While the BCR serves to recognize antigen and transduces an activation signal for B cell expansion, it is also responsible for delivering the bound antigen to specific compartments within the cell where the antigen is processed into peptides that are loaded onto MHC class II (MHC-II) molecules, which are then trafficked to the cell surface for presentation to antigen-specific helper T cells. Accumulating evidence suggests that the initial endocytosis of the antigen and MHC-II-associated presentation of antigenic peptides to T cells are both coordinated within membrane raft domains.
Following internalization, the antigen-BCR complexes are delivered to early endosomes from where they move to late endosomes. The Igα/Igβ components of the BCR were shown to be necessary and sufficient for the initial internalization as well as for sorting to the late endosomes (27
). Igβ-mutant receptors are retained in early endosomes, whereas those containing only Igβ were shown to go straight from early endosomes to terminal lysosomes and undergo degradation without productive loading of peptides onto MHC-II (27
). The contribution of Igβ ITAM residues was further examined more recently in vivo
by exchanging the ITAM tyrosines for alanines by gene targeting. The resulting mice (IgβAA
) showed a normal development for all B cell subtypes, except for B1 cells which were significantly reduced. Purified B cells from the IgβAA
mice showed highly decreased steady state and ligand-mediated BCR internalization. BCR cross-linking resulted in diminished Src family and Syk activation, but elevated and prolonged signaling with respect to Ca2+
flux, total tyrosine phosphorylation, and Akt and Erk activation. This study concluded that the Igβ component of the BCR is responsible for setting a threshold for signaling by regulating receptor internalization, which terminates signaling (29
The Igα chain of the BCR complex also participates in its internalization. Proper sorting of the BCR-antigen complexes requires the recruitment of the tyrosine kinase Syk to phosphorylated ITAMs on Igα (30
). Interestingly, both the ITAM tyrosines (29
) and the non-ITAM tyrosines Y176 and Y204 of Igα participate in coordinating the internalization signals. Receptors bearing tyrosine to phenylalanine mutations in the Igα chain are still internalized but do not co-localize with MHC-II-rich internal compartments and could not facilitate antigen presentation to T cells (32
). The requirement for both ITAM and non-ITAM tyrosine residues on Igα for efficient and productive internalization of the BCR complexes is further supported by extensive mutational analysis reported in a recent study (33
While the BCR complex contains sufficient signals for its own internalization and sorting, recent reports suggest that B cells can endocytose via two pathways, internalization via clathrin-coated pits and via a clathrin-independent pathway involving membrane rafts. A significant amount of clathrin heavy chain was found to be constitutively associated with membrane rafts in B cells (34
), and it becomes tyrosine phosphorylated upon BCR engagement in a Src family kinase-dependent manner (34
). Furthermore, antigen uptake is largely dependent on the association of clathrin with membrane rafts and its phosphorylation at these sites. Chicken DT40 B cells conditionally deficient in clathrin heavy chain showed a marked reduction in their BCR-mediated uptake of antigen. Disruption of membrane rafts by treatment with nystatin caused a reduction similar to that seen with loss of clathrin, whereas treatment with latrunculin A to disrupt the actin cytoskeleton led to a 50% loss in antigen internalization. These data suggest that clathrin, actin and membrane rafts are needed for the most complete endocytosis of BCR-bound antigen, but argue that internalization can still proceed with at least two of these three components intact. However, membrane rafts and the actin cytoskeleton cannot support internalization independently. A complete block in endocytosis of the BCR resulted in increased BCR signaling as assessed by sustained tyrosine phosphorylation and Erk phosphorylation, further supporting the idea that internalization is a means of signal attenuation as well as a means to promote antigen presentation to helper T cells (36
). Therefore, the view that has emerged from these studies is that BCR ligation drives its association with membrane rafts already bearing the clathrin heavy chain and enriched in Src family kinases. Both the BCR and clathrin get phosphorylated in this confined space, the BCR localizes to clathrin-coated pits and internalizes along with membrane rafts (34
In another study, anergic B cells (which are continually binding their antigen) were reported to have an enhanced rate of BCR endocytosis, which was blocked by depleting membrane cholesterol with methyl β-cyclodextrin, suggesting that membrane rafts play a critical role in BCR internalization in these B cells (12
). Further evidence for at least two pathways of BCR internalization has recently been presented. In this study, the pathway of BCR endocytosis, as indicated by its sensitivity to membrane raft- and actin-disrupting agents and its dependence on Src and Syk family kinase signaling, was shown to be governed by the nature of the ligand. The internalization of anti-Ig antibody/BCR complexes was dependent on Src and Syk family kinase signaling, the integrity of the actin cytoskeleton and membrane rafts, and these complexes were delivered to early and recycling endosomes. In contrast, the internalization of a model antigen/BCR complex was independent of signaling, membrane rafts and actin and these complexes trafficked to late endosomes, and were targeted for proteolytic processing (38
A membrane raft-localized transmembrane protein, the linker for activation of B cells/non-T cell activation linker (LAB/NTAL) (40
) also appears to play an important role in BCR internalization. BCR cross-linking led to co-internalization of LAB/NTAL along with the receptor and it is the C-terminal tail of LAB/NTAL that is responsible for this effect. Mouse B cells deficient in LAB/NTAL showed a reduction in the ligation-dependent uptake of the BCR (42
). To the extent that LAB/NTAL is important for BCR internalization, its localization to membrane rafts may contribute to the importance of these domains for BCR-mediated antigen uptake.
Taken together, the available data suggest that BCR endocytosis is a dynamic and complex process comprised of at least two mechanistically distinct pathways for internalization, and includes additionally regulated steps that control delivery to late endosomes where antigen can be processed and loaded onto MHC-II molecules. Resolution of molecular features of this process will depend on getting a closer look at early events that govern the intracellular fate of the BCR and its association with components of membrane rafts.
The endocytosed BCR is delivered to intracellular compartments where its bound antigen is proteolyzed into antigenic peptides. The peptides are then loaded onto MHC-II molecules, and traffic out to the surface of the B cell from where they can be surveyed by CD4+
T cells bearing the cognate T cell receptors (TCRs). Biochemical fractionation and fluorescence microscopy experiments indicate that not only are the MHC-II molecules constitutively associated with membrane rafts, they are also loaded with antigenic peptides in a concentrated membrane raft environment (43
). Indeed, our proteomic analysis of purified membrane rafts from the human B cell line Ramos confirmed that MHC-II molecules associate with membrane rafts and showed that this association is unaffected by ligation of the BCR (35
). Constitutive association of MHC-II with membrane rafts has also been observed in human tonsil B cells, in transformed B cell lines, and in human monocytes (44
). While it is unclear where in the biosynthetic pathway MHC-II molecules get associated with membrane rafts, both microscopic and biochemical determinations suggest that half of the cell surface MHC-II is associated with membrane rafts. The functional relevance of MHC-II association with membrane rafts is suggested by the fact that raft-associated MHC-II/peptide complexes concentrate in the immunological synapse (IS) and facilitate antigen presentation to T cells. Pharmacological disruption of membrane rafts on the antigen presenting B cells abrogated their recruitment to the IS and T cell activation. However, this was only true under conditions when the numbers of MHC-II/peptide complexes were limiting, indicating that the role of membrane rafts is to concentrate rare MHC-peptide complexes at the IS for recognition by T cells (45
). Consistent with this hypothesis, confocal imaging of the B cell side of the IS has revealed that membrane rafts are rapidly enriched in the IS upon B:T conjugation, and that this is an actin-dependent phenomenon. In addition, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments showed that the membrane raft proteins in the IS are highly dynamic and rapidly exchange with other membrane compartments of the B cell (46
Thus, it appears that lipid rafts play a significant role in the entire journey of an antigen, starting with its initial uptake through the BCR, trafficking through intracellular MHC-II-loading compartments, and finally its presentation to T cells in the immunological synapse ().
Membrane rafts in BCR endocytosis and antigen presentation