To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present inBilateria, for instance inDrosophila melanogasterandCaenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands. AIF-1 controls calcium-dependent cytoskeletal rearrangement in innate immune cells by means of its functional EF-hand. We propose that Swiprosin-1/EFhd2 as well is a cytoskeleton associated adaptor protein involved in immune and brain cell function. Pro-inflammatory conditions are likely to modulate expression and function of Swiprosin-1/EFhd2. Swiprosin-2/EFhd1, on the other hand, modulates apoptosis and differentiation of neuronal and muscle precursor cells, probably through an association with mitochondria. We suggest furthermore that Swiprosin-2/EFhd1 is part of a cellular response to oxidative stress, which could explain its pro-survival activity in neuronal, muscle and perhaps some malignant tissues.

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits α-1 and β-2, vimentin, γ-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

The CC‐chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low‐affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6^high B‐cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre‐GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen‐specific IgG1 and IgG2a antibody generation in a B‐cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen‐specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen‐specific pre‐GC B cells into the GC reaction.