CD24 and CD27 resolve ontogeny and gradual differentiation of CD11c+ atypical memory B cells after malaria infection

Resolving the Identity Crisis of Atypical B Cells

Scientists have long struggled to define a mysterious group of immune cells known as atypical B cells (ABCs). While these cells appear consistently during various infections, their exact nature remains a subject of intense debate. Researchers observe them in malaria, HIV, and even COVID-19. Yet, they lack a unified definition.

Current research often treats ABCs as a single, static population. Scientists typically identify them by the loss of certain surface markers like CD21 and CD27. However, this approach is like trying to describe a marathon runner and a sprinter using only the word "athlete." It misses the vital nuances of their speed, stamina, and purpose. Because existing definitions are inconsistent, studies often yield conflicting results. Some report ABCs that produce high levels of antibodies. Others describe them as having almost no response to antigen stimulation.

A new study from the Karolinska Institutet suggests this confusion stems from a misunderstanding of cell identity. The authors find that ABCs are not a single type of cell. Instead, they form a continuous spectrum of development. By tracking specific markers, the authors show that ABCs transition through distinct stages. They move from active antibody producers to specialized scavengers that present antigens to other immune cells.

The limitations of binary gating

The prevailing problem in studying ABCs is the reliance on "binary" gating. This is a method in flow cytometry (a technique used to detect physical and chemical characteristics of cells) that categorizes cells into rigid "yes" or "no" buckets. Most studies define ABCs as $CD21^-$ $CD27^-$. This means they look for cells that have lost these two specific proteins.

The authors report that this narrow focus creates a significant blind spot. By only looking for the "final" state where both markers are gone, researchers exclude cells currently in transition. As shown in [Figure 2G], the researchers found that the ABC population is far more complex than a simple "off" switch. Conventional gating captures terminal, late-stage cells. However, it misses the early and intermediate stages that are still actively participating in the immune response. This fragmentation explains why different labs arrive at different conclusions about ABC functions.

Mapping the differentiation axis

To resolve this, the researchers moved toward a "differentiation continuum." This is a model that views cell types as points on a sliding scale of maturity. They used targeted CITE-seq (a method measuring a cell's transcriptome and its surface proteins simultaneously). This provided a high-resolution map of both what a cell is doing and what it looks like.

The authors' approach follows a logical progression of cellular maturation:

1. Identification of the anchor: They identified CD11c as the essential, minimum common feature across all ABC lineages.
2. Resolution of intermediate states: By analyzing the loss of CD24 and CD27, they identified four distinct stages. These include Early-ABC ($CD24^+ CD27^+$), two intermediate states (Int-ABC1 and Int-ABC2), and Late-ABC ($CD24^- CD27^-$). This is visible in the protein-level mapping in [Figure 3C].
3. Lineage tracing: Using pseudotime analysis (a computational method that reconstructs a cell's developmental path), the authors identified two distinct starting points. One lineage emerges from germinal center-derived memory B cells (cells trained in specialized immune hubs). The other comes from marginal zone-like B cells (rapid responder cells).

As illustrated in [Figure 2F], these two paths eventually converge on the same late-stage phenotype. This creates a shared destination for cells that started in very different places.

Evidence of functional reprogramming

The most significant finding is that cell jobs change as they move along the CD24/CD27 axis. The authors report a profound "functional dichotomy." This term describes how the cells perform two very different roles depending on their stage.

In the early stages, the cells behave like traditional defenders. The study finds that Early-ABCs possess the highest capacity for cytokine production and antibody secretion. In a single-cell culture assay, the authors measured antibody production. They found that 37.7% of wells from Early-ABCs produced antibodies. In contrast, only 14.7% of wells from Late-ABCs produced antibodies [Figure 7E].

As the cells progress to the late stage, they undergo "reprogramming." Their biological priorities shift. The researchers report that Late-ABCs acquire enhanced capabilities for phagocytosis (the process of engulfing particles) and antigen presentation. This is supported by transcriptomic module scores in [Figure 7B]. Physical evidence is also seen in [Figure 7F]. Here, Late-ABCs showed the highest association with fluorescent microbial particles. Essentially, the cell shifts from an antibody factory to a scout. It moves from producing weapons to identifying threats for the rest of the immune system.

Assessing the scope of the framework

While this framework is powerful, it has clear boundaries. First, the study was conducted in the context of malaria. This disease serves as a model for intense inflammation. While the authors argue these principles are general, the exact timing of these subsets in other infections is unknown.

Second, the researchers note a limitation in their V(D)J sequencing (which analyzes the genetic sequences of B cell receptors). They did not benchmark these results against classical memory cells. They can see relative differences in mutation levels between subsets. For example, Int-ABC1 had a 5.4% mutation frequency. Meanwhile, Int-ABC2 had only 0.4% [Figure 4B]. However, they cannot definitively state the absolute mutation levels compared to a standard healthy baseline.

Finally, the particle association assay has a technical constraint. It cannot strictly distinguish between a cell sticking to a particle and actually pulling it inside (internalization). Consequently, the "scavenger" role of late ABCs is a highly probable inference. It is not a visually confirmed certainty.

The verdict

The study transforms a fragmented collection of "atypical" cells into a coherent biological process. By replacing binary gates with a continuous axis of CD11c, CD24, and CD27, the authors provide a practical toolkit.

If you are a researcher studying B cell immunology, the decision is clear. Stop using narrow, multi-marker gates that exclude transitioning cells. Instead, adopt the CD11c-based inclusive approach. This allows you to capture the full spectrum of the immune response. The code for their single-cell analysis is reportedly available; see the paper for the canonical link at https://github.com/SundlingLab/ABCdifferentiation. This work explains why these cells behave the way they do.

Figures from the paper