Multiple studies have suggested the importance of synergistic engagement of TLR9 and the BCR in hyper B cells responses in autoimmunity (Leadbetter et al., 2002
; Viglianti et al., 2003
). A growing number of studies indicate that TLRs play important roles in defining B cell responses to both T-dependent and T-independent antigens and that both DNA- and RNA-containing antigen complexes stimulate autoantibody production in a BCR and TLR9-or TLR7-dependent manner (Leadbetter et al., 2002
; Viglianti et al., 2003
). However, the mechanism by which these spatially segregated receptors interact in response to DNA- and RNA- containing antigens has not been previously addressed. Earlier reports suggested that the BCR might function as a shuttle for the delivery of DNA and RNA to the TLR9- and TLR7-containing compartments, however, no evidence was provided for such a mechanism (Leadbetter et al., 2002
; Viglianti et al., 2003
The results presented here provide evidence for a novel pathway by which B cells regulate responses to DNA-containing antigens, namely, by governing the subcellular location of TLR9. In our study we show that in resting B cells TLR9 is localized in punctate, small endosomal compartments and signals in these compartments to MAP kinase activation following stimulation by CpG alone. However, the location of TLR9 is dramatically altered in cells in which the BCR is crosslinked by antigen. Following BCR crosslinking the BCR initiates signaling at the plasma membrane and then traffics to LAMP-1-positive autophagosome-like compartments where the BCR signals to MAP kinase activation. TLR9 is recruited to these compartments where it colocalizes with the BCR. The recruitment of TLR9 to the autophagosome is required for enhanced signaling as the hyperactivation of MAP kinases to the anti-IgM and CpG DNA was not observed when TLR9 recruitment was blocked by the tubulin-depolymerizing agent, nocodazole.
A number of earlier studies indicated that TLR9 is located in late endosomes or lysosomes where it signals upon CpG DNA stimulation, consistent with the endosomal location of TLR9 in primary splenic B cells presented here (Ahmad-Nejad et al., 2002
; Tabeta et al., 2006
). However, a more detailed analysis provided evidence that TLR9 was concentrated in the endoplasmic reticulum (ER) of dendritic cells, macrophages and a B cell line and that upon stimulation with CpG DNA, TLR9 redistributed from the ER to CpG-DNA containing endosomes (Latz et al., 2004
; Leifer et al., 2004
). The mechanism underlying this redistribution of TLR9 is not known, however, a relationship between the ER and endosomes in TLR9 signaling was also suggested by the finding that a mutation in the gene encoding the 12-membrane spanning protein, UNC93B, a highly conserved resident ER protein, resulted in a complete deficiency in signaling via TLRs 3, 7 and 9 but did not alter their sub-plasmalemmal vesicular compartmentalization (Tabeta et al., 2006
). The UNC93B mutation also resulted in deficiencies in antigen cross presentation, a process that involves both autophagy and endocytic trafficking. Moreover, recently it has been shown that TLR3, 7 and 9 physically interact with UNC93B, and this interaction is mediated through the transmembrane domain of at least TLR3 and 9 (Brinkmann et al., 2007
). Taken together these findings suggest a link between the ER and endosomal TLR9 signaling that will be of interest to explore.
The results provided here indicate that the antigen bound BCR and TLR9 colocalize and signal from autophagosome-like compartments. Recently evidence was provided that autophagosomes play an important role in intracellular TLR signaling. In pDCs TLR7 gains access to single strand RNA viruses and viral replication intermediates through autophagy (Lee et al., 2007
). In B cells autophagosomes have been less well characterized, however, recent results showed that a relationship exists between the class II-loading compartments and autophagosomes that results in the delivery of cytosolic antigens to class II-loading compartments (Schmid et al., 2007
). It remains to be determined if similar mechanisms are at play for the relocalization of TLR9 to class II-loading compartments where BCR traffics DNA-containing antigens. It will also be of interest to determine if BCR-induced relocalization of TLR9 to autophagosome-like compartments is limited to TLR9 or is also observed with other intracellular TLRs. We provide evidence here that the recruitment of TLR9 was selective to the extend that TfR-positive endosomes were not recruited along with TLR9 positive endosomes. However, the regulation of the location of the TLR3 and 7 is of interest. Indeed, TLR3 that recognizes dsRNA is constitutively present in multivesicular bodies of a B cell line (Matsumoto et al., 2003
The studies presented here support a model for B cell hyperresponses to DNA-containing antigen in which the antigen-engaged, internalized BCR signals to recruit TLR9 to autophagosome-like compartments to allow TLR9 to survey the antigen for its DNA ligand. If the ligand is present in the BCR-bound antigen, TLR9 is engaged and activated. How does this process result in autoantibody production? The enhanced signaling from autophagosomes through the BCR and TLR9 may contribute to autoantibody production by reducing the threshold for B cell activation. Alternatively, the subcellular location of TLR9 could influence its specificity for ODNs as suggested by the observation that a chimeric TLR9 receptor that localized to the cell surface rather than endosomes responded to both self and nonself DNA (Barton et al., 2006
). In addition, in plasmacytoid dendritic cells the subcellular location to which CpG is delivered dictates the outcome of MyD88-IRF7 signaling (Honda et al., 2005
). Additional studies indicated that crosslinking the BCR that we now know recruits TLR9 to autophagosomes resulted in the B cells’ ability to respond to a broader array of CpG-containing ODNs, including methylated DNA as occur in self DNA (Goeckeritz et al., 1999
). Thus, it is possible that TLR9 recruitment to the autophagosomes by the BCR may have a significant effect on the ability of the BCR to discriminate self and nonself DNA-containing antigens.
Another important aspect of the recruitment of TLR9 vesicles to BCR-containing autophagosome-like compartments is the possible need to regulate recruitment. Presumably, the TLR9 is induced to locate to the class II loading compartments rather than being constitutively present in that compartment to allow regulation of the TLR amplification of BCR signaling. It will be of interest to determine if BCR coreceptors such as the inhibitory FcγRIIB that has emerged as a key factor in autoimmunity (Schmidt and Gessner, 2005
), regulates TLR recruitment by the BCR. In addition, since multivesicular bodies in B cells are the subcellular sites in which the MHC class II molecules are assembled with peptides derived from the BCR-bound antigen, recruitment of TLR9 into the class-II loading compartments along with the antigen might influence the ability of the BCR to present DNA-containing antigens to helper T cells.
Lastly, an important aspect of the studies presented here is the evidence that the BCR continues to signal after its internalization to class II loading compartments and moreover, that the intracellular signaling is required for TLR9 recruitment. A number of cell surface receptors have been shown to continue to signal as they are internalized and that intracellular signaling was essential for the function of the receptor (Grimes et al., 1996
; Vieira et al., 1996
; Wang et al., 2002
). To date, the intracellular signaling of the immune receptors has not been investigated in detail and a few studies suggested that signaling was extinguished once the receptor was internalized. Here we provide evidence that the BCR-initiated phosphorylation of p38 continues as the BCR is internalized into the cells, first into small endosomal vesicles then into class-II loading compartments. Thus, the spatial segregation of the BCR’s activation of p38 may play an important role in facilitating interaction with TLR9. It is also possible that the intracellular location of late BCR signaling may play additional roles in regulating B cell activation.