B lymphocyte-typing for prediction of clinical response to rituximab
© Brezinschek et al.; licensee BioMed Central Ltd. 2012
Received: 10 February 2012
Accepted: 6 July 2012
Published: 6 July 2012
The prediction of therapeutic response to rituximab in rheumatoid arthritis is desirable. We evaluated whether analysis of B lymphocyte subsets by flow cytometry would be useful to identify non-responders to rituximab ahead of time.
Fifty-two patients with active rheumatoid arthritis despite therapy with TNF-inhibitors were included in the national rituximab registry. DAS28 was determined before and 24 weeks after rituximab application. B cell subsets were analyzed by high-sensitive flow cytometry before and 2 weeks after rituximab administration. Complete depletion of B cells was defined as CD19-values below 0.0001 x109 cells/liter.
At 6 months 19 patients had a good (37%), 23 a moderate (44%) and 10 (19%) had no EULAR-response. The extent of B lymphocyte depletion in peripheral blood did not predict the success of rituximab therapy. Incomplete depletion was found at almost the same frequency in EULAR responders and non-responders. In comparison to healthy controls, non-responders had elevated baseline CD95+ pre-switch B cells, whereas responders had a lower frequency of plasmablasts.
The baseline enumeration of B lymphocyte subsets is still of limited clinical value for the prediction of response to anti-CD20 therapy. However, differences at the level of CD95+ pre switch B cells or plasmablasts were noticed with regard to treatment response. The criterion of complete depletion of peripheral B cells after rituximab administration did not predict the success of this therapy in rheumatoid arthritis.
The use of monoclonal antibodies (mAbs) against cytokines or lymphocyte surface molecules has opened new therapeutic options for patients with rheumatoid arthritis (RA) . 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 . 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 . 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 [4–6]. Furthermore, subsequent articles indicated that complete depletion is also a prognostic factor for re-treatment  and efficacy of the rituximab therapy .
Several articles have analyzed the changes in B-cell subsets following depletion therapy with rituximab [7–9]. 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-, CD24hi, and CD38hi) , 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++) [11–13] 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 , and CD95, which had been correlated with disease activity in systemic lupus erythematosus (SLE) .
Materials and methods
This work was funded by an unrestricted grant from Roche (Vienna, Austria). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Patients and controls
Baseline characteristics of patients included in this study
Age in years, mean ± SE
62.7 ± 1.9
60.8 ± 3.1
Female gender, percentage
Disease duration in years, mean ± SE
11.7 ± 1.3
16.2 ± 3.9
ESRc, mean ± SE
39.2 ± 3.8
43.3 ± 11.8
DAS28-ESR, mean ± SE
5.9 ± 0.2
4.9 ± 0.3
Lymphocytesd, mean ± SE
2,138 ± 174
3,083 ± 23
Concomitant MTX usage, percentage
Previous sDMARD, mean ± SE
2.6 ± 0.1
2.0 ± 0.3
Previous TNF inhibitors, mean ± SE
1.1 ± 0.1
1.4 ± 0.4
No previous biologics, percentage
One previous biologic, percentage
Two previous biologics, percentage
Three previous biologics, percentage
Systemic steroids, percentage
Peripheral blood samples were drawn before and 15 days after the first rituximab infusion. Peripheral mononuclear cells were prepared as described  and stained with the following antibodies: fluorescein isothiocyanate-labeled IgD, phycoerythrin (PE)-conjugated CD24, allophycocyanin (APC)-conjugated CD27, PE-Cy7-labeled CD38, APC-H7-conjugated CD45, horizon Blue-labeled CD19, pyridine-chlorophyll-protein (PerCP)-conjugated CD3 and CD14, and PE-labeled CD80 and CD95 (all mAbs were obtained from BD Biosciences, Schwechat, Austria). Five hundred thousand CD45+ cells were acquired and analyzed by using a seven-channel flow cytometry (BD Canto II cytometer, Software FACSDiva; BD Biosciences). According to their surface staining in the CD27/IgD blot, B cells were classified as naïve (CD19+, IgD+, and CD27-), pre-switch memory (CD19+, IgD+, and CD27+), post-switch memory (CD19+, IgD-, and CD27+), and double-negative (CD19+, IgD-, and CD27-) cells. Plasmablasts were classified as CD19+, IgD-, and CD27++ [12, 16]. In addition, the intensity of the CD38 expression was determined in the post-switch memory and plasmablast populations since this molecule was recently shown not to be constantly expressed within the populations [16, 17]. The distribution of co-stimulatory molecules and the FAS receptor on the different B-cell subsets was determined by replacing CD24 with CD80 or CD95. Complete depletion of B cells was defined as CD19 values of below 0.0001 × 109 cells per liter. The gating strategy and representative dot blots can be found in Figure S1 of Additional file 1.
Since 41% of the blood samples coming from peripheral hospitals had to be transported for more than 12 hours, we compared 15 samples of patients with RA in a pair-wise fashion (2 and 24 hours after blood letting) and found no significant change in absolute counts of B-lymphocyte subsets after this time.
Data were analyzed for normal distribution in order to decide whether to use parametric or non-parametric tests. Values are expressed as the mean ± standard error of the mean or as median (interquartile range) and were calculated by using GraphPad Prism (version 5.0b; GraphPad Software, La Jolla, CA, USA). Baseline clinical variables and B-cell subsets as predictors of response to the first cycle of rituximab were compared with non-responders by using univariate logistic regression. P values of less than 0.05 were considered significant.
Characterization of rheumatoid arthritis patients
B-cell depletion and clinical response
B-cell subpopulations and clinical response
At baseline, the differences in the frequency of naïve, double-negative, pre-switch memory, and post-switch memory B cells between EULAR responders and non-responders did not reach significance. In addition, the expressions of CD80 or CD95 on the B-cell subsets were similar between RA patients and healthy controls. Only a few naïve B cells in patients with RA expressed the co-stimulation and activation markers (1.8% (1.1% to 3.5%) and 4.9% (3.0% to 8.5%), respectively), whereas the highest frequency was found in post-switch memory B cells (58.0% (42.0% to 94.1%) and 65.8% (55.1% to 79.0%), respectively). In all populations analyzed, there was a significant correlation between the frequency of CD95+ and CD80+ B-cell subsets. In responders, this correlation was highest in the double-negative subset (R2 = 0.71; P ≤ 0.0001) but was lowest in non-responders and controls (R2 = 0.520; P ≤ 0.0437 and R2 = 0.384; P ≤ 0.0080, respectively).
The total cohort of patients with RA had a significantly lower frequency of CD38hi B cells within the post-switch memory subset (P ≤ 0.0001). Thus, the median (interquartile range) frequencies of this B-cell subset were 0.9% (0.3% to 2.1%) in responders, 1.3% (0.3% to 3.9%) in non-responders, and 18.2% (14.7% to 34.5%) in controls (Figure 3c). Furthermore, the frequency of CD38hi B cells within plasmablasts was significantly diminished in EULAR responders compared with controls: 28.1% (9.6% to 54.3%) versus 61.1% (44.1% to 66.0%), P ≤ 0.001 (Figure 3d). In contrast, only non-responders had a significantly higher frequency of CD95+ pre-switch memory B cells than controls: 52.6% (37.9% to 62.6%) versus 37.9% (26.5% to 49.3%), P ≤ 0.023 (Figure 3e). The frequency of CD95+ cells in the other B-cell subsets is depicted in Figure S2 of Additional file 2. As indicated in Figure 3, no significant differences were found between EULAR responders and non-responders.
Prediction of first-cycle response
Baseline clinical variables and B-cell subsets in responders and non-responders, as determined by univariate logistic regression
Clinical variable or B-cell subset
OR (95% CI)
Age in years, median (interquartile range)
sDMARD use, median (interquartile range)
Prior anti-TNF use, median (interquartile range)
ESRa, median (interquartile range)
Rheumatoid factor, number (percentage)
B-cell subset, median (interquartile range)
B-cell depletion, number (percentage)
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 [4–6]. 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 [4–6].
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 . 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 . 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 . 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 ), disease activity did not correlate with CD95 expression on double-negative B cells (data not shown).
In all patients with RA, CD38hi 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 . 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 [19, 20] but this molecule reappears when B cells develop into plasmablasts and plasma cells . Plasmablasts can be separated further into CD38- early plasmablasts and CD38++ late plasmablasts . Interestingly, in synovial tissue of patients with RA, CD38- B cells appear to serve as immunoglobulin-producing effector B cells . 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 .
Recently, treatment with tumor necrosis factor (TNF) blockers was shown to alter the distribution of peripheral blood B cells . 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  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.
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.
disease activity score using 28 joint counts
European League Against Rheumatism
systemic lupus erythematosus
tumor necrosis factor.
We give special thanks to Irene Holzer, Carinna Köhler, and Veronika Krischan for their excellent technical work and Erich Kvas for the statistical analysis. We thank our study coordinator, Saelde Baumgartner, and Barbara Nussbaumer and all colleagues who participated in the study and provided the clinical data and samples.
- Furst DE, Keystone EC, Braun J, Breedveld FC, Burmester GR, De Benedetti F, Dorner T, Emery P, Fleischmann R, Gibofsky A, Kalden JR, Kavanaugh A, Kirkham B, Mease P, Sieper J, Singer NG, Smolen JS, Van Riel PL, Weisman MH, Winthrop K: Updated consensus statement on biological agents for the treatment of rheumatic diseases, 2010. Ann Rheum Dis. 2011, 70 (Suppl 1): i2-36. 10.1136/ard.2010.146852.View ArticlePubMedGoogle Scholar
- Tak PP, Kalden JR: Advances in rheumatology: new targeted therapeutics. Arthritis Res Ther. 2011, 13 (Suppl 1): S5-PubMed CentralPubMedGoogle Scholar
- Breedveld F, Agarwal S, Yin M, Ren S, Li NF, Shaw TM, Davies BE: Rituximab pharmacokinetics in patients with rheumatoid arthritis: B-cell levels do not correlate with clinical response. J Clin Pharmacol. 2007, 47: 1119-1128. 10.1177/0091270007305297.View ArticlePubMedGoogle Scholar
- Dass S, Rawstron AC, Vital EM, Henshaw K, McGonagle D, Emery P: Highly sensitive B cell analysis predicts response to rituximab therapy in rheumatoid arthritis. Arthritis Rheum. 2008, 58: 2993-2999. 10.1002/art.23902.View ArticlePubMedGoogle Scholar
- Vital EM, Dass S, Rawstron AC, Buch MH, Goeb V, Henshaw K, Ponchel F, Emery P: Management of nonresponse to rituximab in rheumatoid arthritis: predictors and outcome of re-treatment. Arthritis Rheum. 2010, 62: 1273-1279. 10.1002/art.27359.View ArticlePubMedGoogle Scholar
- Vital EM, Rawstron AC, Dass S, Henshaw K, Madden J, Emery P, McGonagle D: Reduced-dose rituximab in rheumatoid arthritis: efficacy depends on degree of B cell depletion. Arthritis Rheum. 2011, 63: 603-608. 10.1002/art.30152.View ArticlePubMedGoogle Scholar
- Roll P, Palanichamy A, Kneitz C, Dorner T, Tony HP: Regeneration of B cell subsets after transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis. Arthritis Rheum. 2006, 54: 2377-2386. 10.1002/art.22019.View ArticlePubMedGoogle Scholar
- Rehnberg M, Amu S, Tarkowski A, Bokarewa MI, Brisslert M: Short- and long-term effects of anti-CD20 treatment on B cell ontogeny in bone marrow of patients with rheumatoid arthritis. Arthritis Res Ther. 2009, 11: R123-10.1186/ar2789.PubMed CentralView ArticlePubMedGoogle Scholar
- Moller B, Aeberli D, Eggli S, Fuhrer M, Vajtai I, Vogelin E, Ziswiler HR, Dahinden CA, Villiger PM: Class-switched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis. Arthritis Res Ther. 2009, 11: R62-10.1186/ar2686.PubMed CentralView ArticlePubMedGoogle Scholar
- Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE: Identification and characterization of circulating human transitional B cells. Blood. 2005, 105: 4390-4398. 10.1182/blood-2004-11-4284.PubMed CentralView ArticlePubMedGoogle Scholar
- Anolik JH, Looney RJ, Lund FE, Randall TD, Sanz I: Insights into the heterogeneity of human B cells: diverse functions, roles in autoimmunity, and use as therapeutic targets. Immunol Res. 2009, 45: 144-158. 10.1007/s12026-009-8096-7.View ArticlePubMedGoogle Scholar
- Sanz I, Wei C, Lee FE, Anolik J: Phenotypic and functional heterogeneity of human memory B cells. Semin Immunol. 2008, 20: 67-82. 10.1016/j.smim.2007.12.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Jacobi AM, Reiter K, Mackay M, Aranow C, Hiepe F, Radbruch A, Hansen A, Burmester GR, Diamond B, Lipsky PE, Dorner T: Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95. Arthritis Rheum. 2008, 58: 1762-1773. 10.1002/art.23498.View ArticlePubMedGoogle Scholar
- Rau FC, Dieter J, Luo Z, Priest SO, Baumgarth N: B7-1/2 (CD80/CD86) direct signaling to B cells enhances IgG secretion. J Immunol. 2009, 183: 7661-7671. 10.4049/jimmunol.0803783.PubMed CentralView ArticlePubMedGoogle Scholar
- Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, Stevens RM, Shaw T: Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med. 2004, 350: 2572-2581. 10.1056/NEJMoa032534.View ArticlePubMedGoogle Scholar
- Avery DT, Ellyard JI, Mackay F, Corcoran LM, Hodgkin PD, Tangye SG: Increased expression of CD27 on activated human memory B cells correlates with their commitment to the plasma cell lineage. J Immunol. 2005, 174: 4034-4042.View ArticlePubMedGoogle Scholar
- Shubinsky G, Schlesinger M: The CD38 lymphocyte differentiation marker: new insight into its ectoenzymatic activity and its role as a signal transducer. Immunity. 1997, 7: 315-324. 10.1016/S1074-7613(00)80353-2.View ArticlePubMedGoogle Scholar
- Buffa S, Bulati M, Pellicano M, Dunn-Walters DK, Wu YC, Candore G, Vitello S, Caruso C, Colonna-Romano G: B cell immunosenescence: different features of naive and memory B cells in elderly. Biogerontology. 2011, 12: 473-483. 10.1007/s10522-011-9353-4.View ArticlePubMedGoogle Scholar
- Lagresle C, Bella C, Defrance T: Phenotypic and functional heterogeneity of the IgD- B cell compartment: identification of two major tonsillar B cell subsets. Int Immunol. 1993, 5: 1259-1268. 10.1093/intimm/5.10.1259.View ArticlePubMedGoogle Scholar
- Pascual V, Liu YJ, Magalski A, de Bouteiller O, Banchereau J, Capra JD: Analysis of somatic mutation in five B cell subsets of human tonsil. J Exp Med. 1994, 180: 329-339. 10.1084/jem.180.1.329.View ArticlePubMedGoogle Scholar
- Mei HE, Yoshida T, Muehlinghaus G, Hiepe F, Dorner T, Radbruch A, Hoyer BF: Phenotypic analysis of B-cells and plasma cells. Methods Mol Med. 2007, 136: 3-18. 10.1007/978-1-59745-402-5_1.View ArticlePubMedGoogle Scholar
- Reparon-Schuijt CC, van Esch WJ, van Kooten C, Ezendam NP, Levarht EW, Breedveld FC, Verweij CL: Presence of a population of CD20+, CD38- B lymphocytes with defective proliferative responsiveness in the synovial compartment of patients with rheumatoid arthritis. Arthritis Rheum. 2001, 44: 2029-2037. 10.1002/1529-0131(200109)44:9<2029::AID-ART352>3.0.CO;2-2.View ArticlePubMedGoogle Scholar
- Snapper CM, Rosas FR, Zelazowski P, Moorman MA, Kehry MR, Bravo R, Weih F: B cells lacking RelB are defective in proliferative responses, but undergo normal B cell maturation to Ig secretion and Ig class switching. J Exp Med. 1996, 184: 1537-1541. 10.1084/jem.184.4.1537.View ArticlePubMedGoogle Scholar
- Souto-Carneiro MM, Mahadevan V, Takada K, Fritsch-Stork R, Nanki T, Brown M, Fleisher TA, Wilson M, Goldbach-Mansky R, Lipsky PE: Alterations in peripheral blood memory B cells in patients with active rheumatoid arthritis are dependent on the action of tumour necrosis factor. Arthritis Res Ther. 2009, 11: R84-10.1186/ar2718.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodriguez-Bayona B, Ramos-Amaya A, Perez-Venegas JJ, Rodriguez C, Brieva JA: Decreased frequency and activated phenotype of blood CD27 IgD IgM B lymphocytes is a permanent abnormality in systemic lupus erythematosus patients. Arthritis Res Ther. 2010, 12: R108-10.1186/ar3042.PubMed CentralView ArticlePubMedGoogle Scholar
- Siegel RM, Chan FK, Chun HJ, Lenardo MJ: The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity. Nat Immunol. 2000, 1: 469-474. 10.1038/82712.View ArticlePubMedGoogle Scholar
- Peng SL: Fas (CD95)-related apoptosis and rheumatoid arthritis. Rheumatology (Oxford). 2006, 45: 26-30. 10.1093/rheumatology/kei113.View ArticleGoogle Scholar
- Rodriguez-Bayona B, Perez-Venegas JJ, Rodriguez C, Brieva JA: CD95-Mediated control of anti-citrullinated protein/peptides antibodies (ACPA)-producing plasma cells occurring in rheumatoid arthritis inflamed joints. Rheumatology (Oxford). 2007, 46: 612-616.View ArticleGoogle Scholar
- Agematsu K, Nagumo H, Oguchi Y, Nakazawa T, Fukushima K, Yasui K, Ito S, Kobata T, Morimoto C, Komiyama A: Generation of plasma cells from peripheral blood memory B cells: synergistic effect of interleukin-10 and CD27/CD70 interaction. Blood. 1998, 91: 173-180.PubMedGoogle Scholar
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