Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.DOI: http://dx.doi.org/10.7554/eLife.07218.001

The cells of both the adaptive and innate immune systems express a dizzying array of receptors that transduce and integrate an enormous amount of information about the environment that allows the cells to mount effective immune responses. Over the past several years, significant advances have been made in elucidating the molecular details of signal cascades initiated by the engagement of immune cell receptors by their ligands. Recent evidence indicates that immune receptors and components of their signaling cascades are spatially organized and that this spatial organization plays a central role in the initiation and regulation of signaling. A key organizing element for signaling receptors appears to be cholesterol- and sphingolipid-rich plasma membrane microdomains termed lipid rafts. Research into the molecular basis of the spatial segregation and organization of signaling receptors provided by rafts is adding fundamentally to our understanding of the initiation and prolongation of signals in the immune system.

Antibody affinity maturation, a hallmark of adaptive immune responses, results from the selection of B cells expressing somatically hypermutated B cell receptors (BCRs) with increased affinity for antigens. Despite the central role of affinity maturation in antibody responses, the molecular mechanisms by which the increased affinity of a B cell for antigen is translated into a selective advantage for that B cell in immune responses is incompletely understood. We use high resolution live-cell imaging to provide evidence that the earliest BCR-intrinsic events that follow within seconds of BCR–antigen binding are highly sensitive to the affinity of the BCR for antigen. High affinity BCRs readily form oligomers and the resulting microclusters grow rapidly, resulting in enhanced recruitment of Syk kinase and calcium fluxes. Thus, B cells are able to read the affinity of antigen by BCR-intrinsic mechanisms during the earliest phases of BCR clustering, leading to the initiation of B cell responses.

Binding of antigen to the B cell antigen receptor (BCR) triggers signaling that ultimately leads to B cell activation. Using quantitative fluorescence resonance energy transfer imaging, we provide evidence here that the BCR is a monomer on the surface of resting cells. Binding of multivalent antigen clustered the BCR, resulting in the simultaneous phosphorylation of and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation required immunoreceptor tyrosine-activation motif and continuous Src family kinase activity but not binding of the kinase Syk. Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by Src family kinase activity.

Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21−CD27− ‘atypical’ memory B cells (MBCs) that exhibit reduced B cell receptor (BCR) signaling and effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet, and that T-bet expression correlates inversely with BCR signaling and skews toward IgG3 class switching. Moreover, a longitudinal analysis of a subset of children suggested a correlation between the incidence of febrile malaria and the expansion of T-bethi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs plus BCR cross linking induced T-bet expression in naïve B cells that was abrogated by neutralizing IFN-γ or blocking the IFN-γ receptor on B cells. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsillar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data provide new insight into the mechanisms underlying atypical MBC differentiation.

Summary Memory B cells express high affinity, immunoglobulin B cell receptors (IgG-BCRs) that enhance B cell responses giving rise to the rapid production of high affinity, IgG antibodies. Despite the central role of IgG-BCRs in memory responses, the mechanisms by which the IgG-BCRs function to enhance B cell responses are not fully understood. Using high-resolution live-cell imaging we showed that independent of affinity, IgG1-BCRs dramatically enhanced the earliest BCR-intrinsic events that followed within seconds of B cells’ encounter with membrane bound antigen including BCR oligomerization and BCR microcluster growth, leading to Syk kinase recruitment and calcium responses. The enhancement of these early events was dependent on a membrane proximal region of the IgG1 cytoplasmic tail not previously appreciated to play a role in IgG1-BCR signaling. Thus, intrinsic properties of the IgG1-BCR enhance early antigen-driven events that ultimately translate into heightened signaling.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.

Correspondence to Susan K. Pierce: spierce@nih.gov Abbreviations used in this paper: APC, antigen-presenting cell; BCR, B cell receptor; FI, fl uorescence intensity; FRET, fl uorescence resonance energy transfer; ICAM-1, intercellular adhesion molecule-1; ITAM, immunoreceptor tyrosinebased activation motif; LynFL, full-length Lyn; PC, phosphorylcholine; TIRFM, total internal refl ection fl uorescence microscopy.The online version of this article contains supplemental material.