Earlier studies revealed that the physiological mechanism that triggers BCR activation was thought to be physically crosslinking by soluble multivalent antigen (13). Later on, evidence was provided that physically crosslinking is not a prerequisite for BCR signaling triggered by a membrane anchored antigen (14). This is in agreement with observations that, indeed, B-cells encounter and respond to antigen both in solution and on so-called antigen-decorated cells, like conventional antigen presenting cells (APCs) (15, 16) or follicular dendritic cells.
Moreover, both mono- and multivalent antigen incorporated into fluid bilayers were demonstrated to induce microcluster formation and signaling of cognate BCR (82).
Our findings suggest that molecules capable of clustering Ii during antigen detection by the BCR influence the early events of BCR induced B-cell activation by bringing both BCR and Ii in close proximity. Macrophage migration inhibitory factor (MIF) has been shown to serve as a physiological ligand of invariant chain (83, 84) in addition to the chemokine receptors CXCR2 and CXCR4 (85). There is only a very small fraction of Ii present at the cell surface (41, 86). It has been assumed that surface Ii is modified by a proteoglycan (87), associated with CD44 (88,89), CXCR2 (85) and CXCR4(90). However, there are no quantitative data on what fraction of surface Ii is complexed by these or other molecules. Earlier work has revealed that a substantial amount of newly synthesized Ii-MHC II complexes is cycling back and forth between the plasma membrane and early endosomal compartments before its retrieval to later endocytic compartments (41). As shown in this work (Fig. 1), only about 3 % of surface invariant chain was present as free invariant chain trimer in NHHT-39 B-cells, whereas the remaining material was in fact complexed to MHC II. We neither found evidence for substantial amounts of other association products of invariant chain at the plasma membrane, nor did we detect evidence for a high molecular weight form of Ii by immunoprecipitation of velocity gradient fractions (Fig. 1). Although this analysis does not rule out that complexes of surface Ii with other proteins or high molecular weight modification products of Ii exist in NHHT-39 cells, it shows that such complexes can only be present in very small quantities, if at all. The most likely binding partners for MIF are
5. Discussion
29 therefore either MHC II-Ii-complexes or the small quantity of free Ii that was detected at the surface of NHHT-39 cells (Fig. 1)
The mere binding of MIF did not lead to its recruitment to BCR clusters. Instead, independent clustering of both, BCR and MIF (bound to Ii) was required (90) to induce co-localization of the surface of NHHT-39 cells. This result was similar to what we found for the receptor of MIF, Ii, when this molecule was clustered by antibodies (91). It was surprising that a trimeric ligand bound to its trimeric receptor did not trigger receptor oligomerization and subsequent co-clustering with patched BCR by itself. Possible explanations for this failure could be i) an excess of MIF in our experimental setting, ii) a possible 1:1 binding of a MIF trimer to a trimer of Ii or iii) an inability of Ii-MHC II to bind more than 1 MIF trimer. The binding site of MIF has been shown to be contained in a soluble fragment of Ii (Ii73-232) comprising the MHC II binding site and the C-terminal trimerization domain (83, 84). Binding in this region of Ii would be incompatible with an 1:1 stoichiometry, unless it occurred right on the top end of the Ii molecule. This, however, appears to be unlikely, since recent investigations showed that partial, soluble MHC II molecules consisting of linked α1, β1 subunits and antigenic peptide interfered with the interaction of MIF and Ii (92, 93). If confirmed for intact MHC II, these results would suggest that binding of MIF to invariant chain is on the lateral side of the Ii trimer (close to the MHC II binding site) and therefore should occur in a stoichiometry of more than one MIF trimer per trimer of Ii. Their results furthermore suggest that nonameric Ii-MHC II complexes do not interact with MIF. Binding of MIF to Ii should therefore to be expected to occur either on the small population of MHC II-free Ii trimers (about 3 % of the surface Ii molecules, see Fig. 1) or to Ii-MHC II complexes that contain fewer than three MHC II molecules per Ii trimer. Such complexes could offer 1-2 MIF binding sites per Ii trimer, while the rest would be blocked by bound MHC II. Evidence for such substoichiometric Ii-MHC II complexes is still weak (40, 94) but also see comments by Lindner and Cresswell (Plos ONE, 2011). Our finding that the two Ii-MHC II species on the surface of NHHT-39 cells sedimented much slower than expected for an Ii-MHC II nonamer (Fig. 1) support a scenario with unsaturated Ii-MHC II complexes offering free binding sites for MIF. Further work is required to clarify the structure of Ii-MHC II complexes on the surface of B-cells and to elucidate their role as receptors of MIF.
By contrast to clustering of Ii-MHC II, the oligomerization of the BCR by polyvalent antigen is a well-studied process that bears physiological relevance for B-cell activation by soluble
5. Discussion
antigens (13, 14). Recent evidence, however, reveals that other pathways for the delivery of antigens to B-cells exist in vivo. They involve the display of non-degraded antigen on the surface of DCs (16), macrophages (15) or follicular dendritic cells (95). On their itinerary through the lymphatic system, B-cells pass these so-called antigen-decorated cells and become activated once they detect their cognate antigen on the surface of the displaying cell (18). Extensive work by the laboratories of Batista/Neumann and Pierce has shown that the recognition of membrane-bound monomeric antigen also results in BCR clustering, subsequent triggering of the signaling cascade and finally in endocytosis (reviewed in (18)).
We therefore asked ourselves whether a physiologic setting like this could induce the oligomerization of Ii-MHC II and its co-clustering with BCR patches. In support of such a scenario, the chemokine receptors CXCR2 and CXCR4 have recently been described as alternative receptors for MIF (85), which may function as display receptors for MIF on the surface of antigen-decorated cells (ADCs). In fact, cell-bound MIF has been shown to be able to activate CXCR2 and CXCR4-expressing cells in trans (85). Since the chemokine receptors appear to bind to a different part of the MIF molecule than Ii (90) it is conceivable that chemokine receptor-bound MIF on an ADC might interact with Ii on a B-cell during an episode of cell contact. In analogy to the BCR, such an event might trigger the clustering of Ii and this in turn should lead to the coalescence of the BCR and Ii, if also the BCR had been clustered by cell-bound antigen (as demonstrated by Batista and Neuberger (14)). Our considerations are summarized in a hypothetical model depicted below (Fig. 3):
5. Discussion
31 Fig. 3: Hypothetical model for the simultaneous presentation of membrane-bound antigen and MIF by an
antigen-decorated cell (ADC) to a B-cell. Recognition of antigen by the B-cell receptor (BCR) and of MIF by an (substoichiometric) Ii-MHC II complex leads to microcluster formation and convergence in a common lipid raft in the B-cell membrane. Co-endocytosis and subsequent co-targeting to MHC-II peptide loading compartments as well as augmented signaling facilitate the processing and the presentation of cognate antigen by the B-cell.