31 and our unpublished results). cells compared with B-2 cells. This is the first definitive demonstration of a B cell-intrinsic function for an NFAT family transcription element. The signaling events that lead to the differentiation of CM-579 B-1a cells are not well recognized. B-1a cells are phenotypically and functionally unique from the bulk of B cells (e.g., B-2, B-0, and follicular) (examined in refs. 1 and 2); they may be long-lived cells that communicate CD5, CD43, and high IgM together with little or no IgD and low CD45(B220). In mice, B-1a cells are known to play an important part in the response to T-cell-independent type-2 antigens (3) and produce the bulk of natural serum IgM (4-7). These antibodies help obvious bacterial toxins (8) and are critically important for effective resistance to some pathogens (9). B-1a cells will also be the source of many of the gut-associated plasma cells that create IgA (7). These are important in keeping homeostasis with intestinal flora (10). B-1a cells have a restricted repertoire, reactive with many common bacterial and self-antigens. Much evidence indicates that this is a consequence of the positive selection by self-antigens of cells into the B-1a subset (examined in ref. 1). We while others (11, 12) previously showed that Rabbit polyclonal to ZNF200 crosslinking of the B cell receptor (BCR) on splenic B cells, to partially mimic positive selection by self-antigen, induces several aspects of the B-1a phenotype, including the manifestation of CD5. We consequently demonstrated that this induction of CM-579 CD5 is definitely cyclosporin-sensitive and requires an enhancer located 2 kb upstream of the CD5 coding sequences (13, 14). This enhancer consists of two practical nuclear element of triggered T cells (NFAT) sites (14) and is highly conserved in humans (15). NFAT was originally identified as a Ca2+-inducible transcription element necessary for the manifestation of IL-2 in T cells (16). It was consequently identified that there is a family of four related proteins, encoded by four genes, that have comparable properties and function similarly when ectopically expressed and in binding assays (17-21). Individual NFAT proteins are expressed in a variety of cell types, within and outside of the immune system, and they can bind to regulatory sequences of many different genes (examined in refs. 22-24). Three Ca2+-sensitive members of the NFAT transcription factor family, NFATc1 (NFAT2, NFATc), NFATc2 (NFAT1, NFATp), and NFATc3 (NFAT4, NFATx), are expressed in B cells (21, 25). B cell NFAT is usually activated by BCR crosslinking or CD40 ligation and can transactivate NFAT-dependent reporter constructs (26-28). The physiological functions of NFAT in B cells remain unclear. Targeted disruption of NFATc2 was reported to result in a slightly increased B cell proliferative response to CM-579 BCR or CD40 signaling (29). NFATc1-/- mice exhibit reduced levels of serum IgG1 (30, 31) and IgE (31), but this is apparently the result of impaired IL-4 production by T cells (30, 31). B cells in these mice were hyporesponsive to BCR or CD40 ligation effects of this are not known (30, 31). These decreased responses may reflect a B cell-intrinsic function of NFATc1 or CM-579 an effect on B cell development of the absence of NFATc1 in another cell type. In mice made up of targeted disruptions of both NFATc2 and NFATc1 there was overproduction of IgG1 and IgE and a polyclonal plasma cell infiltration of end organs (32). The overproduction of IgG1 and IgE was impartial of IL-4 and was observed with isolated B cells. Because NFAT appears to be necessary for induction of CD5, the hallmark of B-1a cells, we initiated a study of B-1 development in NFAT-deficient mice. Materials and Methods Mice. NFATc2-/-, NFATc1-/+ CM-579 double knockout mice on a BALB/c background were obtained from Laurie Glimcher (Harvard University or college School of General public Health, Boston). NFATc2-/- and NFATc1-/+ single knockout mice were obtained by breeding these mice with BALB/cByJ mice obtained from The Jackson Laboratory. Some NFATc2-/- mice were from.