Summary Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies had identified bone marrow and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM secreting cells in spleen, and for the first time in the bone marrow as IgM+ IgDlo/−CD19hi CD43+ CD5+/− B-1 cells. The newly identified population of bone marrow B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and bone marrow B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Together the study identifies populations of non-terminally differentiated B-1 cells in spleen and bone marrow as the most significant producers of natural IgM.

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To define the role of alpha/beta interferons (IFN-α/β) in simian immunodeficiency virus (SIV) infection, IFN-α and IFN-β mRNA levels and mRNA levels of Mx, an antiviral effector molecule, were determined in lymphoid tissues of rhesus macaques infected with pathogenic SIV. IFN-α/β responses were induced during the acute phase and persisted in various lymphoid tissues throughout the chronic phase of infection. IFN-α/β responses were most consistent in tissues with high viral RNA levels; thus, IFN-α/β responses were not generally associated with effective control of SIV replication. IFN-α/β responses were differentially regulated in different lymphoid tissues and at different stages of infection. The most consistent IFN-α/β responses in acute and chronic SIV infection were observed in peripheral lymph nodes. In the spleen, only a transient increase in IFN-α/β mRNA levels during acute SIV infection was observed. Further, IFN-α and IFN-β mRNA levels showed a tissue-specific expression pattern during the chronic, but not the acute, phase of infection. In the acute phase of infection, SIV RNA levels in lymphoid tissues of rhesus macaques correlated with mRNA levels of both IFN-α and IFN-β, whereas during chronic SIV infection only increased IFN-α mRNA levels correlated with the level of virus replication in the same tissues. In lymphoid tissues of all SIV-infected monkeys, higher viral RNA levels were associated with increased Mx mRNA levels. We found no evidence that monkeys with increased Mx mRNA levels in lymphoid tissues had enhanced control of virus replication. In fact, Mx mRNA levels were associated with high viral RNA levels in lymphoid tissues of chronically infected animals.

Rapidly induced, specific Ab generated in extrafollicular foci are important components of early immune protection to influenza virus. The signal(s) that prompt B cells to participate in extrafollicular rather than germinal center responses are incompletely understood. To study the regulation of early B-cell differentiation events following influenza infection, we exploited earlier findings of a strong contribution of C12 idiotype-expressing B cells to the primary HA-specific response against influenza A/PR/8/34. Using an idiotype-specific mAb to C12 and labeled HA, in conjunction with multicolor flow cytometry, we followed the fate of C12Id-expressing influenza HA-specific B cells in WT BALB/c mice, requiring neither genetic manipulation nor adoptive cell transfer. Our studies demonstrate that HA-specific C12Id 1 B cells are phenotypically indistinguishable from follicular B cells. While they induced both extrafollicular and germinal center responses, extrafollicular responses were strongly predominant. Provision of increased HA-specific T-cell help increased the magnitude of the extrafollicular response, but did not shift the C12Id 1 response toward germinal center formation. Collectively the data are consistent with the hypothesis that B-cell fate determination following activation is a stochastic process in which infectioninduced innate signals might drive the preferential expansion of the early extrafollicular response. Key words: B cells . Cell differentiation . Immune responses . Immune regulation IntroductionInfluenza virus infection-induced anti-viral Ab can contribute to survival from primary and secondary infection [1][2][3][4][5][6][7]. Rapid B-cell responses in the local respiratory tract draining mediastinal LN (MedLN) are induced as early as 48-72 h after infection [8]. They contribute to viral clearance during primary infection by neutralizing the virus and reducing influenza virus spread [2,5]. Therefore the events that govern early B-cell activation following influenza virus infection are crucial for ameliorating disease outcome. The mechanisms underlying early B-cell activation, however, are incompletely understood.Rapid Ab production originates from extrafollicular foci developing at the edges of the T-and B-cell zones in secondary lymphoid tissues following antigen exposure. These responses are thought to generate primarily short-lived plasma cells [9]. Rapid Ab production at extrafollicular sites is attributed to T cell-independent as well as T-dependent responses [10,11]. In contrast, the slower intrafollicular germinal center reactions require cognate CD4 T-cell-B-cell interactions [12,13]. They are regarded as the birthplace of long-lived humoral immunity, providing both memory B cells and long-lived plasma cells [11,13]. Both extra and intra-follicular responses develop strongly in the regional LN following influenza virus infection [14].The selection events that underlie the establishment of extrafollicular versus germinal center B-cell responses are important events in the initiation ...

Background: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Methods: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. Results: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying