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

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

Genes Differentially Expressed between Ctrl-M^hi and Cre-M^lo Cell Populations

<p>Shown are representative genes that were generally either (A) up-regulated or (B) down-regulated in Cre-M^lo cells compared with Ctrl-M^hi cells. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030082#pbio-0030082-g002" target="_blank">Figure 2</a> legend for details.</p

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