Rituximab Seen to Affect How B-cells Return After Treatment Stops

Rituximab, an investigational B-cell depletion therapy that target CD20 to treat people with multiple sclerosis (MS), has significant effects on the characteristics of B-cells that return after treatment is stopped, with cells being less mature yet more activated toward a pro-inflammatory state, a study showed.

Treatment also affected immune system T-cells and myeloid cells. 

Whether these changes impact patients clinically is unknown, its researchers said, and these findings may assist in further developing B-cell treatment approaches. 

The study, “B cells reappear less mature and more activated after their anti-CD20–mediated depletion in multiple sclerosis,” was published in the journal Proceedings of the National Academy of Sciences. 

MS, an autoimmune disease, is caused by the immune system mistakenly attacking the protective myelin sheath that covers nerve fibers. B-cells are a type of immune cell that drives inflammation  in MS and immune attacks against myelin. 

Ocrevus (ocrelizumab, marketed by Genentech) is an antibody-based therapy approved to treat people with relapsing MS and primary progressive MS (PPMS), as is Kesimpta (ofatumumab), by Novartis, recently approved in the U.S. for relapsing MS. Known as B-cell depleting therapies, they work by targeting a protein on the B-cell surface called CD20, which leads to the death of B-cells. 

Rituximab (also developed by Genentech) is a B-cell depleting therapy currently being used off-label in MS.

However, as B-cells play an essential role in modulating immune responses, the impact of these B-cell depleting therapies on other parts of the immune system has not been fully explored.

“Little is known about the maturation/activation stage of the returning B cell population after treatment cessation and the wider effects on other immune cells,” the researchers wrote.

Researchers at the University Medical Center Göttingen in Germany examined immune cells isolated from blood samples collected from 15 relapsing-remitting MS (RRMS) patients before and after treatment with rituximab. 

Patients’ mean age (eight women and seven men) was 35.7, and their average disease duration was 10.7 years. All patients received 1,000 mg doses of rituximab. Blood samples were collected at visits before starting the anti-CD20 antibody therapy, and at several times later over 24 months. 

Before rituximab treatment (baseline measure), all 15 patients had mainly two types of B-cells: naive and memory B-cells. Naive B-cells are those that have not been activated by an invading pathogen, such as a virus or bacteria; in contrast, memory B-cells have been triggered by infection, and maintain immunology memory to protect against future infections. 

Despite previous treatments with different disease-modifying therapies, the baseline results showed only minor variations in B-cell counts and characteristics between patients.

Anti-CD20 treatment was “effective” in these patients, the researchers noted, with only one having a relapse six months after starting treatment and another disease worsening 19 months after treatment initiation, as measured by the expanded disability status scale (EDSS) score — an indicator of disability.

Characteristics of the B-cells that reappeared after stopping rituximab treatment were similar across all patients. Their B-cell population was strongly diminished in memory B-cells (36.7% before vs. 8.9% after treatment) and enriched in transitional B-cells (10.1% before vs. 58.8% after) . These are the least mature type of B-cell, marking an early stage of development before the naive B-cell stage.

The frequency of mature naive B-cells was also reduced, dropping from 45.5% pre-treatment to 25.1% post-treatment.

Next, the team examined the state of activation on reappearing B-cells. Before treatment, patients’ B-cells showed a relatively low production (expression) of two proteins — CD25 and CD69 — that are markers for B-cell growth and activation. 

In contrast, returning B-cells consistently showed a significantly higher production of these markers, indicating the cells were more activated than before treatment. 

Reappearing B-cells also showed increased secretion of a pro-inflammatory signaling protein (cytokine) called interleukin-6 (IL-6), which suggested that “reoccurring B cells have a reinforced pro-inflammatory capacity,” the researchers wrote. 

How rituximab’s use affected two other important immune cells, known as T helper cells (CD4⁺) and cytotoxic T-cells (CD8⁺), was also analyzed before and after treatment.

While the total number of T helper and cytotoxic T-cells did not change significantly after treatment, there was a significant increase in the ratio between T helper and cytotoxic T-cells, with T helper cells dominating. 

Both kinds of T-cells also showed a loss of the ability to respond to stimulus or effector function, and a type of memory T-cell called terminally differentiated T-cells were fewer.

Patients with more memory B-cells before treatment showed greater differences in T-cell characteristics after treatment than did those with more naive B-cells at baseline. These patients showed a partly opposite effect on T-cell traits. 

Finally, the impact of treatment on myeloid cells, a cell that is a precursor to many types of blood cells, including red blood cells and immune macrophages, was investigated. These cells were activated by rituximab, suggesting that “B cell depletion results in cessation of B cell anti-inflammatory regulation of myeloid cells … As the majority of these changes are transient, one could see the return of B cell anti-inflammatory regulation of myeloid cells,” the researchers wrote.

Overall, based on the results, the team noted that the “process of therapeutically removing CD20-positive cells in patients with MS and related diseases is immunologically more complex than previously thought,” as “besides unselectively removing B cells, anti-CD20 treatment is associated with profound changes in the T cell compartment.”

Upon stopping rituximab “B cells reappear in a relatively immature status yet with a substantial incline in the expression of activation markers and in the release of pro-inflammatory cytokines,” the researchers wrote. 

“While it remains to be determined whether this change in our conception of anti-CD20 is of direct clinical impact, it may be instructive in the development of … B cell-directed treatment strategies beyond continuous anti-CD20 therapies,” they added.

While neither Ocrevus nor Kesimpta were part of this study, the team also noted that the “reoccurrence of B cells is extremely rare with ocrelizumab [Ocrevus] due to the usually constant and relatively short treatment interval. Nevertheless, as rituximab and other anti-CD20 deplete the identical population of B cells, it is reasonable to assume that ocrelizumab and ofatumumab [Kesimpta] might have similar effects.”