T cell-independent and toll-like receptor-dependent antigen-driven activation of autoreactive B cells.

On the lupus-prone MRL-lpr/lpr (MRL-lpr) background, AM14 rheumatoid factor (RF) B cells are activated, differentiate into plasmablasts, and undergo somatic hypermutation outside of follicles. Using multiple strategies to impair T cells, we found that such AM14 B cell activation did not require T cells but could be modulated by them. In vitro, the signaling adaptor MyD88 is required for IgG anti-chromatin to stimulate AM14 B cell proliferation when T cells are absent. However, the roles of Toll-like receptors (TLRs) in AM14 B cell activation in vivo have not been investigated. We found that activation, expansion, and differentiation of AM14 B cells depended on MyD88; however, mice lacking either TLR7 or TLR9 displayed partial defects, indicating complex roles for these receptors. T cell-independent activation of certain autoreactive B cells, which gain stimuli via endogenous TLR ligands instead of T cells, may be the initial step in the generation of canonical autoantibodies.

Anti-chromatin antibodies drive in vivo antigen-specific activation and somatic hypermutation of rheumatoid factor B cells at extrafollicular sites.

A dominant type of spontaneous autoreactive B cell activation in murine lupus is the extrafollicular generation of plasmablasts. The factors governing such activation have been difficult to identify due to the stochastic onset and chronic nature of the response. Thus, the ability to induce a similar autoreactive B cell response with a known autoantigen in vivo would be a powerful tool in deciphering how autoimmune responses are initiated. We report here the establishment and characterization of a system to initiate autoreactive extrafollicular B cell responses, using IgG anti-chromatin antibodies, that closely mirrors the spontaneous response. We demonstrate that exogenously administered anti-chromatin antibody, presumably by forming immune complexes with released nuclear material, drives activation of rheumatoid factor B cells in AM14 Tg mice. Anti-chromatin elicits autoreactive B cell activation and development into antibody-forming cells at the T zone/red pulp border. Plasmablast generation occurs equally in BALB/c, MRL/+ and MRL/lpr mice, indicating that an autoimmune-prone genetic background is not required for the induced response. Importantly, infused IgG anti-chromatin induces somatic hypermutation in the absence of a GC response, thus proving the extrafollicular somatic hypermutation pathway. This system provides a window on the initiation of an autoantibody response and reveals authentic initiators of it.

Regulation of intracellular pH in human melanoma: potential therapeutic implications.

Melanoma cells in vivo maintain intracellular pH (pHi) in a viable range despite an extracellular tumor pH (pHe) that is typically below 7.0. In general, three families of transporters are capable of removing metabolic protons, but the specific transporters responsible for the maintenance of pHi at low pHe in melanomas have not been identified. Although the transporters exist in most cells, an inhibitor would be predicted to have selectivity for cells located in an acidic tumor bed because cells in that environment would be expected to have transporters chronically activated. In this report, the levels and extent of expression of the Na+/H+ exchanger (NHE-1) and two of the H+-linked monocarboxylate transporters (MCTs) were evaluated in three melanoma cell lines. The effects of inhibitors of each transporter were tested at an extracellular pH (pHe) of 7.3, 6.7, or 6.5 in melanoma cells that were grown at pHe 7.3 or 6.7. The activity of MCT isoform 1 (MCT-1) was up-regulated in three melanoma cell lines at low pHe, but that of NHE-1 was not. Furthermore, NHE-1 activity was lower in the melanomas than in other normal and malignant cell lines that were tested. Reverse transcription-PCR using primers specific for MCT-1, MCT-4, and NHE-1 showed that expression of none of these transporters was reproducibly up-regulated at the level of transcription when cells were grown at pHe 6.7 instead of pHe 7.3. Ex vivo experiments using DB-1 human melanoma xenografts grown in severe combined immunodeficient mice found that MCT-1 and not NHE-1 was a major determinant of DB-1 tumor cell pHi. Taken together, the data indicate that MCTs are major determinants of pH regulation in melanoma. In contrast, keratinocytes and melanocytes under low pHe conditions relied on NHE-1. Inhibitors of MCTs thus have great potential to improve the effectiveness of chemotherapeutic drugs that work best at low pHi, such as alkylating agents and platinum-containing compounds, and they should be selective for cells in an acidic tumor bed. In most tissues, it is proposed that the NHE-1 could compensate for an inhibited MCT to prevent acidification, but in melanoma cells this did not occur. Therefore, MCT inhibitors may be particularly effective against malignant melanoma.