Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms

Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71

The Cre/loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP-flanked DNA segments. While Cre-mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site-directed mutagenesis yielded a pair of asymmetric loxP sites (lox66 and lox71) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66/lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type-specific cre-transgenes in vivo

The B-cell antigen receptor integrates adaptive and innate immune signals

B cells respond to antigens by engagement of their B-cell antigen receptor (BCR) and of coreceptors through which signals from helper T cells or pathogen-associated molecular patterns are delivered. We show that the proliferative response of B cells to the latter stimuli is controlled by BCR-dependent activation of phosphoinositidyl 3-kinase (PI-3K) signaling. Glycogen synthase kinase 3 beta and Foxo1 are two PI-3K-regulated targets that play important roles, but to different extents, depending on the specific mitogen. These results suggest a model for integrating signals from the innate and the adaptive immune systems in the control of the B-cell immune response

Inducible Cre-Mediated Deletion of the B1-8f HC Allele Leads to Loss of Surface Ig from Immature B Cells

<div><p>(A) Flow cytometry showing surface phenotype of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM after 5-d IL-7 culture.</p> <p>(B) Flow cytometric analysis of B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre BM culture cells incubated with IFNαβ (1,000 or 5,000 units/ml), in the absence of IL-7, for 1, 2, or 3 d. The cell populations shown are gated on lymphocytes by forward and side scatter, and then for B220. At the end of the 5-d IL-7 culture, more than 90% of the cells were viable and B220⁺. The numbers shown indicate the percentage of gated cells.</p></div

Microarray Gene Expression Analysis Demonstrates Co-Clustering of Cre-Deleted IgM^lo Cells with IgM⁻ Cell Populations

<div><p>(A) FACS sorting strategy for Ctrl-M^hi, Cre-M^hi, and Cre-M^lo immature B cells incubated with IFNαβ (1,000 units/ml) for 2 d. The numbers shown indicate the percentage of gated cells.</p> <p>(B) Affymetrix microarrays were used to identify genes differentially expressed between IFN-treated Ctrl-M^hi and Cre-M^lo cells. Analysis identified 327 transcripts that met the following criteria: 2-fold or greater change in mean expression level, a more than 200-unit difference in mean expression values, and Student's t-test <i>p</i> < 0.01. Individual expression values for each gene were divided by the mean of expression levels for three control IgM⁺ cell populations: IFN-treated Ctrl-M^hi; IgM^hi cells sorted from 5-d IL-7 HEL-lg BM cultures (HEL-M^hi); and sorted from normal Balb/c BM (FxE). Other populations included Cre-M^hi, Hardy Fraction D pre-B cells (FxD) sorted from normal Balb/c BM, and lgM⁻ cells sorted from 5-d IL-7 cultures of control B6 BM (B6-M⁻). Data were transformed into log₂ space, and represent fold-differences relative to the IgM⁺ cell populations (see scale bar). Data from 293 transcripts (duplicates and all but four representative Ig HC and LC transcripts removed) were clustered and visualized using CLUSTER and TREEVIEW [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-b51" target="_blank">51</a>]. Red represents genes expressed at higher levels, while green represents genes expressed at lower levels, than the mean of IgM⁺ cells. Each column represents an individual sorted cell population.</p></div

Protein Expression Confirms Reversal of Development in Immature B Cells Losing Surface IgM

<p>B1-8f/3-83κ control and B1-8f/3-83κ/Mx-Cre cell populations were harvested at the end of a 3- or 4-d culture with IFNαβ (3,000 units/ml), stained with mAbs for cell surface proteins, and analyzed by flow cytometry. Shown are the expression levels of B220-gated Ctrl-M^hi (thick line) and Cre-M^lo (thin line) cells at the end of the culture period. Essentially identical results were observed when Cre-M^hi and Cre-M^lo cells were compared (data not shown).</p

Immature B Cells That Lose Basal Signaling Show Induction of LC Rearrangements

<div><p>(A) PCR analysis of endogenous Ig light chain rearrangements (V-Jλ3, V-Jλ1, RS, and V-Jκ1) in genomic DNA of FACS-sorted HEL-Ig/Rag2-GFP BM cells incubated with medium alone or with 300 ng/ml herbimycin A for 24 h. IgM^a+GFP⁺ cells were sorted from herbimycin A-treated cultures, and IgM^a+GFP⁻ were sorted from control cultures. Data are from three independent experiments. CD14 is a loading control. −, negative control (C57Bl/6J tail DNA); +, positive control (C57Bl/6J spleen DNA).</p> <p>(B) Genomic DNA from the same cell populations described in (A) was subjected to ligation-mediated PCR to detect double-strand signal end DNA breaks at Jκ1. Controls in right three lanes of blot: H₂O, control; −, negative control (S17 stroma); +, positive control (C57Bl/6 BM).</p> <p>(C) Genomic DNA was extracted from B1-8f/3-83κ and B1-8f/3-83κ/Mx-Cre immature B cells 3 d following incubation with IFNαβ. Quantitative PCR analysis was used to determine the fold-induction of LC rearrangements in B1-8f/3-83κ/Mx-Cre immature B cells treated with IFN compared to medium alone. Data represent the mean ± standard deviation of two (V-Jλ1), three (V-Jλ3), or four experiments (V-Jκ1, RS).</p></div