinants of T-cell receptor bias in immunity","volume":"6","author":"Turner","year":"2006","journal-title":"Nat. Rev. Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"D413","DOI":"10.1093\/nar\/gku1056","article-title":"IMGT\u00ae, the international ImMunoGeneTics information system\u00ae 25 years on","volume":"43","author":"Lefranc","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"101810","DOI":"10.1016\/j.smim.2023.101810","article-title":"Numbers and odds: TCR repertoire size and its age changes impacting on T cell functions","volume":"69","author":"Weng","year":"2023","journal-title":"Semin. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1111\/imr.12667","article-title":"Human adaptive immune receptor repertoire analysis-Past, present, and future","volume":"284","author":"Nielsen","year":"2018","journal-title":"Immunol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Six, A., Mariotti-Ferrandiz, M.E., Chaara, W., Magadan, S., Pham, H.P., Lefranc, M.P., Mora, T., Thomas-Vaslin, V., Walczak, A.M., and Boudinot, P. (2013). The past, present, and future of immune repertoire biology\u2014The rise of next-generation repertoire analysis. Front. Immunol., 4.","DOI":"10.3389\/fimmu.2013.00413"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rosati, E., Dowds, C.M., Liaskou, E., Henriksen, E.K.K., Karlsen, T.H., and Franke, A. (2017). Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol., 17.","DOI":"10.1186\/s12896-017-0379-9"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1038\/s41375-019-0496-7","article-title":"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","volume":"33","author":"Knecht","year":"2019","journal-title":"Leukemia"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1038\/s41408-020-00377-0","article-title":"Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma","volume":"10","author":"Medina","year":"2020","journal-title":"Blood Cancer J."},{"key":"ref_12","first-page":"38","article-title":"T cell receptor sequencing in autoimmunity","volume":"2","author":"Mitchell","year":"2020","journal-title":"J. Life Sci."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Nakayama, M., and Michels, A.W. (2021). Using the T Cell Receptor as a Biomarker in Type 1 Diabetes. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.777788"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1038\/s41525-019-0084-9","article-title":"RNA sequencing identifies clonal structure of T-cell repertoires in patients with adult T-cell leukemia\/lymphoma","volume":"4","author":"Farmanbar","year":"2019","journal-title":"NPJ Genomic Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"eaah6109","DOI":"10.1126\/sciimmunol.aah6109","article-title":"Characterization of T and B cell repertoire diversity in patients with RAG deficiency","volume":"1","author":"Lee","year":"2016","journal-title":"Sci. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1007\/s10875-021-01178-1","article-title":"T Cell Repertoire Abnormality in Immunodeficiency Patients with DNA Repair and Methylation Defects","volume":"42","author":"Fang","year":"2022","journal-title":"J. Clin. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e149080","DOI":"10.1172\/jci.insight.149080","article-title":"Clinical and basic implications of dynamic T cell receptor clonotyping in hematopoietic cell transplantation","volume":"6","author":"Pagliuca","year":"2021","journal-title":"JCI Insight"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4485","DOI":"10.1182\/bloodadvances.2020004073","article-title":"Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI","volume":"5","author":"Kuhlmann","year":"2021","journal-title":"Blood Adv."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1038\/s41409-022-01885-2","article-title":"Potential of TCR sequencing in graft-versus-host disease","volume":"58","author":"Goel","year":"2023","journal-title":"Bone Marrow Transplant."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1567","DOI":"10.1016\/j.bbmt.2020.04.026","article-title":"T Cell Clonal Dynamics Determined by High-Resolution TCR-\u03b2 Sequencing in Recipients after Allogeneic Hematopoietic Cell Transplantation","volume":"26","author":"Leick","year":"2020","journal-title":"Biol. Blood Marrow Transplant."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2582","DOI":"10.1038\/s41409-021-01354-2","article-title":"T cell receptor beta locus sequencing early post-allogeneic stem cell transplant identifies patients at risk of initial and recurrent cytomegalovirus infection","volume":"56","author":"Kuzich","year":"2021","journal-title":"Bone Marrow Transplant."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Milano, F., Emerson, R.O., Salit, R., Guthrie, K.A., Thur, L.A., Dahlberg, A., Robins, H.S., and Delaney, C. (2020). Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.583349"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1422","DOI":"10.1038\/s41375-019-0654-y","article-title":"Genetic T-cell receptor diversity at 1 year following allogeneic hematopoietic stem cell transplantation","volume":"34","author":"Buhler","year":"2020","journal-title":"Leukemia"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"812","DOI":"10.1182\/bloodadvances.2022007270","article-title":"Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing","volume":"7","author":"Huisman","year":"2023","journal-title":"Blood Adv."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1111\/bjh.16053","article-title":"T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy","volume":"187","author":"Keller","year":"2019","journal-title":"Br. J. Haematol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"572.e1","DOI":"10.1016\/j.jtct.2023.06.001","article-title":"Virus-Specific T Cells From Cryopreserved Blood During an Emergent Virus Outbreak for a Potential Off-the-Shelf Therapy","volume":"29","author":"Enrich","year":"2023","journal-title":"Transplant. Cell Ther."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"610","DOI":"10.1182\/blood.2019000923","article-title":"Immune dysregulation in patients with RAG deficiency and other forms of combined immune deficiency","volume":"135","author":"Delmonte","year":"2020","journal-title":"Blood"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2680","DOI":"10.1038\/ncomms3680","article-title":"Using synthetic templates to design an unbiased multiplex PCR assay","volume":"4","author":"Carlson","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Tan, K.T., Ding, L.W., Sun, Q.Y., Lao, Z.T., Chien, W., Ren, X., Xiao, J.F., Loh, X.Y., Xu, L., and Lill, M. (2018). Profiling the B\/T cell receptor repertoire of lymphocyte derived cell lines. BMC Cancer, 18.","DOI":"10.1186\/s12885-018-4840-5"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1038\/nmeth.3364","article-title":"MiXCR: Software for comprehensive adaptive immunity profiling","volume":"12","author":"Bolotin","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1038\/nbt.3979","article-title":"Antigen receptor repertoire profiling from RNA-seq data","volume":"35","author":"Bolotin","year":"2017","journal-title":"Nat. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5005","DOI":"10.4049\/jimmunol.1600005","article-title":"Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians","volume":"196","author":"Britanova","year":"2016","journal-title":"J. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Yang, L., Jin, R., Lu, D., and Ge, Q. (2020). T cell Tolerance in Early Life. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.576261"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1230","DOI":"10.4049\/jimmunol.148.4.1230","article-title":"Developmental regulation of D beta reading frame and junctional diversity in T cell receptor-beta transcripts from human thymus","volume":"148","author":"George","year":"1992","journal-title":"J. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1385\/IR:26:1-3:265","article-title":"Regulation and chance in the ontogeny of B and T cell antigen receptor repertoires","volume":"26","author":"Zemlin","year":"2002","journal-title":"Immunol. Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"13694","DOI":"10.1073\/pnas.1107594108","article-title":"Nonrandom attrition of the naive CD8+ T-cell pool with aging governed by T-cell receptor:pMHC interactions","volume":"108","author":"Rudd","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2689","DOI":"10.4049\/jimmunol.1302064","article-title":"Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence profiling","volume":"192","author":"Britanova","year":"2014","journal-title":"J. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/s12979-020-00195-9","article-title":"Genetic and environmental determinants of human TCR repertoire diversity","volume":"17","author":"Krishna","year":"2020","journal-title":"Immun. Ageing"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e158122","DOI":"10.1172\/JCI158122","article-title":"Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets","volume":"132","author":"Sun","year":"2022","journal-title":"J. Clin. Investig."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Zhuo, Y., Yang, X., Shuai, P., Yang, L., Wen, X., Zhong, X., Yang, S., Xu, S., Liu, Y., and Zhang, Z. (2022). Evaluation and comparison of adaptive immunity through analyzing the diversities and clonalities of T-cell receptor repertoires in the peripheral blood. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.916430"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.imlet.2021.10.002","article-title":"Age-related changes in the TRB and IGH repertoires in healthy adult males and females","volume":"240","author":"Gong","year":"2021","journal-title":"Immunol. Lett."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Ma, L., Yang, L., Shi, B., He, X., Peng, A., Li, Y., Zhang, T., Sun, S., Ma, R., and Yao, X. (2016). Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS. Sci. Rep., 6.","DOI":"10.1038\/srep29544"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1159\/000445656","article-title":"Analysis of the Repertoire Features of TCR Beta Chain CDR3 in Human by High-Throughput Sequencing","volume":"39","author":"Hou","year":"2016","journal-title":"Cell Physiol. Biochem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e15419","DOI":"10.1111\/tan.15419","article-title":"Development and characterization of a cell donor registry for virus-specific T cell manufacture in a blood bank","volume":"103","author":"Rudilla","year":"2024","journal-title":"HLA"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9590\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T11:11:44Z","timestamp":1759317104000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9590"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,1]]},"references-count":44,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms26199590"],"URL":"https:\/\/doi.org\/10.3390\/ijms26199590","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,1]]}}}