Receptors, subcellular compartments and the regulation of peripheral B cell responses: the illuminating state of anergy

Abstract

Signals through the B cell antigen receptor (BCR) are necessary but not sufficient for cellular activation. Co-stimulatory signals must be provided through other immune recognition receptor systems, such as MHC class II/CD40 and the toll-like receptor (TLR) 9 that can only productively acquire their ligands in the processive environment of specialized late endosomes (MHC class II containing compartment or MIIC). It has long been appreciated that the BCR, by effectively capturing complex antigens and delivering them to late endosomes, is the link between activation events on the cell surface and those dependent on late endosomes. However, it has become increasingly apparent that the BCR also directs the translocation of MHC class II and TLR9 into the MIIC and that the endocytic flow of these receptors coincides with that of the BCR. This likely ensures close apposition of receptor complexes within the MIIC and the efficient transfer of ligands from the BCR to MHC class II and TLR9. This complex orchestration of receptor endocytic movement is dependent upon the quality of signals elicited through the BCR. Failure to activate specific signaling pathways, such as occurs in anergic B cells, prevents the entry of the BCR and TLR9 into the MIIC and abrogates TLR9 activation. Like anergy, this block in endocytic trafficking is rapidly reversible. These findings indicate that cellular responsiveness can be determined by mechanisms that control the subcellular location of important immune recognition receptors.

Figure 1. Decision points along the endocytic pathway to the MIIC

(A) Following recognition of antigen, only a small subfraction of surface BCR complexes are tyrosine phosphorylated (PTyr) and these assemble a signalsome at the cell surface. The majority of BCR complexes remain unphosphorylated and therefore are targeted for internalization through tyrosine-based internalization motifs within the cytosolic tails of Igα and Igβ. (B) The second decision point occurs in early endosomes. Receptors that are ubiquitinylated (Ub) are sorted towards late endosomes while those that are not ubiquitinylated recycle back to the cell surface. (C) Analysis of anergic B cells has revealed a third decision point along the endocytic pathway, at entry into the MIIC. BCR signals also control the transit of TLR9 and the block induced in anergy affects entry of both the BCR and TLR9 into the MIIC. These and other observations indicate that the BCR and TLR9 share a common final pathway into the MIIC and that, in wild-type cells, they enter together.

Figure 2. Selection of BCR complexes for internalization

Clustering of the BCR induces the assembly of a signalsome at the receptor containing Syk, BLNK and BLNK-associated molecules. The complex, in conjunction with the adaptor LAB activates signaling cascades that include the molecules Vav, Rac and Src-family kinases (such as fyn and blk). These signaling pathways activate endocytic machinery (the adaptor AP2, clathrin and actin) such that they can select cargo (receptors) for endocytosis and entry into the endocytic pathway. In our model, BCR complexes are selected for inclusion in clathrin-coated pits if they have AP2 docking sites available (YXXL). If these motifs are blocked because they are phosphoryated, or they are obscured by recruited signaling molecules, the receptor is retained on the cell surface. Through this mechanism, BCR complexes involved in signal propagation are retained on the plasma membrane and are not targeted for degradation.