Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders
© Lehmann-Horn et al; licensee BioMed Central Ltd. 2011
Received: 24 August 2011
Accepted: 26 October 2011
Published: 26 October 2011
Clinical trials evaluating anti-CD20-mediated B-cell depletion in multiple sclerosis (MS) and neuromyelitis optica (NMO) generated encouraging results. Our recent studies in the MS model experimental autoimmune encephalomyelitis (EAE) attributed clinical benefit to extinction of activated B-cells, but cautioned that depletion of naïve B-cells may be undesirable. We elucidated the regulatory role of un-activated B-cells in EAE and investigated whether anti-CD20 may collaterally diminish regulatory B-cell properties in treatment of neuroimmunological disorders.
Myelin oligodendrocyte glycoprotein (MOG) peptide-immunized C57Bl/6 mice were depleted of B-cells. Functional consequences for regulatory T-cells (Treg) and cytokine production of CD11b+ antigen presenting cells (APC) were assessed. Peripheral blood mononuclear cells from 22 patients receiving anti-CD20 and 23 untreated neuroimmunological patients were evaluated for frequencies of B-cells, T-cells and monocytes; monocytic reactivity was determined by TNF-production and expression of signalling lymphocytic activation molecule (SLAM).
We observed that EAE-exacerbation upon depletion of un-activated B-cells closely correlated with an enhanced production of pro-inflammatory TNF by CD11b+ APC. Paralleling this pre-clinical finding, anti-CD20 treatment of human neuroimmunological disorders increased the relative frequency of monocytes and accentuated pro-inflammatory monocyte function; when reactivated ex vivo, a higher frequency of monocytes from B-cell depleted patients produced TNF and expressed the activation marker SLAM.
These data suggest that in neuroimmunological disorders, pro-inflammatory APC activity is controlled by a subset of B-cells which is eliminated concomitantly upon anti-CD20 treatment. While this observation does not conflict with the general concept of B-cell depletion in human autoimmunity, it implies that its safety and effectiveness may further advance by selectively targeting pathogenic B-cell function.
Keywordsmultiple sclerosis neuromyelitis optica anti-CD20 B-cell regulation monocytes experimental autoimmune encephalomyelitis
Accumulating evidence suggests that in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO), B-cells, plasma cells and self-reactive antibodies play an essential pathogenic role. In MS, an oligoclonal antibody response generated by a limited repertoire of activated B-cells remains a hallmark diagnostic finding in the cerebrospinal fluid (CSF). While target and pathogenic relevance of this humoral response is still under debate , autoantibodies against aquaporin-4 (AQP-4) allow to distinguish NMO from other central nervous system (CNS) demyelinating conditions, promote development of NMO-like lesions in animal models  and may correlate with progression of NMO itself . Besides developing into plasma cells secreting self-reactive antibodies, antigen-activated B-cells may directly contribute to development of neuroimmunological disease by transporting, processing and presenting antigen to self-reactive T-cells. As activated T-cells in return promote differentiation of B-cells and isotype switching of plasma cells, the interaction of auto-reactive B- and T-cells may foster each other's development in progression of CNS autoimmune disease.
Based on these pathogenic B-cell properties, substantial interest has developed for testing anti-CD20 antibodies (rituximab, ocrelizumab, ofatumumab) in MS and NMO. These antibodies deplete immature and mature B-cells, but spare CD20-negative plasma cells. The retrospective analysis of 25 NMO patients receiving rituximab demonstrated a reduction in attack frequency with subsequent clinical stabilization . While one study suggested that clinical benefit may relate to a decline in anti-AQP-4 antibody titers , it is unclear whether depletion of CD20+ AQP4-specific plasma cell precursors provides the sole and entire basis for therapeutic benefit of anti-CD20 in NMO . Clinical trials testing anti-CD20 rituximab in MS generated encouraging results as well. In relapsing-remitting MS, treatment with rituximab or its humanized successor ocrelizumab led to a rapid decline in newly developing inflammatory CNS lesions [7, 8]; in treatment of primary progressive MS, rituximab reduced lesion formation in a subgroup of younger patients with active CNS inflammation . Immunological analyses revealed that anti-CD20 B-cell depletion diminished proliferation and pro-inflammatory differentiation of peripheral T-cells ; further, rituximab-treatment was associated with a reduced number of B-cells, but also of T-cells within the CSF of patients with relapsing-remitting (RR)-MS . Together, these findings highlight abrogation of B-cell-mediated T-cell activation as an important mechanism for the prompt effect of anti-CD20 treatment in CNS demyelinating disorders.
Notwithstanding these encouraging results, not all CD20+ B-cells may actively contribute to progression of autoimmune disease. Animal models of human autoimmunity suggest that through provision of anti-inflammatory IL-10, naïve B-cells in contrast regulate autoimmune responses  and control pro-inflammatory differentiation of other antigen presenting cells (APC) . Accumulating evidence suggests that equivalent regulatory B-cell properties exist in humans . In a recent report, Iwata and colleagues described a subset of regulatory IL-10 producing B-cells in various autoimmune conditions, including MS with an overall frequency and IL-10 production comparable to healthy individuals . Functionally, these regulatory B-cells inhibited TNF-release of monocytes isolated from the identical patient, further fueling the concept that regulatory B-cell subsets control pro-inflammatory activity of other APC populations.
Our recent study testing anti-CD20 treatment in an animal model of MS, revealed that B-cell depletion exacerbated experimental autoimmune encephalomyelitis (EAE) induced by the short T-cell determinant myelin-oligodendrocyte glycoprotein (MOG) peptide (p)35-55, a setting in which B-cells are not required or involved in a pathogenic manner . One aim of our current investigation was thus to elucidate the immunological mechanisms for deterioration of EAE in this setting. We demonstrate that EAE-exacerbation upon depletion of un-activated B-cells closely correlates with an enhanced production of pro-inflammatory TNF by CD11b+ APC. In light of these preclinical findings and the newly established role of B-cell subsets in regulation of human autoimmunity, we further investigated whether anti-CD20 treatment may collaterally abolish B-cell regulatory properties in human neuroimmunological disorders. Paralleling our findings in EAE, we report that anti-CD20 treatment of MS and NMO is associated with an accentuation of pro-inflammatory monocyte function, providing the first evidence that besides abrogation of pathogenic B-cell function, anti-CD20 diminishes B-cell regulation of myeloid APC.
Subjects and specimens
Characteristics of patients with neuroimmunological disorders and analysis of peripheral blood mononuclear cells.
number of subjects
mean (min. - max.)
α-CD20 treatment duration [months]
mean (min. - max.)
CD19 + of all PBMCs
[mean % +/- SEM]
CD4 + of all PBMCs
CD8 + of all PBMCs
CD14 + of all PBMCs
CD4 + of all CD4 + /CD8 +
CD4 + of all CD4 + /CD8 + /CD14 +
CD8 + of all CD4 + /CD8 + /CD14 +
CD14 + of all CD4 + /CD8 + /CD14 +
CD25 + CD127 - of all CD4 +
[median % with 20/80% percentile]
FACS staining of leucocyte subpopulations and monocytic activation
PBMCs were stained for CD19, CD4, CD14, CD25, CD127, SLAM/CD150 (all BD Bioscience) or CD8a (eBioscience). FACS staining was analyzed on a Cyan ADP9C using software Summit 4.3 (Beckmann Coulter). PBMCs were stimulated with lipopolysaccharid (LPS) and SLAM-expression of CD14+ monocytes was evaluated 24 hours thereafter. Frequency of CD14+ monocytes expressing SLAM was determined as shown in additional file 2.
Analysis of TNF-producing monocytes
Magnetically activated cell sorting (MACS)-separated monocytes (positive selection using CD14 antibodies, Miltenyi Biotec; purity >90%) were plated in TNF capture antibody-precoated Multi-Screen Filter Plates (Millipore) in triplicates (3,000 cells/well) and stimulated with LPS for 18 hours. Plates were washed and incubated successively with TNF detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT substrate. Plates were analyzed with an automated imaging system and software (AID EliSpot reader and software, Autoimmun Diagnostika).
Mice, EAE induction and depletion of B-cells and regulatory T-cells
All murine experiments were carried out as approved by the government of Upper Bavaria (protocol number 55.2-1-54-2531-67-09). C57BL/6 female mice were immunized with 100 μg MOG p35-55 (Auspep, Australia) in Complete Freund's Adjuvant (CFA) followed by 200 ng of pertussis toxin (PTX) i.p. at the day of immunization and 2 days thereafter. Mice were assessed for signs of EAE as described previously . Mice received weekly i.p. injections of 200μg of murine anti-CD20 or isotype-control starting 21 days prior to immunization (provided by Genentech, South San Francisco, USA) and 500μg of anti-mouse CD25 antibody (BioXcell, West Lebanon, USA) or isotype control 5 and 3 days prior to EAE induction. In unimmunized mice, anti-CD25 antibodies are commonly used to deplete regulatory T cells as they represent the majority of CD25+ cells in naïve mice. Results are representative of 3 separate experiments.
Detection of TNF produced by murine monocytes
12 days after immunization, MACS-purified splenic monocytes (positive selection using CD11b antibodies, Miltenyi Biotec; purity >90%) were stimulated with the indicated concentrations of LPS. After 24 hours, supernatants were collected and analyzed for murine TNF by ELISA (R&D Systems). Plates were read at 450 nm wavelength by a Tecan Genios plate reader and analyzed using Magellan6 software.
As frequency of regulatory T-cells followed a skewed distribution, the Mann-Whitney U-Test was used for comparisons. Frequency of monocytes was distributed normally and analyzed by t-Test. Variability of monocytic SLAM expression was compared using the Siegel-Tukey test, capable to deal with non-normal data. Variability of TNF-producing monocytes in anti-CD20 treated vs. untreated patients was compared using the F-Test based on a normal distribution of values. All statistical tests were two-sided and conducted in an explorative manner on a 5% level of significance. Descriptive statistics for continuous, normally distributed data are given by the mean, its standard error (SEM) or the range (min. - max.). Skewed data is presented by the median as well as 20% and 80% percentiles. Categorical data is summarized by absolute and relative frequencies.
Results and Discussion
We investigated next whether alternatively, elimination of B-cell-mediated regulation of APC activity may account for anti-CD20-associated worsening of peptide-induced EAE. CD11b+ APC were isolated from all four groups of mice and evaluated for production of the pro-inflammatory hallmark cytokine TNF. As indicated in Figure 1c, in all mice depleted of B-cells, remaining CD11b+ cells produced increased levels of pro-inflammatory TNF. This effect was further accelerated when mice were in addition depleted of Treg, resulting in a close correlation between the relative increase in monocytic TNF release and the extent of clinical deterioration. In our previous study, elevated TNF production by CD11b+ cells resulted in an enhanced ability of these APC to generate encephalitogenic Th1 and Th17 cells . TNF was further shown to direct migration of these cells within the CNS, facilitating early initiation of CNS autoimmune disease . Collectively, these findings support the conclusion that in EAE, naive B-cells regulate CD11b+ APC and highlight an enhanced pro-inflammatory APC function as explanation for exacerbation of CNS autoimmune disease upon depletion of naïve B-cells.
While the majority of patients with neuroimmunological disorders clearly benefit from anti-CD20 treatment [5, 7, 8], few cases have been reported in which autoimmune disease progression appeared to be promoted. In a patient with anti-MAG polyneuropathy disability worsened within weeks following anti-CD20 treatment ; in a small study with individuals with anti-MAG polyneuropathy, 8 patients clinically stabilized or improved while one patient markedly deteriorated upon B-cell depletion . In another report, a patient with myasthenia gravis developed ulcerative colitis while on anti-CD20 treatment . A patient with anti-MAG polyneuropathy and secondary-progressive MS showed an improvement of polyneuropathy symptoms, but experienced 2 persistently disabling MS relapses ; another patient with NMO severely progressed while on anti-CD20 therapy . In light of our new findings, and having in mind that monocytic TNF and SLAM expression strongly varied among anti-CD20-treated patients with only few individuals displaying substantially elevated levels, it will be crucial to investigate whether such assumed occasional promotion of autoimmunity may correlate with an enhanced pro-inflammatory APC activity upon anti-CD20 treatment.
In conclusion, we herein provide novel evidence that besides abrogation of pathogenic B-cell function, anti-CD20 treatment eliminates preexisting B-cell regulation in human autoimmunity. In treatment of NMO and MS, this observation in conjunction with our EAE findings could indicate that individual patients with minor counter-balancing pathogenic B-cell involvement may not benefit or even deteriorate upon pan-B-cell depletion via CD20. Whereas our study does not conflict with the projected general potential of B-cell depletion in treatment of autoimmune disorders, it cautions that its indication should be assessed individually and supports further development of this therapeutic approach to selectively target pathogenic B-cell function.
List of abbreviations
antigen presenting cell
Complete Freund's Adjuvant
clinically isolated syndrome
central nervous system
experimental autoimmune encephalomyelitis
enzyme linked immunosorbent assay
enzyme linked immuno spot technique
fluorescence activated cell sorting
magnetically activated cell sorting
myelin associated glycoproteins
myelin oligodendrocyte glycoprotein
Peripheral blood mononuclear cell
relapsing-remitting multiple sclerosis
standard error of the mean
signalling lymphocytic activation molecule
tumor necrosis factor.
This study was supported by a pilot grant of the National Multiple Sclerosis Society (NMSS) to M.S.W. (PP 1660). M.S.W. received further grant support from the Else Kröner Fresenius Stiftung (A69/2010), TEVA and the Deutsche Forschungsgemeinschaft (DFG; WE 3547/4-1). M.S.W. and K. L.-H. are supported by the Kommission für Klinische Forschung (KKF) of the Technische Universität München. R.H. is supported by the Deutsche Forschungsgemeinschaft (SFB 571, A1) and KKNMS (BMBF). B.H. was supported by a grant from the German Ministry for Education and Research (BMBF, "German Competence Network Multiple Sclerosis" (KKNMS), Control-MS, 01GI0917) and the DFG (He2386/7-1). We thank Dr. Uwe Thiel (Department of Pediatrics, Technische Universität München, Munich, Germany) for providing valuable expertise in using his ELISPOT reader.
- von Budingen HC, Gulati M, Kuenzle S, Fischer K, Rupprecht TA, Goebels N: Clonally expanded plasma cells in the cerebrospinal fluid of patients with central nervous system autoimmune demyelination produce "oligoclonal bands. J Neuroimmunol. 2010, 218 (1-2): 134-9. 10.1016/j.jneuroim.2009.10.005.View ArticlePubMedGoogle Scholar
- Owens GP, Bennett JL, Lassmann H, O'Connor KC, Ritchie AM, Shearer A, Lam C, Yu X, Birlea M, DuPree C, Williamson RA, Hafler DA, Burgoon MP, Gilden D: Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid. Ann Neurol. 2009, 65 (6): 639-49. 10.1002/ana.21641.PubMed CentralView ArticlePubMedGoogle Scholar
- Bennett JL, Lam C, Kalluri SR, Saikali P, Bautista K, Dupree C, Glogowska M, Case D, Antel JP, Owens GP, Gilden D, Nessler S, Stadelmann C, Hemmer B: Intrathecal pathogenic anti-aquaporin-4 antibodies in early neuromyelitis optica. Ann Neurol. 2009, 66 (5): 617-29. 10.1002/ana.21802.PubMed CentralView ArticlePubMedGoogle Scholar
- Jarius S, Aboul-Enein F, Waters P, Kuenz B, Hauser A, Berger T, Lang W, Reindl M, Vincent A, Kristoferitsch W: Antibody to aquaporin-4 in the long-term course of neuromyelitis optica. Brain. 2008, 131 (Pt 11): 3072-80.PubMed CentralView ArticlePubMedGoogle Scholar
- Jacob A, Weinshenker BG, Violich I, McLinskey N, Krupp L, Fox RJ, Wingerchuk DM, Boggild M, Constantinescu CS, Miller A, De Angelis T, Matiello M, Cree BA: Treatment of neuromyelitis optica with rituximab: retrospective analysis of 25 patients. Arch Neurol. 2008, 65 (11): 1443-8. 10.1001/archneur.65.11.noc80069.View ArticlePubMedGoogle Scholar
- Pellkofer HL, Krumbholz M, Berthele A, Hemmer B, Gerdes LA, Havla J, Bittner R, Canis M, Meinl E, Hohlfeld R, Kuempfel T: Long-term follow-up of patients with neuromyelitis optica after repeated therapy with rituximab. Neurology. 2011, 76 (15): 1310-5. 10.1212/WNL.0b013e3182152881.View ArticlePubMedGoogle Scholar
- Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ, Bar-Or A, Panzara M, Sarkar N, Agarwal S, Langer-Gould A, Smith CH: B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med. 2008, 358 (7): 676-88. 10.1056/NEJMoa0706383.View ArticlePubMedGoogle Scholar
- Kappos C, O'Connor , Bar-Or , Li , Barkhof , Yin , Glanzman , Tinbergen : Hauser Efficacy and safety of ocrelizumab in patients with relapsing-remitting multiple sclerosis:results of a phase II randomised placebo-controlled multicentre trial presented at the Congress of the European Comittee for Treatment and Research in Multiple Sclerosis. 2010Google Scholar
- Hawker K, O'Connor P, Freedman MS, Calabresi PA, Antel J, Simon J, Hauser S, Waubant E, Vollmer T, Panitch H, Zhang J, Chin P, Smith CH: Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebo-controlled multicenter trial. Ann Neurol. 2009, 66 (4): 460-71. 10.1002/ana.21867.View ArticlePubMedGoogle Scholar
- Bar-Or A, Fawaz L, Fan B, Darlington PJ, Rieger A, Ghorayeb C, Calabresi PA, Waubant E, Hauser SL, Zhang J, Smith CH: Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?. Ann Neurol. 2010, 67 (4): 452-61. 10.1002/ana.21939.View ArticlePubMedGoogle Scholar
- Cross AH, Stark JL, Lauber J, Ramsbottom MJ, Lyons JA: Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol. 2006, 180 (1-2): 63-70. 10.1016/j.jneuroim.2006.06.029.PubMed CentralView ArticlePubMedGoogle Scholar
- Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM: B cells regulate autoimmunity by provision of IL-10. Nat Immunol. 2002, 3 (10): 944-50. 10.1038/ni833.View ArticlePubMedGoogle Scholar
- Moulin V, Andris F, Thielemans K, Maliszewski C, Urbain J, Moser M: B lymphocytes regulate dendritic cell (DC) function in vivo: increased interleukin 12 production by DCs from B cell-deficient mice results in T helper cell type 1 deviation. J Exp Med. 2000, 192 (4): 475-82. 10.1084/jem.192.4.475.PubMed CentralView ArticlePubMedGoogle Scholar
- Mauri C, Blair PA: Regulatory B cells in autoimmunity: developments and controversies. Nat Rev Rheumatol. 2010, 6 (11): 636-43. 10.1038/nrrheum.2010.140.View ArticlePubMedGoogle Scholar
- Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, Szabolcs PM, Bernstein SH, Magro CM, Williams AD, Hall RP, St Clair EW, Tedder TF: Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood. 2011, 117 (2): 530-41. 10.1182/blood-2010-07-294249.PubMed CentralView ArticlePubMedGoogle Scholar
- Weber MS, Prod'homme T, Patarroyo JC, Molnarfi N, Karnezis T, Lehmann-Horn K, Danilenko DM, Eastham-Anderson J, Slavin AJ, Linington C, Bernard CC, Martin F, Zamvil SS: B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity. Ann Neurol. 2010, 68 (3): 369-83. 10.1002/ana.22081.PubMed CentralView ArticlePubMedGoogle Scholar
- Matsushita T, Horikawa M, Iwata Y, Tedder TF: Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis. J Immunol. 2010, 185 (4): 2240-52. 10.4049/jimmunol.1001307.PubMed CentralView ArticlePubMedGoogle Scholar
- Sfikakis PP, Souliotis VL, Fragiadaki KG, Moutsopoulos HM, Boletis JN, Theofilopoulos AN: Increased expression of the FoxP3 functional marker of regulatory T cells following B cell depletion with rituximab in patients with lupus nephritis. Clin Immunol. 2007, 123 (1): 66-73. 10.1016/j.clim.2006.12.006.View ArticlePubMedGoogle Scholar
- Stasi R, Cooper N, Del Poeta G, Stipa E, Laura Evangelista M, Abruzzese E, Amadori S: Analysis of regulatory T-cell changes in patients with idiopathic thrombocytopenic purpura receiving B cell-depleting therapy with rituximab. Blood. 2008, 112 (4): 1147-50. 10.1182/blood-2007-12-129262.View ArticlePubMedGoogle Scholar
- Saadoun D, Rosenzwajg M, Landau D, Piette JC, Klatzmann D, Cacoub P: Restoration of peripheral immune homeostasis after rituximab in mixed cryoglobulinemia vasculitis. Blood. 2008, 111 (11): 5334-41. 10.1182/blood-2007-11-122713.View ArticlePubMedGoogle Scholar
- Dalakas MC, Rakocevic G, Salajegheh M, Dambrosia JM, Hahn AF, Raju R, McElroy B: Placebo-controlled trial of rituximab in IgM anti-myelin-associated glycoprotein antibody demyelinating neuropathy. Ann Neurol. 2009, 65 (3): 286-93. 10.1002/ana.21577.View ArticlePubMedGoogle Scholar
- Aversa G, Chang CC, Carballido JM, Cocks BG, de Vries JE: Engagement of the signaling lymphocytic activation molecule (SLAM) on activated T cells results in IL-2-independent, cyclosporin A-sensitive T cell proliferation and IFN-gamma production. J Immunol. 1997, 158 (9): 4036-44.PubMedGoogle Scholar
- Broglio L, Lauria G: Worsening after rituximab treatment in anti-mag neuropathy. Muscle Nerve. 2005, 32 (3): 378-9. 10.1002/mus.20386.View ArticlePubMedGoogle Scholar
- Renaud S, Gregor M, Fuhr P, Lorenz D, Deuschl G, Gratwohl A, Steck AJ: Rituximab in the treatment of polyneuropathy associated with anti-MAG antibodies. Muscle Nerve. 2003, 27 (5): 611-5. 10.1002/mus.10359.View ArticlePubMedGoogle Scholar
- El Fassi D, Nielsen CH, Kjeldsen J, Clemmensen O, Hegedus L: Ulcerative colitis following B lymphocyte depletion with rituximab in a patient with Graves' disease. Gut. 2008, 57 (5): 714-5. 10.1136/gut.2007.138305.View ArticlePubMedGoogle Scholar
- Benedetti L, Franciotta D, Vigo T, Grandis M, Fiorina E, Ghiglione E, Roccatagliata L, Mancardi GL, Uccelli A, Schenone A: Relapses after treatment with rituximab in a patient with multiple sclerosis and anti myelin-associated glycoprotein polyneuropathy. Arch Neurol. 2007, 64 (10): 1531-3. 10.1001/archneur.64.10.1531.View ArticlePubMedGoogle Scholar
- Capobianco M, Malucchi S, di Sapio A, Gilli F, Sala A, Bottero R, Marnetto F, Doriguzzi Bozzo C, Bertolotto A: Variable responses to rituximab treatment in neuromyelitis optica (Devic's disease). Neurol Sci. 2007, 28 (4): 209-11. 10.1007/s10072-007-0823-z.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.