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Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and\/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and\/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.<\/jats:p>","DOI":"10.4049\/jimmunol.2300330","type":"journal-article","created":{"date-parts":[[2023,7,19]],"date-time":"2023-07-19T13:35:07Z","timestamp":1689773707000},"page":"743-754","source":"Crossref","is-referenced-by-count":7,"title":["<i>N<\/i>-Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201"],"prefix":"10.1093","volume":"211","author":[{"given":"Anastasia","family":"Iatrou","sequence":"first","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]},{"name":"Department of Molecular Biology and Genetics, Democritus University of Thrace , Alexandroupolis ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Gounari","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Electra","family":"Sofou","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0207-257X","authenticated-orcid":false,"given":"Laura","family":"Zaragoza-Infante","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ioannis","family":"Markopoulos","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3545-0330","authenticated-orcid":false,"given":"Ioannis","family":"Sarrigeorgiou","sequence":"additional","affiliation":[{"name":"Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute , Athens ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8822-0141","authenticated-orcid":false,"given":"Georgios","family":"Petrakis","sequence":"additional","affiliation":[{"name":"Pathology Department, Medical School, Aristotle University of Thessaloniki , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolaos","family":"Pechlivanis","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8690-6473","authenticated-orcid":false,"given":"Maria","family":"Roumeliotou-Dimou","sequence":"additional","affiliation":[{"name":"Hematology Section of the First Department of Propedeutic Internal Medicine, Laikon University Hospital , Athens ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panagiotis","family":"Panayiotidis","sequence":"additional","affiliation":[{"name":"Hematology Section of the First Department of Propedeutic Internal Medicine, Laikon University Hospital , Athens ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Basile","family":"Stamatopoulos","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Cell Therapy, Jules Bordet Institute, Free University of Brussels , Brussels ,","place":["Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Gkanidou","sequence":"additional","affiliation":[{"name":"Blood Transfusion Department, G. Papanikolaou Hospital , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Sandaltzopoulos","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Democritus University of Thrace , Alexandroupolis ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0787-1883","authenticated-orcid":false,"given":"Massimo","family":"Degano","sequence":"additional","affiliation":[{"name":"Biocrystallography Unit, Division of Immunology, Transplantation, and Infectious Diseases, IRCCS Scientific Institute San Raffaele , Milan ,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4435-3532","authenticated-orcid":false,"given":"Triantafyllia","family":"Koletsa","sequence":"additional","affiliation":[{"name":"Pathology Department, Medical School, Aristotle University of Thessaloniki , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4693-789X","authenticated-orcid":false,"given":"Peggy","family":"Lymberi","sequence":"additional","affiliation":[{"name":"Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute , Athens ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0222-4273","authenticated-orcid":false,"given":"Fotis","family":"Psomopoulos","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3750-7342","authenticated-orcid":false,"given":"Paolo","family":"Ghia","sequence":"additional","affiliation":[{"name":"Division of Experimental Oncology, IRCCS Scientific Institute San Raffaele , Milan ,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8467-7945","authenticated-orcid":false,"given":"Andreas","family":"Agathangelidis","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]},{"name":"Department of Biology, School of Science, National and Kapodistrian University of Athens , Athens ,","place":["Greece"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia","family":"Chatzidimitriou","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]},{"name":"Department of Molecular Medicine and Surgery, Karolinska Institute , Stockholm ,","place":["Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kostas","family":"Stamatopoulos","sequence":"additional","affiliation":[{"name":"Institute of Applied Biosciences, Centre for Research & Technology Hellas , Thessaloniki ,","place":["Greece"]},{"name":"Department of Molecular Medicine and Surgery, Karolinska Institute , Stockholm ,","place":["Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,7,19]]},"reference":[{"key":"2025020406090866200_r1","doi-asserted-by":"crossref","first-page":"2879","DOI":"10.1182\/blood-2004-01-0132","article-title":"Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia","volume":"104","author":"Tobin","year":"2004","journal-title":"Blood"},{"key":"2025020406090866200_r2","doi-asserted-by":"crossref","first-page":"1678","DOI":"10.1182\/blood-2004-07-2606","article-title":"Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: the lesson of the IGHV3-21 gene","volume":"105","author":"Ghia","year":"2005","journal-title":"Blood"},{"key":"2025020406090866200_r3","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1182\/blood-2006-03-012948","article-title":"Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations","volume":"109","author":"Stamatopoulos","year":"2007","journal-title":"Blood"},{"key":"2025020406090866200_r4","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1111\/j.1365-2141.2008.07469.x","article-title":"Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study","volume":"144","author":"Bomben","year":"2009","journal-title":"Br. 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