Anu Cherukuri

The CD19/CD21 Complex Functions to Prolong B Cell Antigen Receptor Signaling from Lipid Rafts

The CD19/CD21 complex functions to significantly enhance B cell antigen receptor (BCR) signaling in response to complement-tagged antigens. Recent studies showed that following antigen binding the BCR translocates into plasma membrane lipid rafts that serve as platforms for BCR signaling. Here, we show that the binding of complement-tagged antigens stimulates the translocation of both the BCR and the CD19/CD21 complex into lipid rafts, resulting in prolonged residency in and signaling from the rafts, as compared to BCR cross-linking alone. When coligated to the BCR, the CD19/CD21 complex retards the internalization and degradation of the BCR. The colocalization and stabilization of the BCR and the CD19/CD21 complex in plasma membrane lipid rafts represents a novel mechanism by which a coreceptor enhances BCR signaling.

The Role of the CD19/CD21 Complex in B Cell Processing and Presentation of Complement-Tagged Antigens

The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.

The Tetraspanin CD81 Is Necessary for Partitioning of Coligated CD19/CD21-B Cell Antigen Receptor Complexes into Signaling-Active Lipid Rafts

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.

Rafts and synapses in the spatial organization of immune cell signaling receptors

The multichain immune recognition receptors (MIRRs), including the T cell and B cell antigen receptors and the high affinity receptor for IgE, play an important role in immune cell signaling. The MIRRs have no inherent kinase activity, but rather associate with members of the Src‐family kinases to initiate signaling. Although a great deal is understood about the biochemical cascades triggered by MIRRs, the mechanism by which signaling is initiated was not known. The evidence now indicates that the Src‐family kinases are concentrated in cholesterol‐ and sphingolipid‐rich membrane microdomains, termed lipid rafts, that exclude the MIRRs. Upon ligand‐induced crosslinking the MIRRs translocate into rafts where they are phosphorylated. The MIRRs subsequently form highly ordered, polarized structures termed immunological synapses that provide for prolonged signaling. An understanding of the biochemical composition of rafts and synapses and the mechanisms by which these form should lend insight into the regulation of immune cell activation.

Floating the Raft Hypothesis for Immune Receptors: Access to Rafts Controls Receptor Signaling and Trafficking

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand‐binding domains associated with signal‐transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src‐family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen‐trafficking functions of the BCR are regulated by cholesterol‐ and sphingolipid‐rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src‐family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co‐receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling‐competent microdomains may represent a novel mechanism to initiate and regulate immune‐cell activation.

CD21/CD19 Coreceptor Signaling Promotes B Cell Survival during Primary Immune Responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2−/− MD4 B cells do not. In contrast, Cr2−/− MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.

Isolation of Lipid Rafts From B Lymphocytes

Recent advances in cell biology have provided evidence that the plasma membrane is not a homogeneous lipid bilayer but rather contains within it sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, which serve as platforms for both receptor signaling and trafficking. In B lymphocytes lipid rafts appear to play a key role in the initiation of B-cell antigen receptor (BCR) signaling. Current methods to isolate lipid rafts rely on the relative detergent insolubility of lipid rafts as compared to the nonraft, glycerophospholipid bilayer. Here a method to isolate and characterize lipid rafts from B lymphocytes is described. Particular emphasis is given to the potential artifacts inherent in current procedures that rely on detergents to isolate lipid rafts and alternative technologies that may circumvent these.