Most Marginal Zone B Cells in Rat Express Germline Encoded Ig VH Genes and Are Ligand Selected

Abstract

The present study was performed to analyze whether marginal zone B (MZ-B) cells in nondeliberately immunized adult rats are selected on basis of the specificity of their B cell receptor, and to determine to what extent memory B cells contribute to the MZ-B cell subset. To this end, the Ig PC7183 VH gene repertoire was studied among VHDJH-μ transcripts expressed in four sequential stages of B cell development, of two individual untreated adult rats. B cell subsets, i.e., pro/pre-B cells and newly formed B (NF-B) cells from bone marrow, and recirculating follicular B cells and MZ-B cells from spleen were sorted by flow cytometry. In addition, from one these rats, cells were microdissected from follicular and MZ areas of the spleen and productive PC7183 VH gene rearrangements were analyzed for the presence of somatic mutations. Sequence analysis reveals that most MZ-B cells in the adult rat, either defined by flow cytometry or by their anatomical location in the spleen, express germline encoded VH genes (naive MZ-B cells) and a minor fraction (about 20%) of the MZ-B cells carry somatic mutations (memory MZ-B cells). In addition, we show that naive MZ-B cells are a selected population of cells, both based on PC7183 VH gene repertoire and on the length of the Ig heavy (H) chain complementarity-determining region 3 (H-CDR3) region, i.e., PC7183 VHDJH-μ transcripts of MZ-B cells carry significantly shorter H-CDR3 regions than other B cell subsets.

Categorical selection of the antibody repertoire in splenic B cells

“…We show here that this process of CDR-H3 selection continues in the spleen and that B cells enriched for specific CDR-H3 sequence categories appear to either gain ready access to or are excluded from individual splenic B cell compartments. Our findings also confirm and extend previous observations that selection of B cells into the various compartments of the spleen can be influenced by V usage [17][18][19]. Collectively, these observations support the hypothesis that entry to the various peripheral B cell compartments is influenced by ligand selection [17,18,20,21] and further suggest that selective pressure may be exerted at the level of categories of antigenic epitopes.…”

“…Our findings also confirm and extend previous observations that selection of B cells into the various compartments of the spleen can be influenced by V usage [17][18][19]. Collectively, these observations support the hypothesis that entry to the various peripheral B cell compartments is influenced by ligand selection [17,18,20,21] and further suggest that selective pressure may be exerted at the level of categories of antigenic epitopes. V H usage in the bone marrow mature B cell subset was highly similar to that observed in progenitor immature B cells, but differs from that observed in all of the splenic subsets that we examined, including the transitional T1 population.…”

“…A previously recognized feature of MZ is enrichment for short CDR-H3 [18,19]. Deconstruction of the sequences we obtained in this study revealed that shorter MZ CDR-H3 length cannot be attributed solely to an increased prevalence of sequences bearing the hallmarks of neonatal origin [24].…”

“…Moreover, the shorter lengths of these MZ CDR-H3 also reflect increased exonucleolytic nibbling and altered use of shorter J H gene segments. Although it is possible that these are features of an as yet unrecognized bone marrow progenitor pool, there is a reasonable likelihood that they are a manifestation of ligand selection for a specific set of antigen binding site paratopes, and that this selection process could serve as a motive force for entry into the marginal zone [17,18,20,21].…”

Vaccination against encapsulated bacteria in humans: paradoxes

“…In rodents in which these responses have [2] been mostly studied, immunization do not induce an extensive proliferation within B cell follicles, neither the formation of germinal centers, and are for most of them taken care by unmutated germline antibodies . In humans on the contrary, the splenic marginal zone [3][4][5] contains B cells with mutated Ig receptors and mutated antibodies are raised in responses to encapsulated bacteria, these mutations [6][7][8] being responsible for the antibodies avidity for the immunizing antigen , . In all species, these TI responses do not trigger any [9 10] memory, the B cells engaged being able very rapidly to switch isotype and to secrete large amount of antibodies.…”