Multimodal single-cell analysis following SARS-CoV-2 breakthrough infection reveals distinct B cell populations

Abstract

B cells underpin durable immunity by generating long-lived memory B cells and antibody-secreting cells (ASCs) that re-engage upon antigen re-encounter. How human IgG memory B cells are organized to establish, maintain, and reactivate humoral immunity remains incompletely understood. Here, we combine multimodal single-cell profiling with longitudinal characterization of antigen-specific memory B cells following SARS-CoV-2 infection and vaccination to define the developmental relationships and biological functions of human IgG memory B cell subsets. We identify a coordinated activation-memory cycle shared by germinal center- and extrafollicular-derived memory B cells marked by differential CD45RB expression. Within this cycle, activated B cells represent specialized effector cells that acquire inflammatory responsiveness, migratory capacity, and differentiation potential toward ASCs. Activated memory B cells persist as an intermediate that retains migratory capacity while shifting toward homeostatic survival, preserving recall competence and contributing to the regeneration of long-lived resting memory. The resting memory compartment comprises two complementary populations: CD73 resting memory B cells form the principal long-lived recall reservoir, whereas CD24 resting memory B cells adopt a more regulatory resting state and shows limited participation in the SARS-CoV-2 recall response. We further identify a CD24 intermediate population as the earliest transitional state emerging upon memory B cell reactivation, bridging resting memory and the effector recall response. Together, these findings establish the human IgG memory B cell compartment as a dynamic activation-memory cycle rather than a collection of static subsets, providing a framework for understanding humoral immunity and interpreting B cell responses in vaccination, infection, and immune-mediated disease. One sentence summaryOur results provide novel insights on B cell recall responses after SARS-CoV-2 infection or vaccination, formulating distinct classical and non-classical re-activation trajectories from a resting memory state through intermediate phenotypes towards an activated state.