Human germinal center B cells differ from naive and memory B cells by their aggregated MHC class II-rich compartments lacking HLA-DO

Abstract

To generate memory B cells bearing high-affinity antibodies, naive B cells first encounter antigen in the T cell-rich areas of secondary lymphoid organs. There, they are activated by antigen-specific T cells and become germinal center (GC) founder B cells. GC founders enter the GC to become centroblasts that proliferate and mutate their BCR. Centroblasts differentiate into centrocytes that undergo selection, which requires both the recognition/capture of antigen on follicular dendritic cells and the presentation of processed antigen to GC T cells. Because at each stage of differentiation B cells act as antigen-presenting cells, we analyzed their content of HLA-DR+-rich compartments (MIIC), as well as their expression of HLA-DM, which catalyzes peptide loading of class II molecules, and HLA-DO, which interacts with HLA-DM and focuses MHC class II peptide loading on antigens internalized by the BCR. Naive and memory B cells concentrate HLA-DR, -DM and -DO into compartments dispersed under the cell surface, which are identified by their expression of lysosome-associated membrane protein (Lamp)-1 as late endosomes/lysosomes. GC founders and GC B cells express larger Lamp-1+DR+ compartments that are concentrated in the juxta-nuclear region. These compartments express lower levels of HLA-DM and virtually no HLA-DO. Upon induction of a GC founder phenotype through the prolonged (days) co-ligation of BCR and CD40, the naive B cell's peripheral DR+DM+Lamp-1+ compartments aggregate in a polar fashion close to the nucleus. Furthermore, HLA-DO expression virtually disappears, whereas low levels of HLA-DM remain co-localized with HLA-DR. Anti-κ/λ antibodies, used as surrogate antigens, are promptly (minutes) endocytosed in naive, memory and GC B cells. Then, naive and memory B cells target the surrogate antigen to their peripheral HLA-DO+ MIIC, while GC B cells target it to their HLA-DO- MIIC aggregates. Taken together, our results show that human GC B cells differ from naive and memory B cells by their aggregated MIIC that lack HLA-DO.