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

Fine-Mapping Chromosome 20 in 230 Systemic Lupus Erythematosus Sib Pair and Multiplex Families: Evidence for Genetic Epistasis with Chromosome 16q12

The presence of systemic lupus erythematosus (SLE) susceptibility genes on chromosome 20 is suggested by the observation of genetic linkage in several independent SLE family collections. To further localize the genetic effects, we typed 59 microsatellites in the two best regions, as defined by genome screens. Genotypes were analyzed for statistical linkage and/or association with SLE, by use of a combination of nonparametric linkage methods, family-based tests of association (transmission/disequilibrium and pedigree disequilibrium tests), and haplotype-sharing statistics (haplotype runs test), in a set of 230 SLE pedigrees. Maximal evidence for linkage to SLE was to 20p12 (LOD=2.84) and 20q13.1 (LOD=1.64) in the white pedigrees. Subsetting families on the basis of evidence for linkage to 16q12 significantly improved the LOD scores at both chromosome 20 locations (20p12 LOD=5.06 and 20q13 LOD=3.65), consistent with epistasis. We then typed 162 single-nucleotide polymorphism markers across a 1.3-Mb candidate region on 20q13.1 and identified several SNPs that demonstrated significant evidence for association. These data provide additional support for linkage and association to 20p12 and 20q13.1 in SLE and further refine the intervals of interest. These data further suggest the possibility of epistatic relationships among loci within the 20q12, 20q13, and 16q12 regions in SLE families

Genetic Association of the R620W Polymorphism of Protein Tyrosine Phosphatase PTPN22 with Human SLE

We genotyped 525 independent North American white individuals with systemic lupus erythematosus (SLE) for the PTPN22 R620W polymorphism and compared the results with data generated from 1,961 white control individuals. The R620W SNP was associated with SLE (genotypic P=.00009), with estimated minor (T) allele frequencies of 12.67% in SLE cases and 8.64% in controls. A single copy of the T allele (W620) increases risk of SLE (odds ratio [OR]=1.37; 95% confidence interval [CI] 1.07–1.75), and two copies of the allele more than double this risk (OR=4.37; 95% CI 1.98–9.65). Together with recent evidence showing association of this SNP with type 1 diabetes and rheumatoid arthritis, these data provide compelling evidence that PTPN22 plays a fundamental role in regulating the immune system and the development of autoimmunity

Visualizing Human Leukocyte Antigen Class II Risk Haplotypes in Human Systemic Lupus Erythematosus

Human leukocyte antigen (HLA) class I and class II alleles are implicated as genetic risk factors for many autoimmune diseases. However, the role of the HLA loci in human systemic lupus erythematosus (SLE) remains unclear. Using a dense map of polymorphic microsatellites across the HLA region in a large collection of families with SLE, we identified three distinct haplotypes that encompassed the class II region and exhibited transmission distortion. DRB1 and DQB1 typing of founders showed that the three haplotypes contained DRB1*1501/ DQB1*0602, DRB1*0801/ DQB1*0402, and DRB1*0301/DQB1*0201 alleles, respectively. By visualizing ancestral recombinants, we narrowed the disease-associated haplotypes containing DRB1*1501 and DRB1*0801 to an ∼500-kb region. We conclude that HLA class II haplotypes containing DRB1 and DQB1 alleles are strong risk factors for human SLE

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

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

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

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