Examination of Antigen-Induced Endocytic Structure Formation in B Lymphocytes

Abstract

B lymphocytes are an essential component of the adaptive immune response against pathogens. The ability of a B cell lineage to adapt to better recognize a given antigen depends critically upon the ability of each B cell to bind and internalize antigen through its B cell receptor (BCR). The mechanism of antigen internalization by B cells appears to require cooperation between BCR signaling, the actomyosin network and clathrin-mediated endocytic processes. However, it remains unclear how these actors are coordinated to achieve efficient and controlled uptake of structurally heterogeneous antigens. To better understand this process, we set out to directly observe antigen-induced endocytic structures in the human DG-75 B cell line using a combination of conventional and super-resolution microscopy techniques including confocal laser scanning microscopy (CLSM), total-internal reflected fluorescence microscopy (TIRF-M), and correlative super-resolution light and electron microscopy (CLEM). Cells expressing GFP-tagged BCR and red fluorescent protein-tagged endocytic proteins were stimulated with anti-human IgM Fab’2 to model BCR clustering and internalization. We observed recruitment of early adaptors of clathrin mediated endocytosis – FCHO1, Eps15, Epsin, CALM, and clathrin itself – to Fab’2-induced BCR clusters by TIRF-M and CLSM. CLEM revealed that BCR clusters are heterogeneous in size and frequently partition with clathrin on larger membrane invaginations. This work reveals novel structural features of antigen-induced endocytic structures and expands our understanding of the mechanism by which B cells may accommodate antigen of varying size during internalization.