Induction of Ig Somatic Hypermutation and Class Switching in a Human Monoclonal IgM+ IgD+ B Cell Line In Vitro: Definition of the Requirements and Modalities of Hypermutation (original) (raw)
Related papers
1998
B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted C H regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and recombination of the Ig H locus in sorted CL-01 cells suggest that Ig class switching begins in centroblasts, it extends to all isotypes in centrocytes, and it is extinct in memory B cells. Thus, we have induced coordinated Ig class switching, progression through germinal center phenotypic stages, and differentiation to memory B cells and plasma cells at the level of a single B clonotype. Our data suggest that these processes are likely regulated by a common maturation program, the activation of which may require CD40 ligand, IL-4, IL-10, and IL-6 only.
Journal of immunology (Baltimore, Md. : 1950), 1998
B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and ...
Proceedings of the National Academy of Sciences, 2001
Somatically mutated IgM ؉ -only and IgM ؉ IgD ؉ CD27 ؉ B lymphocytes comprise Ϸ25% of the human peripheral B cell pool. These cells phenotypically resemble class-switched B cells and have therefore been classified as postgerminal center memory B cells. Xlinked hyper IgM patients have a genetic defect characterized by a mutation of the CD40L gene. These patients, who do not express a functional CD40 ligand, cannot switch Ig isotypes and do not form germinal centers and memory B cells. We report here that an IgM ؉ IgD ؉ CD27 ؉ B cell subset with somatically mutated Ig receptors is generated in these patients, implying that these cells expand and diversify their Ig receptors in the absence of classical cognate T-B collaboration. The presence of this sole subset in the absence of IgM ؉ -only and switched CD27 ؉ memory B cells suggests that it belongs to a separate diversification pathway.
European Journal of Immunology, 2001
Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD -IgM + (IgM-only) and IgD + IgM + cells, and that together they form the CD27 + compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27 + (CD40L stimulation) B cells. Already switched IgG + B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD + CD27 + switched as much as IgD + CD27 -B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD + CD27 + human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel crossreactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.
The Journal of Immunology, 2000
The human bcl-6 proto-oncogene has been found to be mutated in both neoplastic and normal B cells. We used CL-01 cells, our monoclonal model of germinal center differentiation, and normal human B cells to explore the induction requirements and the modalities of bcl-6 hypermutation. As we have previously shown, CL-01 cells are IgM+ IgD+ and effectively mutate the expressed Ig VHDJH and VλJλ genes and switch to IgG, IgA, and IgE upon B cell receptor engagement and contact with CD4+ T cells through CD40:CD154 and CD80:CD28 coengagement. In this paper we showed that the same stimuli induce somatic hypermutation of bcl-6 in CL-01 and normal IgM+ IgD+ B cells. bcl-6 hypermutation was not accompanied by translocation of this proto-oncogene or hypermutation of the β-actin gene, and it did mimic Ig hypermutation. It was associated with transcription initiation, in that it targeted the first exon and a 696-bp sequence immediately downstream (∼0.6 kb) of the transcription initiation site while...
T Cell-Independent Somatic Hypermutation in Murine B Cells with an Immature Phenotype
Immunity, 2004
We asked whether somatic hypermutation contributes to the diversity of the repertoire of murine immunoglobulin genes and whether AID is expressed early dur-In order to address these questions, we studied so-Program in Immunology matic hypermutation in light chain genes from mice Tufts University School of Medicine and with a limited Ig repertoire. This strategy was based Sackler School of Graduate Biomedical Sciences on the assumption that a limited primary Ig repertoire Boston, Massachusetts 02111 provides an experimental system in which normally rare 2 The CBR Institute for Biomedical Research and somatic variants will be expanded because of strong Department of Pediatrics selective pressure. Indeed, receptor editing in the early Harvard Medical School stages of B cell development, another mechanism of Boston, Massachusetts 02115 diversification of the repertoire, was first discovered in a similar experimental situation (Gay et al., 1993; Kleinfield et al., 1986; Tiegs et al., 1993). Summary Four models were chosen for analysis: Ϫ/Ϫ mice, QM mice, QM C4 Ϫ/Ϫ mice, and QM C4 Ϫ/Ϫ CD3⑀ Ϫ/Ϫ mice. Ϫ/Ϫ Somatic hypermutation contributes to the generation mice have a -restricted repertoire (Chen et al., 1993; of antibody diversity and is strongly associated with