The use of monoclonal antibodies (mAbs) against cytokines or lymphocyte surface molecules has opened new therapeutic options for patients with rheumatoid arthritis (RA) 1. By the prediction of a clinical response, these drugs, which are expensive and have the potential for serious toxicity, could be allotted to those patients who would benefit most 2. B-cell monitoring has been extensively used recently to assess the effect of B cell-directed therapies and the reconstitution of the peripheral blood B-cell repertoire after treatment with the B cell-depleting mAb rituximab. Initially, the clinical response to this therapy was thought not to be correlated to B-cell subset distribution or depletion 3. This view has been challenged by using high-sensitivity flow cytometry, a technique originally developed to detect small numbers of residual malignant cells. Thus, complete depletion of B cells 2 weeks after the first infusion has been suggested to be an indicator for therapy responsiveness 456. Furthermore, subsequent articles indicated that complete depletion is also a prognostic factor for re-treatment 5 and efficacy of the rituximab therapy 6.

Several articles have analyzed the changes in B-cell subsets following depletion therapy with rituximab 789. In most articles, B cells were characterized by the surface markers IgD, CD27, CD38, and CD24, which allow separation of newly generated 'transitional' (IgD⁺, CD27^-, CD24^hi, and CD38^hi) 10, naïve (IgD⁺ and CD27^-), pre-switch (IgD⁺ and CD27⁺) and post-switch (IgD^- and CD27⁺) memory, and double-negative B (IgD^- and CD27^-) cells and plasmablasts (IgD^- and CD27⁺⁺) 111213 in the peripheral blood.

We set out to further delineate B-cell subsets by using high-sensitivity flow cytometry that might help to characterize RA patients who would benefit from rituximab therapy. We expanded our analysis to the co-stimulatory marker CD80, which had been shown to be a potent regulator of IgG secretion by previously activated B cells 14, and CD95, which had been correlated with disease activity in systemic lupus erythematosus (SLE) 13.

In our small registry cohort of patients with routinely treated RA, complete depletion of B cells 15 days after the first rituximab infusion was not a prognostic factor for clinical response. We did not find technical explanations for this discrepancy with previous reports 456. The time lag between blood letting and analysis was proven not to be a valid explanation since no significant difference was found between samples that were analyzed within 6 or 24 hours (Figure S3 of Additional file 3). We cannot exclude biological differences between our cohort and the patients in previous reports 456.

Interestingly, in the regression analysis, the results for baseline distribution of B-cell subsets in responders and non-responders (Table 2) were similar to those reported by Vital and colleagues 5. Thus, in both studies, fewer plasmablasts were significantly associated with response to rituximab. These B-cell populations were also significantly less frequent in responders than in healthy controls (Figure 3b, d).

Analyzing the frequency of the major B-cell subsets (that is, naïve, pre-switch and post-switch memory, and double-negative B cells and plasmablasts) in our RA patients before rituximab treatment, we and others did not find a significant difference with healthy age-matched controls 11. Interestingly, separating the RA population in EULAR responders and non-responders revealed a significantly higher percentage of double-negative (IgD^-/CD27^-) B cells in the former group (Figure 3a). In a recent study in healthy older people, this population of B cells was shown to be enriched in exhausted cells 18. In patients with RA, the humoral immune system already seems to be overstimulated, driving more B cells into the double-negative subset in comparison with healthy controls (Figure 3). The significant result for EULAR responders might be related primarily to the higher number of patients in this group. In these patients (in contrast to patients with SLE 13), disease activity did not correlate with CD95 expression on double-negative B cells (data not shown).

In all patients with RA, CD38^hi post-switch memory B cells were significantly reduced in comparison with healthy age-matched controls (Figure 3c). CD38 is a 45-kDa transmembrane glycoprotein expressed on different human cells, including T and B lymphocytes 17. Activation of naïve B cells leads to an upregulation of this molecule, and transition of activated B lymphocytes into memory cells is characterized by loss of CD38 1920 but this molecule reappears when B cells develop into plasmablasts and plasma cells 21. Plasmablasts can be separated further into CD38^- early plasmablasts and CD38⁺⁺ late plasmablasts 16. Interestingly, in synovial tissue of patients with RA, CD38^- B cells appear to serve as immunoglobulin-producing effector B cells 22. Whether the CD38^-/low post-switch memory B cells in the peripheral blood of patients with RA are similar to the cells described in the synovial tissue cannot be answered yet. Since the latter cells are enriched in the peripheral blood, it is now possible to perform functional assays and determine whether CD38^- B cells are similar to mouse B cells that lack RelB and that are defective in proliferative responses but are able to secrete immunoglobulins and undergo class switching 23.

Recently, treatment with tumor necrosis factor (TNF) blockers was shown to alter the distribution of peripheral blood B cells 24. Thus, infliximab therapy induces an increase in pre-switch memory B cells. Although the majority of our patients have received TNF inhibitors in the past, we did not find this difference. In contrast, our patients exhibited similarly low median percentages of IgD⁺CD27⁺ memory B cells (8.6% and 6.8% for responders and non-responders, respectively) as described for patients with long-standing RA (10.4%). In addition, the frequencies of these B-cell subsets in the control groups in this study and in the article by Souto-Carneiro and colleagues 24 were almost identical (14.9% and 15.1%, respectively). It is fascinating to speculate whether the missing increase in pre-switch memory B cells is a surrogate marker for TNF failure, but further studies have to confirm these results.

Although pre-switch memory B cells are reduced in our RA cohort, this subset is significantly enriched in CD95⁺ cells in non-responders (Figure 3e). Similar results were found in patients with SLE 25. It has been suggested that, in SLE, the pre-switch B-cell population is enriched in autoantibody-producing B cells that are constantly recruited into lymphoid tissue and that therefore are found less often in the peripheral blood. Under physiological conditions, expression of CD95 is important in maintaining peripheral self-tolerance by inducing apoptosis upon engagement with its ligand, CD178 26. In RA, defects of this pathway have been demonstrated 27, and accumulation of autoantibody-secreting plasma cells in synovium has been suggested to be the result of the lack of this regulatory mechanism 28. Our results suggest that an increased frequency of activated (that is, CD95⁺ pre-switch memory) B cells is associated with poor response to rituximab. Figure 4 summarizes the alterations in the different B-cell subsets between EULAR responders and non-responders compared with healthy age-matched controls. Despite the great efforts made, neither certain cell populations nor serum markers that might help to choose a specific therapy have been found so far. The heterogeneity of the patients with RA (for example, disease duration, previous medication, or co-medication or a combination of these) could skew the results.

In summary, even in this small cohort, the frequency of plasmablast seems to be the best predictor for response to a B cell-depleting therapy. Still, prospective studies have to confirm this finding. High-sensitivity flow cytometry appears to be very helpful to narrow further candidates.

APC: allophycocyanin; DAS28: disease activity score using 28 joint counts; EULAR: European League Against Rheumatism; mAb: monoclonal antibody; PE: phycoerythrin; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; TNF: tumor necrosis factor.

The authors declare that they have no competing interests.

HPB and WBG conceived the study and participated in its design and coordination and wrote the manuscript. FR and KB undertook recruitment of patients and collection of clinical data. All authors read and approved the final manuscript.