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Evdoshenko et al.

Cellular Therapy and Transplantation (CTT), Vol. 2, No. 7

Please cite this article as follows: Evdoshenko EP, Alekseev S, Stankevich Y, Babenko E, Afanasyev BV. В lymphocytes as a therapeutic target in multiple sclerosis. Cell Ther Transplant. 2011;2:e.000067.01. doi:10.3205/ctt-2011-en-000067.01

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Submitted: xx month 2010, accepted: xx month 2010, published: xx month 2011

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В lymphocytes as a therapeutic target in multiple sclerosis

Evgeny P. Evdoshenko2, Sergey Alekseev1, Yulia Stankevich1, Elena Babenko1, Boris V. Afanasyev1

1Pavlov’s State Medical University, Saint-Petersburg, Russia; 2Leningrad Regional Clinical Hospital, Saint-Petersburg, Russia  

Correspondence: Dr. Evgeny P. Evdoshenko, Leningrad Regional Clinical Hospital, Department of Neurology, Lunacharsky av. 47-49, 194291 Saint-Petersburg; Phone: +7-911-740-23-89, Fax: +7(812)-592-78-40, E-mail: centerms@spam is badgmail.comadmin@spam is badantisclerosis.ru

Abstract

This review article is dedicated to analyzing immunological mechanisms of multiple sclerosis and its immunopathogenesis, with special attention to the role of B-lymphocytes for initiation and progression of inflammatory process in central nervous system. It also describes current methods of immune correction implied in multiple sclerosis therapy, first of all, based on specific suppression of B-cells and their activities. A new concept for combined anti-B-cell therapy is proposed by the authors (rituximab combined with mitoxantrone). Some new preliminary data on results of such combined anti-B cell therapy are presented.

Keywords: В-cells, multiple sclerosis, pathogenesis, therapy, rituximab, mitoxantrone



Autoimmunity features in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the CNS, characterized by demyelination, loss of axons, and subsequent neurological symptoms. MS etiology is still unclear, while there are some advances in learning its immunopathology.

The predominant role of autoimmune mechanisms in MS pathogeneses has been outlined in many experimental and clinical works.

First of all, the most exact experimental murine model of MS — allergic encephalomyelitis — is a classic illustration of an autoimmune process and can be caused in mice by injection of basic myelin protein. Additionally, this condition can be transferred to another experimental animal by T-lymphocytes transfection [34,46].

Like many other autoimmune conditions, MS is associated with specific HLA class II loci (HLA DRB1 1501 and DRB1 1503), and the severity of clinical presentation is also affected by the gene dosage effect [5,20]. In immunomorphological studies of MS, associated lesions always reveal inflammatory infiltration, mostly by CD4+ T-cells and CD68+ macrophages, expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin), different chemokines, cytokines, matrix metalloproteinases, and HLA class II antigens. Another argument for the autoimmune pathogenesis of MS is the recently discovered selective accumulation of regulatory T-cells (defined by FOXP3+ CD4+ CD25bright phenotype) in MS patients’ spinal fluid [16]. Regulatory T-cells' focal accumulation has been previously described in many autoimmune conditions, such as rheumatoid arthritis and psoriasis, as a mechanism aimed at the restriction of autoreactive T-cells proliferation [8,22]. Finally, the autoimmune nature of SC is proved by the effectiveness of several immunosuppressive agents, such as glucocorticoids, mitoxantrone, natalizumab (anti - α4 integrin antibody), shown by double-blind placebo-controlled clinical trials [14,19,29,36,39,43].

For a long period of time, CD4+ T-cells and monocyte-macrophage cells were considered the main effectors of an autoimmune reaction, but data has been gradually gathered that points to a prominent role for B-cells as mediators of an autoimmune inflammation in MS. Animal models of allergic encephalomyelitis can not be reproduced after B-cell depletion [31]. In human subjects the importance of antibody-mediated immune reactions is proved by appearance of oligoclonal bands and increased intrathecal synthesis of different immunoglobulin classes (IgG, IgА, IgМ и IgD) [42]. There is some evidence of a positive correlation between liquor oligoclonal band numbers and the rate of MS progression [4].

High levels of IgG and oligoclonal bands in liquor samples are commonly used as criteria for MS diagnostics due to their high sensitivity (>90%) and specificity. They were considered to be the result of increase in autoantibody production. Autoantibodies to myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) are found in peripheral blood and liquor of 40–50% of MS patients. However, these antibodies were also revealed in patients with other autoimmune diseases and healthy controls — these facts give reasons to doubt their specificity [40]. The true role of these antibodies in MS pathogenesis is currently unclear.

It should also be mentioned that detection of autoantibodies to aquaporin-4 is a highly specific diagnostic marker, restricted to Devic opticomyelitis [45].

The role of antibodies in MS pathogenesis is illustrated by characteristic pathomorphology of lesions characterized by accumulation of B-cells and plasma cells [35]; sometimes there is also noticeable diffuse plasma cell infiltration of white matter. In patients with early MS the role of humoral immunity is directly evidenced by detection of immunoglobulin and immune complex deposits, containing activated complement proteins in plaques [17,18,26]. Some investigators found these changes to be associated with severity of demyelination.

Regulatory mechanisms that determine the migration of B-cells to CNS and their activation are currently the subject of research. There is evidence of CXCL13 B-cell chemokine production in perivascular infiltrates located in active demyelination foci (active plaques); high levels of this chemokine in MS patients liquor also correlate with intensity of intrathecal immunoglobulin synthesis and B-cell numbers [23].

The liquor cytokine profile of SC patients is characterized by high levels of BAFF (B-cells activating factor) — antiapoptotic and activating cytokine [24]. This cytokine impairs apoptosis and is needed for sustaining B-cells that express its receptor, br-3. The B-cell also needs BAFF–br-3 interaction to be transformed into plasma cell after recognition of its specific antigen. According to available data, oligodendrocytes serve as the main source of this cytokine.

The basic mechanisms of B-cell involvement in the pathogenesis of MS and some other autoimmune conditions can be divided into two groups: antibody-dependent and antibody-independent. Antibody-dependent mechanisms have already been mentioned above.

Existence of antibody-independent mechanisms was first demonstrated by Shlomchik et al. in a series of in vivo experiments employing B-cells with impaired immunoglobulin production [9]. Presently, there are several antibody-independent B-cell–associated mechanisms proven to play an important role in MS pathogenesis:

1) B-cells involvement in neolymphogenesis processes [27];

2) autoantigen presentation to T-cells [7,38] and activation of autoreactive     

T-cells [7];

3) proinflammatory cytokine production (mostly LTά and TNFα) [13].

General pre-requisites for anti-B-cell therapy in MS

Basic mechanisms mediating the B-cells' involvement in MS pathogenesis are shown in figure1.

Figure 1. The B-cells' involvement in MS pathogenesis
Involvement in neolymphogenesis processes
T-cell activation and antigen presentation
Production of cytokines (proinflammatory)

The B-cells' involvement in neolymphogenesis determines the processes of tertiary lymphoid tissue formation in damaged areas. In brain tissue biopsies of patients with a secondary progressive form of MS, ectopic lymphoid follicles were found in 50% of cases; these changes were associated with an unfavorable prognosis. B-cells are the main production source of lymphotoxin-α (LTα) [25], the principal mediator of neolymphogenesis [44].

Experiments on murine models of B-cell deficiency demonstrated the importance of this cell compartment for T-cell and marginal zones' formation, follicular dendritic cell maturation, organization of germinal centers' structure, and stromal cell–associated production of some chemokines (CCL19, CCL21 and CCL13) that have a determining influence on dendritic cell functions [3,11,28,33].

One of the important aspects for B-cell participation in autoimmune processes' induction and sustaining is their ability to present antigens. Although dendritic cells are currently considered to be the main antigen-presenting cells, B-cells can also effectively prime naive T-cells via antigen presentation [37].

There is also experimental data on CD4+ and CD8+ T-cells activation by B-cells [9]. Finally, one of important stages of MS pathogenesis is associated with change in spectrum of B-cell–associated cytokines. Non-primed naive B-cells produce mostly proinflammatory IL-10, while memory B-cells are characterized by predominant production of TNFα and LTα. Patients with MS demonstrate lowered IL-10 response on activating stimuli and intact TNFα and LTα production [13]. As postulated, this fact is linked to formation of a large pool of memory B-cells during their clonal expansion, associated with MS pathogenesis.

Therefore, there is a considerable body of data on B-cells being one of the key factors of inflammatory process in MS and, consequently, a potential target for therapeutic intervention.

The first and only product used in clinical practice for B-cell depletion is rituximab (Mabthera), developed by F. Hoffman-La Roche Ltd., Genentech and Biogen Idec.

Rituximab is a mouse/human chimeric monoclonal antibody against the B-cell–associated CD20 antigen. It is registered for treatment of some B-cell lymphomas (since 1997) and rheumatoid arthritis resistant to standard therapeutic agents (since 2006).

Its mechanism of action is mediated by binding to CD20 B-cell membrane antigen. This antigen is an integrated protein (33-35 kDa) expressed on B-cells of different maturation stages, but not on stem cells, pre-B lymphocytes, and plasma cells. The main function of this molecule is membrane transport of ionized calcium (calcium channel), which plays an important role in B-cells activation and apoptosis.

Rituximab binding to CD20 antigen activates several mechanisms: complement activation by classical pathway with consequent complement-mediated cytolysis, cell-mediated cytotoxicity, and apoptosis induction. The exact contribution of each mechanism to the therapeutic effect is still unclear. Currently, complement activation seems to be the most important factor for transfusion-related reactions development, while Fc-receptor–mediated cell cytotoxicity and apoptosis are the principal mechanisms of therapeutic action.

Anti-B-cell therapy is now one of the most promising methods for therapy of autoimmune conditions. We have already published a review on current state and perspectives of anti-B-cell therapy of autoimmune conditions, including demyelinating neuropathies, and the safety issues [2].

Current strategies of anti-B-cell treatment in MS

The first attempts at rituximab therapy for MS patients were made by several research groups in 2005. The drug was used in several patients with progressive or primary resistant MS. First treatment results demonstrated the possibility of B-cell depletion in peripheral blood and liquor, and its association with good clinical dynamics [10,30,41]. The researchers also noted lower liquor levels of CD3+ T-cells in patients on rituximab therapy. At the same time, there was no clear correlation between B-cell numbers and immunoglobulin production. Repeated appraisal of liquor B-cell levels 24 weeks after the end of therapy showed a lower level in most patients, but not their full depletion.

This data formed the basis for further investigation within the double-blinded placebo-controlled phase II study HERMES (Helping to Evaluate Rituxan in Relapsing-Remitting Multiple Sclerosis) aimed at assessment of rituximab therapy effectiveness. In the whole study 104 patients were included, randomized for rituximab and placebo groups. The follow-up period amounted to 48 weeks after completion of the therapy course. Results demonstrated the ability of rituximab to control inflammatory process in MS based on MRI data (fewer new foci).

When speaking of B-cell–directed approaches to MS therapy, we should mention one more drug — mitoxantrone. This topoisomerase II inhibitor is the only cytostatic used in MS therapy with an effect proven by controlled studies.

Investigations on murine models of MS have shown a success rate of the mitoxantrone effect to be ten times higher than cyclophosphamide. We assume it to be linked to this drug’s unique mechanism of action. The studies demonstrated the ability of mitoxantrone to induce B-cells apoptosis; the most sensitive population are memory B-cells (CD19+CD27+) [6,12,32]. Furthermore, additional clinical studies have shown correlations between memory B-cells (CD19+CD27+) pool depletion and therapy effectiveness (A. Bar-Or, oral communication). These changes were accompanied by a lowering of TNFα and LTα production and increase in IL-10 level. Here, mitoxantrone’s excellent blood-brain barrier permeability due to high lipophilicity of its molecules should also be noted. Mitoxantrone therapy forms a current standard for secondary-progradient MS therapy.

This leads us to conclusion that mitoxantrone effects are also mediated by its cytostatic influence on B-cells.

As rituximab and mitoxantrone target the same cell type through different mechanisms of action, we assumed the possibility of therapeutic synergism between these two drugs. This synergism was earlier demonstrated in some autoimmune diseases. For example, rituximab combination with methotrexate or cyclophosphamide achieved a better response to therapy [15]. In hematology this synergism is employed in therapy of lymphoproliferative disorders. One of its possible mechanisms is rituximab’s ability to suppress antiapoptotic Bcl-xL protein expression increasing B-cells sensitivity to apoptosis [21].     

We’ve already published our first impressions on Rituximab in combination with mitoxantrone used as salvage therapy [1]. We’ve administered 2000 mgs of rituximab and 20 mgs of mitoxantrone. This therapy scheme allowed a patient with secondary-progradient MS resistant to standard therapy to reach remission. This patient is currently still in remission (2 years after the end of therapy).

At the time of this publication 17 of our MS patients have received combined therapy with rituximab and mitoxatrone. Some of them received it after the failure of standard therapy, others as first-line therapy.

The most noticeable fact in this case is a possibility of reaching remission and long-term stabilization in patients with disease progression and resistance to standard therapy. Here we must add that this therapy scheme proved to be least effective in a patient with primary progressive MS. Five cycles of combined anti-B-cell therapy didn’t cause a prolonged cell depletion in this patient, and were associated with only transitory positive neurological dynamics. Disease stabilization was noted in all patients with initial use of the combined scheme (based on clinical, immunological and radiological data).

This therapy was well-tolerated. Among the adverse events, transitory severe granulocytopenia (in 17 patients — 100%) with episodes of subfebrile fever should be mentioned. In all the cases it lasted for 4–5 days and didn’t require growth factor therapy. No secondary infections were observed.

Of course, we have only preliminary results now. The pilot study we now plan to conduct will also be aimed at initial detection of therapy effectiveness predictors and updating of the security profile to allow further randomized studies.

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© The Authors. This article is provided under the following license:
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Please cite this article as follows: Evdoshenko EP, Alekseev S, Stankevich Y, Babenko E, Afanasyev BV. В lymphocytes as a therapeutic target in multiple sclerosis. Cell Ther Transplant. 2011;2:e.000067.01. doi:10.3205/ctt-2011-en-000067.01

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