In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to TH cells. While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC). Surprisingly, we detected fast targeting of internalised antigen into peripheral acidic compartments that possessed the hallmarks of MIIC and also showed degradative capacity. In these vesicles, internalised antigen converged rapidly with membrane-derived MHCII and partially overlapped with Cathepsin-S and H2-M, both required for peptide loading. These early compartments appeared heterogenous and atypical as they contained a mixture of both early and late markers, indicating specialized endosomal route. Together, our data suggests that, in addition to previously-reported perinuclear late endosomal MIICs, antigen processing and peptide loading could start already in these specialized early peripheral acidic vesicles (eMIIC) to support fast peptide-MHCII presentation.

B lymphocytes form a central part of the adaptive immune system, helping to clear infections by mounting antibody responses and immunological memory. B cell activation is critically controlled by a specific antigen receptor, the B cell receptor (BCR), which triggers a complex, multibranched signaling cascade initiating various cellular changes. While parts of these pathways are reasonably well characterized, we still lack a comprehensive protein-level view of the very dynamic and robust cellular response triggered by antigen engagement. Ability to track, with sufficient kinetic resolution, the protein machineries responding to BCR signaling is imperative to provide new understanding into this complex cell activation event. We address this challenge by using APEX2 proximity labeling technique, that allows capture a major fraction of proteins in a given location with 20nm range and 1min time window, and target the APEX2 enzyme to the plasma membrane lipid raft domain, where BCR efficiently translocates upon activation. Our data provides unprecedented insights into the protein composition of lipid raft environment in B cells, and the changes triggered there upon BCR cross-linking and translocation. In total, we identified 1677 proteins locating at the vicinity of lipid raft domains in cultured mouse B cells. The data includes a majority of proteins known to be involved in proximal BCR signaling. Interestingly, our differential enrichment analysis identified various proteins that underwent dynamic changes in their localization but that had no previously known linkage to early B cell activation. As expected, we also identified, for example, a wealth of proteins linked to clathrin-mediated endocytosis that were recruited to the lipid rafts upon cell activation. We believe that his data serves as a valuable record of proteins involved in BCR activation response and aid various future studies in the field.

The graded expression of transcription factor interferon regulatory factor 4 (IRF4) regulates B cell development and is critical for plasma cell differentiation. However, the mechanisms, by which IRF4 elicits its crucial tasks, are largely unknown. To characterize the molecular targets of IRF4 in B cells, we established an IRF4-deficient DT40 B cell line. We found that in the absence of IRF4, the expression of several molecules involved in BCR signalling was altered. For example, the expression of B cell adaptor for PI3K (BCAP) was upregulated, whereas the SHIP (SH2-containing Inositol 5´-Phosphatase) expression was downregulated. These molecular unbalances were accompanied by increased BCR-induced calcium signalling, attenuated B cell linker protein (BLNK) and ERK activity and enhanced activity of PI3K/protein kinase B (Akt) pathway. Further, the IRF4-deficient cells showed dramatically diminished cytoskeletal responses to anti-IgM cross-linking. Our results show that IRF4 has an important role in the regulation of BCR signalling and help to shed light on the molecular mechanisms of B cell development and germinal centre response.