Understanding Rituximab Treatment in Lupus: A Look Inside Immune Cells

 

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Systemic lupus erythematosus (SLE), often simply called lupus, is a chronic autoimmune disease that can cause inflammation and damage in many parts of the body, like the skin, kidneys, and joints. In lupus, the immune system becomes dysregulated, particularly B cells, which mistakenly produce antibodies against the body's own tissues.

Historically, treating lupus involved broad medications that suppress the whole immune system, often leading to significant side effects. Fortunately, advancements in understanding lupus have led to more targeted therapies. One such therapy is rituximab, a treatment that targets and removes CD20+ B cells. Rituximab is frequently used for lupus, even though it hasn't always met its goals in clinical trials. While it's thought to work by reducing autoantibody production and inflammatory signals, scientists still don't fully understand exactly how it provides symptom relief or why some patients respond well while others don't.

To get a clearer picture, researchers in this study used advanced techniques to look at the effects of rituximab on immune cells in detail, at the single-cell level.

The Study: Tracking Immune Cells Over Time

This research involved following nine patients with lupus who were treated with rituximab. Blood samples were collected at different time points: Before treatment (pretreatment), Soon after treatment (early post-treatment, 1 to 6 months), Much later after treatment (late post-treatment, 7 to 15 months)

These samples were compared to samples from eight healthy individuals.

The scientists used several sophisticated methods to analyse individual immune cells from the blood, including:

• Single-cell RNA sequencing (scRNA-seq): to look at gene activity in each cell.

• Surface protein profiling (CITE-seq): to measure protein levels on the cell surface.

• B cell receptor (BCR) and T cell receptor (TCR) sequencing: to study the unique immune receptors on B and T cells.

• Bulk BCR sequencing: another way to study the overall collection of B cell receptors.

In total, data was collected from 169,513 individual immune cells. By looking at gene activity and surface proteins, the researchers could identify 31 different types of immune cells.

Rituximab's Impact on B Cells: Depletion and Return

As expected, rituximab treatment led to a significant drop in the numbers of certain types of B cells soon after treatment. The B cells that were significantly depleted included:

• Naïve B cells

• Memory B cells (both non-switched and switched)

• Age-associated B cells (ABCs), which are thought to play a role in autoimmune diseases like lupus.

Other immune cell types generally didn't change much in number early on.

Later, the B cell population started to come back (repopulate). This repopulation was mainly driven by transitional B cells, which are immature B cells. Naïve B cells, non-switched memory B cells, and ABCs also increased again when comparing the late stage to the early stage after treatment.

Changes in the B Cell Receptor Repertoire

The researchers also examined the collection of B cell receptors (BCRs), which are crucial for recognising specific targets (antigens). They found that early after treatment, the diversity of BCRs decreased, and the likelihood of a sequence appearing by random chance also decreased. At the same time, the rate of changes (mutations) in the BCR sequences increased. These changes were less pronounced in patients where B cell depletion wasn't complete. By the later time points, these measures of repertoire diversity and mutation returned closer to what was seen before treatment.

They also looked at the different types of antibodies B cells were set up to make (isotype usage). Early after treatment, there was an increase in sequences related to switched antibodies (like IgG), which is consistent with the depletion of naïve B cells that haven't yet switched. Later, this reduced, aligning with the return of naïve B cells.

Interestingly, the study found "persistent clones" of B cells. These are groups of B cells that seemed to survive the treatment and were present both before and after rituximab. These persistent clones were more likely to be found among switched memory B cells, plasmablasts, and plasma cells. They also had a higher rate of mutations, suggesting they were B cells that had previously encountered antigens. This indicates that rituximab is very effective at removing less mature B cells, while some more experienced cells can remain.

Comparing lupus patients to healthy individuals, even before treatment, the lupus patients' B cells had shorter CDR3 lengths (a key part of the BCR) in transitional and naïve B cells. They also showed some differences in how certain gene segments (IGHJ genes) were used to build the BCR.

Looking Beyond B Cells: Effects on Other Immune Cells

Rituximab's primary target is B cells, but the researchers also investigated whether other immune cells were affected. They found changes in the activity of specific genes (differential expression) in several non-B cell types. Regulatory CD4 T cells (Tregs) and double negative (DN) T cells showed the most gene activity changes early after treatment.

The study also looked at genes related to interferon signaling, which is known to be important in lupus. While some individual interferon-related genes showed decreased activity in certain cell types (like Tregs, CD8 T cells, DN T cells, and NK cells) early after treatment, there wasn't a consistent widespread change in overall interferon pathway activity across all non-B cells. This finding differs from what has been seen with other B cell depletion therapies, possibly because some antibody-producing cells remain after rituximab.

Why Do Some Patients Respond? Looking Closer at Responders vs. Non-Responders

A key part of the study was dividing the lupus patients into those who responded well to rituximab (5 out of 9) and those who didn't (4 out of 9).

Before treatment, most lupus patients had more B cells in their blood compared to healthy individuals. After treatment, everyone had fewer B cells, but non-responders were more likely to have incomplete B cell depletion, meaning a higher percentage of B cells remained in their blood. This incomplete depletion wasn't simply due to non-responders having lower levels of the CD20 protein target on their B cells before treatment.

When comparing the depletion patterns, responders showed significant removal of ABCs and switched memory B cells, which wasn't seen in non-responders. Non-responders, on the other hand, showed a slight increase in plasma cells early after treatment.

The pattern of B cell return also differed. In responders, ABCs and switched memory B cells stayed low at the later time point, while transitional B cells rapidly repopulated. This shift, with more transitional B cells and fewer memory B cells, might be linked to a good response. In non-responders, there wasn't a significant change in the overall numbers of B cell subtypes returning later, and there was a hint that antigen-experienced cells (like those making IgA antibodies) might have survived the treatment.

The researchers then looked at the gene activity in the B cells that came back (repopulated) in responders. Specifically looking at naïve B cells in responders (which were completely depleted early on and then returned), they found significant differences in gene activity compared to naïve B cells before treatment. Pathways related to NF-κB signalling and GPCR signalling were highlighted as changing.

Crucially, the level of NF-κB pathway activity was significantly lower in the repopulated naïve B cells of responders compared to their naïve B cells before treatment. This lower activity in responders' repopulated B cells was similar to the levels seen in healthy individuals. Non-responders, however, had higher NF-κB activity later on. This reduced NF-κB activity in responders seemed to be linked to lower levels of the BAFF-R protein on the surface of their repopulated naïve B cells. BAFF-R is a protein that activates the NF-κB pathway when it interacts with another molecule called BAFF.

Finally, the study found that gene activity changes in non-B cells over time were different depending on whether a patient responded to rituximab. CD4 central memory T cells (CD4 TCMs) and DN T cells showed the most response-dependent gene changes. In general, in responders, genes in these cells tended to show a greater increase in activity, whereas in non-responders, the changes were smaller or even negative. Many of these genes were involved in important immune functions like:

• Cell cytotoxicity (the ability to kill cells)

• MHC class II antigen presentation (showing parts of targets to other immune cells)

• T cell activation

Genes related to these functions often showed increased activity and were expressed in a higher proportion of cells in responders. These findings suggest that the response to rituximab might involve distinct changes in other immune cells, particularly certain T cell types. For example, changes in LGALS1 expression in DN T cells were different between responders and non-responders, and this gene has been linked to B cell programmed cell death and resistance to rituximab treatment in other conditions. This hints that DN T cells might help B cells die off during successful rituximab treatment.

In Conclusion

This study, using detailed single-cell analysis over time, provides valuable insights into how rituximab treatment affects the immune system in lupus patients. It confirms the depletion and subsequent repopulation of B cells, highlights changes in the BCR repertoire, and identifies transcriptomic changes in non-B cells. Importantly, it reveals differences between responders and non-responders, particularly in the dynamics of specific B cell subtypes (like ABCs and transitional B cells), the gene activity in repopulated naïve B cells (lower NF-κB activation and BAFF-R in responders), and response-specific changes in certain T cells involved in cytotoxicity and activation. These detailed findings help improve our understanding of how rituximab works and potentially why responses vary in lupus.

It's worth noting that this study involved a relatively small number of patients, and having more participants or additional sample collection times might provide even more detailed information about different treatment trajectories.

Single-cell level characterization of B cell depletion and repopulation following rituximab in systemic lupus erythematosus. medRixv. (2025). https://www.medrxiv.org/content/10.1101/2025.05.27.25328230v1

Journal information: https://www.medrxiv.org/