Atypical B cells have often been treated as one confusing, exhausted-looking population. This study instead finds a staged differentiation path, with distinct origins converging on a shared late state. Atypical B-cells are observed across infections, yet their ontogeny and differentiation remain unclear.

The study uses high-dimensional flow cytometry, single-cell transcriptomics, V(D)J sequencing, and functional assays in samples from individuals with malaria and healthy donors to identify markers of cellular origin while tracking ABC differentiation. Early ABCs express CD11c and arise predominantly from memory B-cells, while 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, and differentiation culminates in late ABCs lacking CD24, CD27, and CD21 while expressing CD11c, T-bet, and NKG7 at high levels. Early and intermediate stages show greater plasma-cell differentiation potential and cytokine production capacity, whereas later stages acquire signatures of antigen presentation and association with microbial particles.

Recent reviews converge on differentiation as one of the central questions for atypical B cells. ABCs are best understood as activated states that multiple B cell precursors can enter under distinct inflammatory milieus. The unresolved issues include the relative contribution of naive versus memory origins, whether cells arise from germinal center or extrafollicular settings, and which signal combinations imprint CD11c and T-bet programs.

Their temporal fates are also unresolved: these states might be transient effectors, plasmablast precursors, or longer-lived memory across human timescales. Common phenotypic gates such as CD21 low and CD27 negative or CD11c positive and T-bet positive are insufficient to assign origin.

In malaria, ABC frequency peaks around two weeks after symptom onset, declines within six weeks of treatment, and can remain elevated for up to six months. Many ABCs are non-switched, whereas class-switched ABCs are enriched in previously exposed cohorts, consistent with antigen-experienced memory.

Similar CD11c-positive, T-bet-positive ABC patterns occur in tuberculosis, hepatitis C, hantavirus infection, and COVID-19, where they emerge in response to strong inflammatory signals. ABCs also feature in vaccine responses: antigen-specific CD11c-positive, T-bet-positive B cells expand transiently after influenza and SARS-CoV-2 messenger RNA vaccination.

Current hypotheses propose multiple pathways for ABC differentiation, including the splenic marginal zone and germinal center-derived memory B cells. ABCs often carry somatic hypermutation at levels comparable to or lower than conventional memory B cells, suggesting germinal center experience.

Subsets with lower somatic hypermutation have also been identified in mice during several infections, indicating possible germinal center-independent origins. The link between ABC emergence and impaired humoral responses has raised questions about their role in immunity, and their functions remain debated across inflammatory contexts.

ABCs may represent exhausted cells with reduced survival under chronic antigen load. Alternatively, they may act as plasma cell precursors or specialize in antigen presentation by upregulating MHC class two and CD86. Evidence indicates heterogeneity within ABCs, which may explain their diverse functions.

Their origins, relationships between potential subsets, and differentiation pathways remain poorly defined, contributing to variability and conflicting interpretations in the literature. The study combines high-dimensional flow cytometry, single-cell transcriptomics, V(D)J sequencing, and functional assays in human malaria and healthy donors to resolve ABC ontogeny and differentiation.

It proposes that ABCs form a single CD11c, CD24, and CD27 differentiation continuum, with distinct memory and extrafollicular origins converging on a shared late-stage phenotype. Mature B cells from four individuals with recent malaria were assessed for T-bet, CXCR3, CD11c, and FCRL5 expression in recently dividing and non-dividing populations.

Ki67-positive B cells in patients with Plasmodium falciparum infection showed consistently higher expression of these ABC-associated proteins than Ki67-negative cells. Using Boolean gating to identify all combinations of ABC-associated markers, 96.6 percent of Ki67-positive B cells expressed one or a combination of ABC markers.

The predominant subsets were quadruple-positive for T-bet, FCRL5, CXCR3, and CD11c, or triple-positive for T-bet, CD11c, and FCRL5. In panel E, most marker combinations lie above the dashed zero line, showing enrichment among proliferating cells, while a few combinations fall below it.

The strongest positive shifts cluster among combinations containing T-bet and CD11c, whereas the all-negative combination is markedly depleted. ABC markers were strongly associated with B cell proliferation, because nearly all Ki67-positive cells expressed at least one ABC-associated marker.

The combination of markers was heterogeneous within malaria and also with hantavirus infection. To examine how ABC marker heterogeneity relates to B cell transcriptional profiles, the study used a targeted CITE-seq dataset from peripheral blood mononuclear cells of four malaria patients.

The patients were sampled during acute infection, ten days later, and one year after infection; two had primary infections and two had previously been exposed to malaria. The B cell data were extracted and re-clustered with the Seurat pipeline, using messenger RNA data for clustering.

The clustering identified seven distinct clusters, with most evenly distributed between primary and previously infected individuals, except cluster seven, which was enriched in previously infected patients and potentially indicated a memory origin. Two clusters were naive and five were memory-associated; ABC-marker analysis identified one non-switched memory cluster, one switched memory cluster, and three clusters with different ABC-associated marker patterns.

Cluster five expressed ITGAX, or CD11c, and CXCR3 but lacked TBX21, or T-bet, except transiently on day ten, pointing to potential ABC precursors. These potential precursors also retained expression of CD27 and SELL, or CD62L.

Figure three maps sorted CD11c-positive B cells two weeks after malaria, revealing five transcriptional clusters, including four atypical B-cell states and a small CD69-positive memory-like cluster. Violin plots show associated markers such as ITGAX, TBX21, CXCR3, FCRL5, CD24, CD27, ZEB2, and NKG7.

Flow cytometry then resolves four CD24 and CD27 subsets, measures CD11c protein levels, and compares their frequencies between malaria donors and healthy controls, providing protein-level validation across nine malaria and six healthy donors. Late-ABC and intermediate ABC showed enrichment in non-switched BCR isotypes, while Early ABC were enriched in switched isotypes, particularly IgA.

The CD24 and CD27 expression patterns differed: intermediate ABC exhibited high CD24 and low CD27, while Early ABC showed the opposite pattern. Both markers were downregulated in Late-ABC, consistent with a potential late stage of differentiation.

Pseudotime trajectory analysis identified two lineages. Lineage two progressed from naive B cells through non-switched memory B cells and intermediate ABC to Late-ABC, reflecting a differentiation pathway toward progressively later ABCs. Lineage one passed through switched memory B cells and culminated in Early ABC, indicating a distinct route largely influenced by isotype switching, particularly IgA.

These trajectories reflect relationships between transcriptional states and do not imply a single linear differentiation pathway or direct transitions between all populations. Flow cytometry in a larger acute-malaria cohort was used to validate the ABC populations at the protein level and assess their frequency across individuals.

The re-analyzed dataset contained eighteen patients, all processed in a single experiment to reduce batch variation. From each donor, 3500 mature B cells were concatenated, for a total of 63,000 cells, and UMAP was applied for dimensionality reduction. CD27, IgD, CD21, and CD11c were used as defining markers; Late ABCs were identified by low expression of CD21 and CD27.

Protein-level analyses confirmed the RNA-sequencing-derived clusters in a larger patient cohort, highlighting consistency across method and patients. To study B cell differentiation after malaria, the study performed ten-X single-cell RNA sequencing with paired V(D)J sequencing on sorted CD11c-positive B cells ten days after treatment with artemether and lumefantrine.

Five distinct clusters were identified within the CD11c-positive B cell compartment; four expressed multiple ABC markers, while one small cluster potentially represented recently activated memory B cells or cells migrating into tissues. All clusters expressed ITGAX, or CD11c, and also expressed the transcription factor ZEB2.

Early ABC cells expressed high levels of CD24 and CD27, relatively low levels of ITGAX, CXCR3, and FCRL5, and no detectable TBX21, or T-bet, or NKG7. Late ABCs were defined by high TBX21, ITGAX, FCRL5, and NKG7 expression but lacked CXCR3.

Two intermediate clusters were identified: Int-ABC1 was CD24-negative and CD27-positive, while Int-ABC2 was CD24-positive and CD27-negative. The heavy-chain V-gene sequences of each cluster were examined for somatic hypermutation. Int-ABC2 had an average mutation frequency of 0.4 percent, compared with 5.4 percent for Int-ABC1, which was similar to Early-ABC at 4.5 percent.

Late-ABC had an intermediate mutation frequency of 1.6 percent between Int-ABC2 and Int-ABC1. Across isotypes, Int-ABC1 consistently showed mutation levels similar to Early-ABC, including among non-switched cells. In panel A, the orange IGHM sector dominates every donut, while switched-isotype slices remain visibly larger in Early-ABC and Late-ABC than in the two intermediate clusters.

Panel C shows that this contrast is not uniform across isotypes: the box plots shift substantially between clusters, with especially broad spreads and outliers in several switched-isotype categories. The two intermediate subsets showed distinct BCR profiles, potentially indicating different origins.

Int-ABC1 displayed higher mutation frequencies and greater isotype diversity, consistent with having gone through a germinal center reaction. Int-ABC2 was enriched for IGHM with low mutation frequencies, indicating origins from naive or non-switched memory cells generated in extrafollicular reactions.

Late ABCs exhibited intermediate levels of switched isotypes and somatic hypermutation, potentially indicating contributions from both intermediate subsets. Early ABC aligned with the mutated, often class-switched Int-ABC1 axis, whereas Int-ABC2 represented a distinct population with low somatic hypermutation, most consistent with a non-switched, antigen-experienced precursor pool.

The intermediate ABC clusters represent transition states with different origins: Int-ABC1 originates from germinal center-derived switched and IgD-negative, IgM-positive B cells, while Int-ABC2 originates from IgD-positive, IgM-positive marginal zone-like B cells. Both intermediate populations eventually converge into Late-ABC.

The fundamental architecture of ABC subsets is conserved between blood and tonsil and can be captured by CD11c, CD24, and CD27. ABC cell behavior could potentially differ because several proteins associated with cell function and migration were expressed to different extents between tissues.

The ABC differentiation axis is associated with progressive functional reprogramming: early stages retain plasma cell differentiation potential and cytokine secretion capacity consistent with an activated effector-like state. Late stages acquire particle-associating capacity and antigen presentation features.

These data provide functional validation of the transcriptomically inferred module scores and reinforce the biological significance of the CD11c, CD24, and CD27 differentiation framework. The study used malaria as an in vivo model of acute and resolving inflammation, but its conclusions are intended to reflect general principles of ABC differentiation rather than malaria-specific conclusions.

The analyses did not enrich for or directly identify antigen-specific B cells, so they represent the broader pool of responding B cells rather than malaria-specific populations. The inferred differentiation trajectories and functional properties should therefore be interpreted as characteristics of the overall activated B cell compartment during infection.

Naive and classical memory B cells were not included as reference populations in antibody production assays, and somatic hypermutation analyses were not benchmarked against classical memory or germinal center B cells. Sex was not considered as a variable, and sex-dependent differences in ABC differentiation cannot be excluded.

The central takeaway is that CD11c, CD24, and CD27 organize atypical B cells along one differentiation axis, while different precursor routes help explain their changing functions during infection.