Proteomic profiling of isolated immune synapses from primary mouse B cells

Abstract

The immune synapse (IS) is a cell-cell interaction platform critical in lymphocyte activation by specific antigens. Despite of B cells being able to also respond to soluble antigens, in particular the in vivo importance of the IS and surface-tethered antigen recognition has strongly emerged in the recent years. The IS serves as a dynamic hub for multiple cellular actions but the molecular details of these functions, especially beyond the B cell antigen receptor (BCR) signalling, remain poorly understood. Here, to address the lack in the systems level understanding of the IS, we setup methodology for comprehensive investigation of the composition of the primary mouse B cells' IS at proteome level. Utilizing functionalized magnetic beads to mimic antigen presenting cells and trigger IS formation on them, we developed a method to specifically and robustly extract the cell adhesions on the beads, namely the IS or transferrin receptor mediated adhesion as a control. Our data revealed 661 proteins exclusively present in the IS at 15 minutes after BCR engagement, 13 exclusively in the control adhesions and 365 proteins shared between the samples. We got strong coverage of the known components of the IS as well as identified a plethora of unknown proteins and functional pathways with hitherto unknown roles in B cell IS. Thus, in this work, we validated the IS isolation method as a valuable tool to study early B cell activation by surface-bound antigens as well as unveil several novel proteins and pathways suggestive of new functional aspects in the IS.

Endolysosomal vesicles at the center of B cell activation

“…We expect that proteomics could be key in the quest to unravel important new insights into lysosome functions upon B cell activation. Analysis of isolated lysosomes, like in macrophages (Gao et al, 2017), or modern proteomics tools like LysoIP (Laqtom, 2023) and proximity proteomics (similar to Awoniyi et al [2023]), combined, for example, with the information of the overall proteome recruited to the IS upon B cell activation (Cunha et al, 2023aPreprint, 2023b hold great promise for new insights of the lysosomal heterogeneity and LRO functions. Understanding B cell activation at the molecular level would help us to decipher the pathogenesis of various autoimmune conditions or B cell lymphomas and has implications on immunotherapeutic aspects as well.…”