CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.

Here we describe the spatiotemporal architecture, at high molecular resolution, of receptors and signaling molecules during the early events of mouse B cell activation. In response to membrane-bound ligand stimulation, antigen aggregation occurs in B cell antigen receptor (BCR) microclusters containing immunoglobulin (Ig) M and IgD that recruit the kinase Syk and transiently associate with the coreceptor CD19. Unexpectedly, CD19-deficient B cells were significantly defective in initiation of BCR-dependent signaling, accumulation of downstream effectors and cell spreading, defects that culminated in reduced microcluster formation. Hence, we have defined the dynamics of assembly of the main constituents of the BCR 'signalosome' and revealed an essential role for CD19, independent of the costimulatory molecule CD21, in amplifying early B cell activation events in response to membrane-bound ligand stimulation.

The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation.

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

Aquaporin-3 regulates endosome-to-cytosol transfer via lipid peroxidation for cross presentation.

Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells. Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex. Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT.

Studying cell-to-cell interactions: an easy method of tethering ligands on artificial membranes.

Extensive studies have been performed in order to understand the interaction of receptors with soluble ligands. However, we know very little of the parameters that regulate the interaction of receptors with membrane-bound ligands. Artificial lipid bilayers can be used to mimic cell-to-cell interactions, but a major challenge remains how to tether molecules to these membranes. We describe a simple and reliable method to tether ligands on glass-supported artificial bilayers containing biotinylated lipids. In this system, the model antigen hen egg lysozyme (HEL) is tethered through a fluorescently labeled streptavidin-monobiotinylated anti-HEL antibody bridge. This allows us to study the interaction of HEL-specific B cells with the tethered antigen by a variety of microscopy techniques. We recently used this system to study the activation of B cells by membrane antigens.

Early events of B cell activation by antigen.

The activation of B cells confers long-lasting protection from a plethora of infectious diseases through the generation of plasma cells that produce high-affinity antibodies and memory cells. Engagement of the B cell receptor (BCR) with cognate antigen initiates intracellular signaling and subsequent internalization of antigen. Membrane-bound antigens are now considered the predominant forms that initiate B cell activation in vivo. We have shown that upon recognition of antigen on the surface of a presenting cell, the B cell undergoes a dramatic change in morphology characterized by rapid spreading followed by more prolonged contraction along the presenting surface. This two-phase response increases the amount of antigen that the B cell accumulates, internalizes, and subsequently presents to T cells. Thus, the spreading and contraction response shapes the outcome of B cell activation. We used a combination of planar lipid bilayers and total internal reflection fluorescence microscopy to investigate the early events that occur after engagement of the BCR and before B cell spreading. We observed the rapid formation of BCR-antigen microclusters, which we redefine as "microsignalosomes" because they mediate the coordinated recruitment of intracellular effectors, such as the kinases Lyn and Syk, the adaptor Vav, and phospholipase C-gamma2 (PLC-gamma2). We identified an essential role for the co-receptor CD19 in mediating spreading, and thus B cell activation, in response to membrane-bound antigen. Preliminary evidence suggests that the cellular morphology changes described in vitro are likely to occur upon recognition of antigen presented on the surface of macrophages in lymph nodes in vivo.

The role of integrins and coreceptors in refining thresholds for B-cell responses.

Despite compelling evidence that a large proportion of antigens encountered in vivo by B cells are membrane bound, the general view is that B cells are mainly activated by soluble antigens. This notion may have been biased somewhat over the years because the high affinity of the B-cell receptor (BCR) for soluble intact ligands allows efficient B-cell stimulation in vitro. In vivo, however, even soluble antigens are likely to be deposited on the surface of antigen-presenting cells, either by complement or Fc receptors in the form of immune complexes, thus becoming more potent stimulators of B-cell activation. In this framework, the BCR works in a complex environment of integrins and coreceptors, as well as the B-cell cytoskeleton. Over the last few years, we have focused on B-cell membrane-bound antigen recognition. Here, we discuss some of our findings in the context of what is currently known in this exciting new field.

LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation.

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.