title: Characterization of memory B cells responsible for affinity maturation of anti- (4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies.
abstract: We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.
authors.affiliation: Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Nishimura
authors.forename: Akikazu
authors.initials: A
authors.lastname: Murakami
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hara
authors.forename: Takachika
authors.initials: T
authors.lastname: Azuma
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000915:Antibody Affinity / Q000235:genetics; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D002450:Cell Communication / Q000276:immunology; D002469:Cell Separation; D005434:Flow Cytometry; D007114:Immunization; D007156:Immunologic Memory; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000276:immunology; D010648:Phenylacetates / Q000276:immunology; D010798:Phycocyanin / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering; D011947:Receptors, Antigen, B-Cell / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics; D055545:gamma-Globins / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D009596:Nitrophenols; D010648:Phenylacetates; D011947:Receptors, Antigen, B-Cell; C002497:allophycocyanin; D055545:gamma-Globins; C026242:4-hydroxy-5-nitrophenyl acetic acid; D010798:Phycocyanin