B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response

B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. over Zalcitabine time in the adult through self-renewal. However, Lin?CD93+CD19+B220lo bone marrow B1 progenitor cells have been identified (3). In addition, B cell receptor (BCR)6 signal strength appears to be important for B1 cell generation, as strong signals increase B1 cell numbers and weak signals decrease their numbers (4, 5). Because natural antibodies are polyreactive, they also bind to self-antigens and contribute to autoimmunity, suggesting that B1 cells must be tightly regulated during homeostasis. In addition, because they comprise the Zalcitabine first wave of B cell development, B1 cells may be linked to childhood leukemias. Work in recent years have begun to reveal a network of transcriptional Zalcitabine regulators important for B1 cell development and function. Among them, members of the classical NFB pathway (p50, Malt1, Carma1, Ikk complex), downstream of the BCR, have been shown to be essential for B1 cell development (6). The RNA-binding protein Lin28b, and its downstream effectors Let-7 and Arid3a, were revealed to promote fetal B1 cell lymphopoiesis (7, 8). Similarly, Ebf1 is required, and its overexpression induces B1 cell development at the expense of B2 cells (9, 10). In contrast, PU.1 (encoded by gene, is a zinc finger DNA-binding protein, that is a key transcriptional regulator and tumor suppressor in B cells. It is required for the specification and development of all B cell lineages (16, 17), and plays specific roles in pre-pro-B and pre-B Rabbit Polyclonal to PBOV1 cells to activate expression, mediate chromatin accessibility during immunoglobulin gene rearrangement Zalcitabine and allelic exclusion at the locus (18,C23). In mature B2 cells, Ikaros directs class switch recombination (24). It functions both as a transcriptional repressor and activator, and acts at least in part through its association with Polycomb repressive complex 2 (25), NuRD and SWI/SNF complexes (26, 27). In pre-B cells, Ikaros activates the transcription of genes important for pre-BCR and BCR signaling, cell survival, and cell migration, as well as that of B cell regulators like (22, 28). Thus Ikaros modulates B cell function at multiple stages. Here, we reveal a novel function for Ikaros as a major negative regulator of B1 cell development and function in the adult bone marrow and spleen. Experimental Procedures Mice The IkL/L and Ikf/f mouse lines have been described (18, 22). IkL/L mice were backcrossed 10 generations onto the C57Bl/6 background and analyzed at 6C8 weeks of age. Ikf/f mice were crossed with CD21-Cre, CD19-Cre, or R26-CreERT2 tg animals (29,C31). Ikaros was deleted in adult Ikf/f R26-CreERT2+ mice after daily intraperitoneal injections of tamoxifen (50 mg/kg weight of mouse, dissolved in sunflower oil) for 3 days. Female MRL/lpr mice were purchased from Harlan. Cell Culture FO B cells were sorted (B220+CD23hiCD21lo; 98% purity) on a FACSVantage S.E. option DiVa (BD Biosciences, San Jose, CA) or a FACSAria II SORP (BD Biosciences), or enriched by depletion of CD43+ cells followed by positive selection of CD23+ cells with MACS beads ( 90% purity; Miltenyi Biotech, Bergisch Gladbach, Germany). Both methods gave similar results. B1 B cells were sorted (CD19+CD43+) on a FACSAria II SORP (BD Biosciences). For BM cultures, 1 106 CD19+ BM B cells were co-cultured on S17 stromal cells in Iscove’s medium supplemented with 10% FCS, 2 Zalcitabine mm l-glutamine, 1 non-essential amino acids, 50 m 2-mercaptoethanol (2-ME), 1% antibiotics plus cytokines IL-7 (7% of supernatant from mIL-7 cDNA-transfected J558L cells), SCF (10 ng/ml; Peprotech, Rocky Hill, NJ) and Flt-3 ligand (2.5% of supernatant from mFlt3L cDNA-transfected B16 cells). For proliferation assays, cells were labeled with CFSE (5 g/ml; Sigma) and 2.5C3 104 cells were cultured in complete medium (RPMI 1640, 10% FCS, 25 mm HEPES, 1 mm sodium pyruvate, 2 mm l-glutamine, 1 non-essential amino acids, 50 m 2-ME, 1% antibiotics). Cells were stimulated with 10 g/ml goat anti-mouse IgM.