Expression patterns of CD24 and CD27 resolve the origins and gradual differentiation of CD11c

Atypical B-cells (ABCs) are observed across infections, yet their ontogeny and differentiation remain unclear. Using high-dimensional flow cytometry, single-cell transcriptomics, V(D)J sequencing, and functional assays in samples from individuals with malaria and healthy donors, we identify markers of cellular origin while tracking ABC differentiation. Early ABCs express CD11c and arise predominantly from memory B-cells. Two intermediate states originate from germinal center-derived and marginal zone-like B-cells. Pseudotime and lineage analyses reveal trajectories marked by progressive loss of CD24 and CD27, which is recapitulated in vitro, defining bona fide differentiation programs. Differentiation culminates in late ABCs lacking CD24, CD27, and CD21, while expressing CD11c, T-bet, and NKG7 at high levels, exhibiting partial overlap with double-negative-2 B-cells. Early and intermediate stages show greater plasma-cell differentiation potential and cytokine production capacity, whereas later stages acquire transcriptomic signatures of antigen presentation and association with microbial particles, defining stage-dependent functions that reconcile conflicting ABC roles. Comparable ABC populations occur in healthy blood and tonsils, with tissue-associated class switching and CD86 expression consistent with recent T-cell interaction. This framework consolidates ABC definitions along a single differentiation axis and clarifies their cellular origins.