B cells rapidly target antigen and surface-derived MHCII into peripheral degradative compartments

Abstract

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.

A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

“…2A,B). Moreover, in the later time point, the internalised FIP-IgM was found to colocalise with Rab7 + endosomes close to the perinuclear compartment, as previously reported 7,37,38 , indicating once more that the FIP-IgM probe did not perturb normal antigen trafficking.…”

“…In our previous work, we found a considerable overlap of the internalised antigen with both early and late endosomal markers, as well as with Lysotracker, a fluorescent dye that stains acidic compartments, suggesting that antigen is directed to specialised processing vesicles immediately after internalization 7 . However, challenged by the surface-derived antigen signal, we were able to clearly distinguish a few vesicle internalization events because of the overlapping surface-derived signal.…”

“…Lymphocytes are round, non-adherent cells with a small cytoplasmic space, as the nucleus occupies 70% of the cell volume, and a high receptor concentration on their surface (Fig. 1A) [7][8][9]35 . With the aim to find a solution for these obstacles, we set up the SHIP assay to image internalized antigen in B cells.…”

“…In our previous work, we characterised the antigen processing route in B cells by analysing different vesicle markers using spinning disk confocal microscopy (SDCM) 7 . However, unambiguous distinction between membrane BCRs and newly internalised BCRs remains a major obstacle in imaging approaches, as BCR is the most abundant receptor on the B cell surface (100,000-300,000 copies per cell) 8,9 and engaged BCRs often form clusters on the cell membrane.…”

“…Alternatively, the non-internalized pool can be labelled prior to permeabilization to distinguish it from the internalized pool (for example, Fig. S2A-C in Hernández-Pérez et al 7 ). Nevertheless, quantification of the internalised antigen using this approach is time-consuming and not straightforward, and notably, none of these methods is compatible with live imaging.…”