Rheumatoid arthritis is a common and debilitating autoimmune disease whose cause and mechanism remain a mystery. We recently described a T cell receptor transgenic mouse model that spontaneously develops a disease with most of the clinical, histological, and immunological features of rheumatoid arthritis in humans. Disease development in K/BxN mice is initiated by systemic T cell self-reactivity; it requires T cells, as expected, but B cells are also needed, more surprisingly. Here, we have identified the role of B cells as the secretion of arthritogenic immunoglobulins. We suggest that a similar scenario may unfold in some other arthritis models and in human patients, beginning with pervasive T cell autoreactivity and ending in immunoglobulin-provoked joint destruction.

The mb1 gene encodes the Ig-␣ signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.Dnmt1 ͉ SRp20 ͉ loxP ͉ enhanced yellow fluorescent protein ͉ lymphocyte T he bacteriophage recombinase cre can efficiently delete DNA sequences that are flanked by loxP sites (floxed) even in eukaryotic cells (1). This feature has led to the frequent use of transgenic cre mice for the tissue-specific deletion or modification of floxed genes to access the function of a gene in a specific tissue (2).Development of a B lymphocyte can be separated into several ordered steps encompassing commitment to the B lineage, somatic recombination and expression of its heavy chain and light chain Ig genes, and selection of the B cell antigen receptor repertoire (for reviews, see refs. 3-5). In the B cell system there are several transgenic mouse lines available that express cre in defined stages of B lymphocyte development. For example, CD19-cre mice (6) express cre from the pre-B cell stage on, whereas CD21-cre mice (7) express cre only in mature B cells. However, a cre transgenic mouse line with efficient cre-mediated deletion from the earliest pro-B cell stage was missing so far. We asked whether expression of the cre recombinase from the murine mb-1 locus would provide an even more efficient model for studying gene function specifically in B cell precursors. The mb1 gene encodes the Ig-␣ signaling subunit of the B cell antigen receptor (8, 9). It is strongly expressed in the B cell lineage beginning at the very early pro-B cell stage in the bone marrow and continues to be expressed in all later stages except plasma cells (10). The mb1-cre line was tested by intercrossing it to a floxed enhanced yellow fluorescent protein (EYFP) reporter mouse line. The analysis showed a very efficient and B cell-specific recombination. To further test the mb1-cre line, we bred it to several different lines bearing floxed genes, some of which are believed to be essential genes in all cell types. We show results for the splicing factor SRp20 and the DNA methylase Dnmt1. SRp20 belongs to a family of serine-arginine-rich proteins important for a variety of cellular functions surrounding mRNA including constitutive and alternative splicing, transport, translation, an...

The maintenance of mature B cells hinges on signals emitted from the BAFF-R cell-surface receptor, but the nature of these signals is incompletely understood. Inhibition of canonical NF-kappaB transcription factor activity through ablation of the essential scaffold protein NEMO arrests B cell development at the same stage as BAFF-R deficiency. Correspondingly, activation of this pathway by constitutively active IkappaB Kinase2 renders B cell survival independent of BAFF-R:BAFF interactions and prevents proapoptotic PKCdelta nuclear translocation. In addition, canonical NF-kappaB activity mediates differentiation and proper localization of follicular and marginal zone B cells in the absence of BAFF-R, but not CD19. By replacing BAFF-R signals, constitutive canonical NF-kappaB signaling, a hallmark of various B cell lymphomas, causes accumulation of resting B cells and promotes their proliferation and survival upon activation, but does not per se induce lymphomagenesis. Therefore, canonical NF-kappaB activity can substitute for BAFF-R signals in B cell development and pathogenesis.

Mice carrying transgenic rearranged V region genes in their IgH and Igkappa loci to encode an autoreactive specificity direct the emerging autoreactive progenitors into a pre-B cell compartment, in which their receptors are edited by secondary Vkappa-Jkappa rearrangements and RS recombination. Editing is an efficient process, because the mutant mice generate normal numbers of B cells. In a similar nonautoreactive transgenic strain, neither a pre-B cell compartment nor receptor editing was seen. Thus, the pre-B cell compartment may have evolved to edit the receptors of autoreactive cells and later been generally exploited for efficient antibody diversification through the invention of the pre-B cell receptor, mimicking an autoreactive antibody to direct the bulk of the progenitors into that compartment.

Supplemental material is available at http://www.genesdev.org.

Self-reactive B cells specific for ubiquitous membrane-bound autoantigens are eliminated in the bone marrow by two mechanisms of tolerance: receptor editing and clonal deletion. However, the relative contributions of clonal deletion and receptor editing to B cell tolerance in a polyclonal B cell population have not been established. Here we show that tolerance toward a membrane antigen-reactive B cell clone acts by receptor editing with very minimal cell loss. The capacity of receptor editing to rescue almost all autoreactive B cells from deletion relies on the availability of multiple joining light chain gene segments as substrate for secondary immunoglobulin light chain gene rearrangement and is independent of the affinity of the autoantigen and the presence of non-autoreactive B cells. Our data further suggest that clonal deletion is a default pathway that functions only when receptor editing has been exhausted.

Hematologic disorders can be caused by sporadic or inherited mutations. However, the molecular mechanisms that lead to pathogenicity are only partially understood. An accurate method to generate mouse models is conditional gene manipulation facilitated by the Cre-loxP recombination system. To enable identification and genomic manipulation of erythroid progenitor cells, we established a knock-in mouse model (ErGFPcre) that expresses an improved GFPcre fusion protein controlled by the endogenous erythropoietin receptor (EpoR) promoter. We show that IntroductionCells of the hematopoietic system such as erythrocytes have a short half-life and are continuously renewed in a tightly controlled growth process. Dysregulation of erythropoiesis results in erythroleukemias or in anemias. The molecular cause for many of these diseases is still unknown. A key regulator of erythropoiesis is the hormone erythropoietin (Epo) and its cognate receptor, the erythropoietin receptor (EpoR). Besides some recent evidence for EpoR expression in the neuronal system, endothelial cells, and the heart, 1-5 the EpoR is predominantly expressed in the erythroid lineage. The receptor is present from the erythroid burst-forming unit (BFU-E) stage, maximally expressed at the erythroid colonyforming unit (CFU-E) stage and/or proerythroblast stage, and declines thereafter. [6][7][8][9] The essential and nonredundant role of the EpoR for the onset of erythropoiesis was confirmed by genetargeting experiments. 10 A reliable surface marker for the identification and enrichment of early erythroid progenitor cells in the murine system is missing, hence signal transduction through the EpoR has been primarily studied in hematopoietic cell lines. However, to determine the in vivo role of signaling molecules, the regulatory pathways have to be studied in the context of an organism.To introduce precise genetic alterations and modifications in the mouse, gene targeting in murine embryonic stem (ES) cells is used. 11 Although gene inactivation by classical gene knock-out has been highly informative, broad effects in multiple tissues or early embryonic lethality often obscure the role of genes in specific tissues or at later developmental stages. Several of the signaling molecules implicated in EpoR signaling or linked to hematologic disorders showed early embryonic lethality upon homozygous inactivation. Examples are the tyrosine phosphatase SHP-2 12 (Src homology 2 domain containing tyrosine phosphatase-2) and the signal transducer and activator of transcription 3 (STAT3), 13 which is constitutively activated in polycythemia rubra vera, a disease characterized by hyperproliferation of the myeloid, erythroid, and megakaryocytic lineage. 14 Conditional gene-targeting strategies such as the Cre-loxP system have the potential to circumvent these limitations by the induction of temporal and/or cell-type-restricted DNA rearrangements. Cre is a site-specific DNA recombinase of bacteriophage P1 that catalyzes reciprocal recombination between two 34-base pair (bp) DNA...

The clonal selection theory states that B lymphocytes producing high-affinity immunoglobulins are selected from a pool of cells undergoing antibody gene mutation. Somatic hypermutation is a well-documented mechanism for achieving diversification of immune responses in mature B cells. Antibody genes were also found to be modified in such cells in germinal centers by recombination of the variable (V), diversity (D), and joining (J) segments. The ability to alter immunoglobulin expression by V(D)J recombination in the selective environment of the germinal center may be an additional mechanism for inactivation or diversification of immune responses.