Contour plots are from one representative example of each group of mice (= 4). known to induce the death of B-1a cells rather than activation. With that, our data reveal fundamental differences in the response regulation of B-1 and B-2 cells during an infection. B-1 cells are a small subset of B cells that secrete most, if not all, natural antibodies in the apparent absence of antigenic challenge. Natural antibodies are often polyreactive and bind to foreign antigens as well as to self-components (1C4). B-1 cellCderived natural antibodies are crucial for host survival from infections. Defects in their production cause increased deaths after infection with bacteria, such as and (5C7), and viruses, such as vesicular stomatitis virus, lymphocytic choriomeningitis virus, vaccinia virus, and influenza (2, 8). B-1 cells are composed of two sister populations, CD5+ B-1a and CD5? B-1b (9). In addition to their disparate expression of CD5, B-1a and B-1b cells appear also to differ developmentally and functionally (10). Developmental differences between B-1a and B-1b cells were identified by exploiting a hallmark of B-1 GSK1324726A (I-BET726) cells, namely their ability to self-replenish (11). B-1a and B-1b cell subsets are thought to replenish only themselves and not the other sister population. Although the mechanisms underlying B-1 cells ability to self-replenish are not understood, earlier studies by Lalor et al. (12) revealed a homeostatic regulatory mechanism by which the presence of normal numbers of B-1 cells in the peritoneal cavity suppresses further B-1 cell expansion and/or de novo development. The difference in CD5 expression between B-1a and B-1b cells might be a crucial factor determining their in vivo responsiveness to pathogen invasion. CD5 acts as a negative regulator of B cell receptor (BCR)Cmediated activation signals (13) and renders B-1a cells nonresponsive to in vitro BCR cross-linking (14). Consistent with the expression of the inhibitor CD5 on B-1a but not B-1b cells, only CD5? B-1b cells were shown to clonally expand in vivo after infection with challenge (15, 16). Although B-1a cells also contributed to immune protection in that system, this was thought to be mediated via natural GSK1324726A (I-BET726) antibody production. Furthermore, B-1b cells formed strong antiCPPS-3 (pneumococcal polysaccharide-3) immune responses after immunization with either PPS-3 or heat-killed and conferred immunity against after their adoptive transfer into Rag1?/? recipient mice (17). B-1a cells did not mount antiCPPS-3 responses after immunization with heat-killed (17). B-1b cells were also shown to mount long-term antibody responses to TI-2 (thymus-independent type-2) antigen, NP-Ficoll (4-hydroxy-3-nitrophenyl-acetyl conjugated to the polysaccharide Ficoll) (18). Collectively, these data were interpreted as evidence for a mainly passive role for CD5+ B-1a cells as producers of natural antibodies and an active role for B-1b cells (19). However, the results from those recent studies are in apparent contrast to earlier studies that had demonstrated an active participation of B-1a cells to (20, 21). Those earlier studies demonstrated GSK1324726A (I-BET726) that immunization led B-1a cells to generate the robust and dominant T15 idiotype antibody response to phosphocholine (20, 21), which provides immune protection against reinfection with (22). Furthermore, although B-1a cells do not respond to BCR-mediated signals they strongly respond to various innate signals both in vivo and in vitro (13, 23). Natural antibodies produced by B-1 cells are mostly of the IgM isotype (1). Thus, these antibodies can also be transported via the polymeric Ig UDG2 receptor onto mucosal surfaces and contribute to mucosal immune defenses (24, 25). We previously showed that natural IgM secretion by B-1 cells is required for maximal protection against influenza virusCinduced deaths by demonstrating enhanced mortality of mice that lacked B-1 cellCderived IgM but had normal levels of B-2 cell IgM (2, 26). This enhanced mortality after influenza virus infection was partially restored by transfer of natural IgM-containing serum from noninfected WT mice, whereas serum from secretory IgM KO mice (sIgM?/?) had no effect. Lung viral titers were significantly higher in influenza virusCinfected sIgM?/? mice compared with WT controls (26). Because B-1 cellCderived virus-binding serum IgM levels were unaltered after infection, we concluded in our earlier studies that B-1 cells play a mainly passive role in immune protection to influenza (2), which is consistent with the findings of others (8). Given the apparent contradictory data on B-1 cells, in particular B-1a cells, regarding their contributions to the active humoral response, we reexamined the responsiveness of B-1 cells to infection with influenza virus in the physiological context of an intact immune system. Collectively our data show strong.