The B cell antigen receptor (BCR) serves dual, interrelated functions in B cell activation. The first is to initiate signal cascades that result in the transcription of a variety of genes associated with B cell activation. The second is to traffic bound antigen into the cell to deliver the antigen into intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides are assembled into MHC class II-peptide complexes. The MHC class II-peptide complexes are expressed on the B cell surface for recognition by helper T cells that will as a consequence be activated to provide essential growth and differentiation factors to the B cell. The signaling and antigen transport functions of the BCR are interdependent in that the BCR signaling is necessary for the correct and rapid targeting of the antigen. In addition to the transport functions of the BCR, the internalization of the BCR may play an important role in regulating signaling by removing activated receptors from the cell surface. At present it is not known if the antigen targeting and BCR down regulation are one in the same or separable processes. ? ? The long term goal of this program is to define the cellular and molecular mechanism by which the BCR transports antigens to the appropriate intracellular compartments and is removed from the surface to regulate signaling. Concerning the mechanism of BCR transport to the MHC class II compartments we previously showed that the EBV latent membrane protein 2A (LMP2A) blocked BCR trafficking. Genetic analyses of the cytoplasmic domain of LMP2A showed that mutations in the Lyn-binding site allowed internalization and degradation of the BCR but not trafficking to the peptide class II assembly compartment. Recent studies have provided evidence that the LMP2A expressing cells do not activate phospholipase D (PLD) in response to BCR crosslinking suggesting that PLD plays a critical role in targeting the BCR to the class II compartments. Subsequent studies showed that 1-butanol, that blocks PLD-1, blocks targeting of the BCR to the class II compartment as does knocking down PLD-1 expression by siRNA. Thus, PLD-1 appears to play a critical role in the appropriate trafficking of the BCR to class II compartments. ? ? Earlier studies showed that the BCR enters the MHC class II compartment as an intact receptor suggesting that it may continue to signal in the class II compartment and thus regulate antigen processing. Recent studies using a combination of confocal and electron microscopy provided evidence that BCR-signaling complexes that assemble at the plasma membrane contain phosphorylated forms of Syk, Erk and JNK that travel with the BCR into the multivesicular MHC-class II antigen processing compartment. Studies are in progress to determine the influence of this endosomal BCR signaling on antigen processing. ? ? Lastly, B cell responses are enhanced by antigens that contain the CpG oligonucleotide ligands for Toll-like receptor-9 (TLR-9). The response of B cells to CpG containing autoantigens has been postulated to play a significant role in antibody-mediated autoimmune diseases. TLR9 is present in endosomal compartments and it is not known how antigen-CpG complexes bound to the BCR at the plasma membrane engage TLR-9. Over the last reporting period we have used confocal and electron microscopy to follow the intracellular movement of the BCR and TLR-9 following CpG-antigen binding to the BCR. The results of these analyses showed that BCR signaling induces the fusion of TLR-9-containing endosomes with the class II-containing multivesticular endosomes where both the BCR and TLR-9 signal. These results are of interest as they may provide new targets to block B cell responses in autoimmune disease.
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