SUMMARY B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has so far been associated primarily with interleukin (IL)-10 because B cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens1,2. Here, we identify IL-35-producing B cells as novel key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a strikingly improved resistance to infection with the intracellular bacterial pathogen Salmonella typhimurium, as shown by their superior containment of the bacterial growth and their prolonged survival both after primary infection, and upon secondary challenge after vaccination, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an enhanced stimulatory function of B cells as antigen-presenting cells (APC). During Salmonella infection IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM+CD138hiTACI+CXCR4+CD1dintTim1int plasma cells expressing the transcription factor Blimp1. During EAE CD138+ plasma cells were also the major source of B cell-derived IL-35 and IL-10. Collectively, our data unravel the importance of IL-35-producing B cells in regulation of immunity, and highlight IL-35 production by B cells as a novel therapeutic target for autoimmune and infectious diseases. More generally, this study emphasizes the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

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Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

We established microRNA profiles from active and inactive multiple sclerosis lesions. Using laser capture microdissection from multiple sclerosis lesions to pool single cells and in vitro cultures, we assigned differentially expressed microRNA to specific cell types. Astrocytes contained all 10 microRNA that were most strongly upregulated in active multiple sclerosis lesions, including microRNA-155, which is known to modulate immune responses in different ways but so far had not been assigned to central nervous system resident cells. MicroRNA-155 was expressed in human astrocytes in situ, and further induced with cytokines in human astrocytes in vitro. This was confirmed with astrocyte cultures from microRNA-155-|-lacZ mice. We matched microRNA upregulated in phagocytically active multiple sclerosis lesions with downregulated protein coding transcripts. This converged on CD47, which functions as a 'don't eat me' signal inhibiting macrophage activity. Three microRNA upregulated in active multiple sclerosis lesions (microRNA-34a, microRNA-155 and microRNA-326) targeted the 3'-untranslated region of CD47 in reporter assays, with microRNA-155 even at two distinct sites. Our findings suggest that microRNA dysregulated in multiple sclerosis lesions reduce CD47 in brain resident cells, releasing macrophages from inhibitory control, thereby promoting phagocytosis of myelin. This mechanism may have broad implications for microRNA-regulated macrophage activation in inflammatory diseases.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

Understanding the mechanisms of immune cell migration to multiple sclerosis lesions offers significant therapeutic potential. This study focused on the chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1), both of which regulate B cell migration in lymphoid tissues. We report that immunohistologically CXCL12 was constitutively expressed in CNS parenchyma on blood vessel walls. In both active and chronic inactive multiple sclerosis lesions CXCL12 protein was elevated and detected on astrocytes and blood vessels. Quantitative PCR demonstrated that CXCL13 was produced in actively demyelinating multiple sclerosis lesions, but not in chronic inactive lesions or in the CNS of subjects who had no neurological disease. CXCL13 protein was localized in perivascular infiltrates and scattered infiltrating cells in lesion parenchyma. In the CSF of relapsing-remitting multiple sclerosis patients, both CXCL12 and CXCL13 were elevated. CXCL13, but not CXCL12, levels correlated strongly with intrathecal immunoglobulin production as well as the presence of B cells, plasma blasts and T cells. About 20% of CSF CD4+ cells and almost all B cells expressed the CXCL13 receptor CXCR5. In vitro, CXCL13 was produced by monocytes and at much higher levels by macrophages. CXCL13 mRNA and protein expression was induced by TNFalpha and IL-1beta but inhibited by IL-4 and IFNgamma. Together, CXCL12 and CXCL13 are elevated in active multiple sclerosis lesions and CXCL12 also in inactive lesions. The consequences of CXCL12 up-regulation could be manifold. CXCL12 localization on blood vessels indicates a possible role in leucocyte extravasation, and CXCL12 may contribute to plasma cell persistence since its receptor CXCR4 is retained during plasma cell differentiation. CXCL12 may contribute to axonal damage as it can become a neurotoxic mediator of cleavage by metalloproteases, which are present in multiple sclerosis lesions. The strong linkage of CXCL13 to immune cells and immunoglobulin levels in CSF suggests that this is one of the factors that attract and maintain B and T cells in inflamed CNS lesions. Therefore, both CXCL13 and CXCR5 may be promising therapeutic targets in multiple sclerosis.

Axonal injury is considered the major cause of disability in patients with multiple sclerosis (MS), but the underlying effector mechanisms are poorly understood. Starting with a proteomics-based approach, we identified neurofascin-specific autoantibodies in patients with MS. These autoantibodies recognize the native form of the extracellular domains of both neurofascin 186 (NF186), a neuronal protein concentrated in myelinated fibers at nodes of Ranvier, and NF155, the oligodendrocyte-specific isoform of neurofascin. Our in vitro studies with hippocampal slice cultures indicate that neurofascin antibodies inhibit axonal conduction in a complement-dependent manner. To evaluate whether circulating antineurofascin antibodies mediate a pathogenic effect in vivo, we cotransferred these antibodies with myelin oligodendrocyte glycoprotein–specific encephalitogenic T cells to mimic the inflammatory pathology of MS and breach the blood–brain barrier. In this animal model, antibodies to neurofascin selectively targeted nodes of Ranvier, resulting in deposition of complement, axonal injury, and disease exacerbation. Collectively, these results identify a novel mechanism of immune-mediated axonal injury that can contribute to axonal pathology in MS.

We report that B cell–activating factor of the tumor necrosis factor (TNF) family (BAFF) is expressed in the normal human brain at ∼10% of that in lymphatic tissues (tonsils and adenoids) and is produced by astrocytes. BAFF was regularly detected by enzyme-linked immunosorbent assay in brain tissue lysates and in normal spinal fluid, and in astrocytes by double fluorescence microscopy. Cultured human astrocytes secreted functionally active BAFF after stimulation with interferon-γ and TNF-α via a furin-like protease-dependent pathway. BAFF secretion per cell was manifold higher in activated astrocytes than in monocytes and macrophages. We studied brain lesions with B cell components, and found that in multiple sclerosis plaques, BAFF expression was strongly up-regulated to levels observed in lymphatic tissues. BAFF was localized in astrocytes close to BAFF-R–expressing immune cells. BAFF receptors were strongly expressed in situ in primary central nervous system (CNS) lymphomas. This paper identifies astrocytes as a nonimmune source of BAFF. CNS-produced BAFF may support B cell survival in inflammatory diseases and primary B cell lymphoma.