**2. B cells and antibodies in MS**

164 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

the relevance of autoantibodies for the classification of neurological diseases and discuss novel

Prevalence 100 / 100,000 1 / 100,000 1 / 100,000 Female : Male ratio 3 : 1 9 : 1 1 : 2 Disease onset 20-30 years 40 years Childhood

CSF IgG OCB + Frequent (>90%) Rarely Rarely

Diagnostic marker AQP4-IgG MOG-IgG

Table 1. Important human inflammatory demyelinating diseases. IFN-ß = interferon-beta, GA = glatiramer acetate, AZT = azathioprine, MRI = Magnetic resonance imaging, OCB =

Controversial results regarding the detection of autoantibodies to myelin oligodendrocyte glycoprotein (MOG) in patients with MS have confused researchers for several years. Latest findings showed increased anti-MOG antibody titers in a subgroup of patients with acute disseminated encephalomyelitis (ADEM) and childhood MS, but not in adult MS (O'Connor et al., 2007; Brilot et al., 2009; Di Pauli et al., 2011; Lalive et al., 2011). The target antigen MOG is expressed on the outer surface of the myelin sheath (Figure 1) and can only be detected using cell based assays expressing MOG on their surface. Early stratification from MS is of great relevance as ADEM is usually a self-limiting disease. However, due to the high number of anti-MOG antibody negative ADEM patients, early diagnosis remains

An early detection of autoantibodies to the NMDA (*N*-methyl *D*-aspartate) receptor is crucial in anti**-**NMDA**-**receptor encephalitis, an acute form of encephalitis, which can have a neuropsychiatric presentation, seizures, dyskinesias or autonomic instability (Dalmau et al., 2007). This disease is potentially reversible if it is recognized and treated as early as possible. In paraneoplastic courses a removal of the tumour is mandatory, but NMDA-receptor encephalitis can also be non-paraneoplastic and affect both genders. Detection of antibodies to the neuronal cell surface antigen NMDA-receptor (NMDA-R, Figure 1) in serum of patients supported a better understanding of the disease pathomechanism. In this chapter we will report on the latest findings of autoantibodies in CNS diseases, primarily focusing on anti-AQP4 antibodies in NMO, the relevance of anti-MOG antibodies in ADEM and MS

Relapse treatment Intravenous high-dose methylprednisolone, plasma exchange

**MS NMO ADEM** 

Relapsingremitting

myelitis

Normal or atypical for MS

Long-segment

AZT + steroids,

D, smoking Unknown Infection,

(>3) lesions Confluent

Rituximab None

Monophasic, recurrent

Encephalopathy, multifocal

Multifocal large bilateral lesions

vaccination

findings of a potential involvement of T cells in NMO.

Disease course Relapsing-remitting

Brain MRI Multiple white

Spinal MRI Short-segment (<3)

Interval treatment IFN-ß, GA, Tysabri,

Environmental triggers

oligoclonal bands.

challenging in some cases.

progressive

Clinical symptoms Variable Optic neuritis,

matter lesions

Fingolimod, AZT

and antibodies to NMDA-R in anti-NMDA-receptor encephalitis.

Infections, Vitamin-

lesions

MS is the most frequent inflammatory demyelinating disease in young adults with a high risk of future disability and a heterogeneous clinical presentation (Noseworthy et al., 2000). Approximately 2.5 million people are affected, experiencing different disease courses. In the majority of patients (85-90%), the disease follows a relapsing–remitting course (RR-MS), characterized by acute relapses and subsequent complete or incomplete remission (Sospedra & Martin, 2005). RR-MS patients often convert into a secondary progressive disease course (SP-MS) (Sospedra & Martin, 2005). In contrast, a minority of patients suffer from the primary-progressive disease course (PP-MS, 10-15%) with a steady disease progression (Sospedra & Martin, 2005). Although the etiology of MS remains unresolved, currently it is believed that components of the myelin sheath are attacked by autoreactive T cells involving the cellular and humoral immune system (Sospedra & Martin, 2005). This infiltration of inflammatory cells within the CNS results in inflammation, thus leading to demyelination of the myelin sheaths, which cover the nerve fibers. Brain MRI shows typically multiple white matter lesions, with frequent development of new lesions. In the last decade, increasing research is focusing on the relevance of B cells and antibodies in MS, investigating their role and contribution in the initiation and propagation of inflammatory demyelinating processes at different disease stages (Figure 2).

In at least a subset of MS patients, pathogenic antibodies are believed to cause demyelination and axonal loss. This resulted in an extensive research in order to identify the still unknown target antigen. The detection of intrathecal IgG synthesis and the occurrence of oligoclonal bands (OCB) in the CSF of more than 90% of MS patients supports the impact

Relevance of Autoantibodies for the Classification and Pathogenesis of Neurological Diseases 167

Anti-MOG antibodies are pathogenic *in vitro* (Kerlero de Rosbo et al., 1990) and *in vivo*, as immunization with the MOG protein induces severe experimental autoimmune encephalomyelitis (EAE), which is commonly used as animal model for MS (Linington et al., 1993; Amor et al., 1994; Adelmann et al., 1995; Genain et al., 1995; Weissert et al., 1998). Immunization of MOG in adjuvant or adoptive transfer of activated myelin-specific T cells results in a MS like pathology in EAE (Goverman, 2009). Anti-MOG antibodies are proposed to augment disease severity by enhancing T cell and macrophage initiated demyelination (Zhou et al., 2006). In addition to their relevance in animal models, anti-MOG autoantibodies were discovered in active MS brain lesions (Genain et al., 1999), yet their presence in the CSF and serum of MS patients remains controversial and there are ongoing studies trying to solve these conflicting outcomes (Reindl et al., 2006). Following a publication of our group showing that increased serum anti-MOG and anti-MBP IgM antibodies in patients with clinically isolated syndrome (CIS), the most common first manifestation of MS, predict early conversion to clinically definite MS (Berger et al., 2003), numerous other studies were performed with correlations ranging from highly significant (Greeve et al., 2007; Tomassini et al., 2007), significant in a subanalysis (Rauer et al., 2006; Kuhle et al., 2007a) or not significant at all (Rauer et al., 2006; Kuhle et al., 2007a; Kuhle et al., 2007b). Possible explanations for this controversy could be linked to discrepancies in antibody assays, study designs and the investigated populations (Bar-Or & Antel, 2008). Former studies used mainly Western Blot, ELISA and liquid phase assays for the analysis of anti-MOG antibodies, which detect primarily linear epitopes of the MOG protein or partially refolded MOG. These assays were performed using bacterially expressed fragments of the MOG protein (MOG1-125). However, binding of pathogenic serum anti-MOG antibodies might require native MOG with its posttranslational modifications. As the commonly used Western Blot and ELISA techniques led to inconsistent results, new assays were developed detecting conformational epitopes of MOG. Therefore, cell-based assays were used reflecting the correct formation and glycosylation of native MOG in the human CNS. Increased levels of autoantibodies to native MOG were observed in MS patients during relapse and in SP-MS, compared to patients in remission and controls when performing an ELISA coated with native MOG expressed by eukaryotic cells (Gaertner et al., 2004). A higher frequency of antibodies to native MOG was detected using cell lines expressing full

Fig. 3. MOG is expressed on the outer myelin surface.

Fig. 2. Increasing evidence suggests an important role of B cells in MS. Currently, research is focusing on their relevance for antigen presentation and T cell activation, production of cytokines and antibodies.

of humoral immune responses in the pathogenesis of MS (Kabat et al., 1948). This seminal finding in 1948 is still an immunological hallmark for the disease, with the incorporation of OCB as diagnostic marker for MS (Freedman et al., 2005). However, OCBs are not unique for MS, as they are commonly detected in infectious diseases (Freedman et al., 2005), underlining the urgent need for specific biomarkers. A central role for B cells in the disease pathology can be attributed to studies showing a deposition of antibodies and complement in acute MS lesions (Lucchinetti et al., 2000) and histopathological studies confirming an antibody mediated demyelination (Lucchinetti et al., 1996; Storch et al., 1998) . Furthermore, clonally expanded B lymphocytes were discovered in chronic MS plaques and in the CSF of MS patients (Qin et al., 1998; Colombo et al., 2000; Owens et al., 2003). Latest studies highlight the role of B cells in the disease pathogenesis, as B cell depletion with the chimeric anti-CD20 monoclonal antibody rituximab had an impact on reduced inflammatory brain lesions in MS patients (Hauser et al., 2008). Nevertheless, there is a lack of unique biomarkers for MS, although numerous studies have focused on the presence of autoantibodies against potential antigens of the myelin sheath (Figure 3) and infectious agents in serum and CSF of patients (Reindl et al., 2006).

#### **2.1 Anti-MOG antibodies and MS**

One promising potential candidate as target antigen for MS is the myelin oligodendrocyte glycoprotein (MOG), a CNS specific antigen, which has been studied for several decades now. This transmembrane protein is localized on the outer membrane of myelin sheaths and oligodendrocytes (Figure 3) (Brunner et al., 1989).

Fig. 3. MOG is expressed on the outer myelin surface.

Fig. 2. Increasing evidence suggests an important role of B cells in MS. Currently, research is focusing on their relevance for antigen presentation and T cell activation, production of

of humoral immune responses in the pathogenesis of MS (Kabat et al., 1948). This seminal finding in 1948 is still an immunological hallmark for the disease, with the incorporation of OCB as diagnostic marker for MS (Freedman et al., 2005). However, OCBs are not unique for MS, as they are commonly detected in infectious diseases (Freedman et al., 2005), underlining the urgent need for specific biomarkers. A central role for B cells in the disease pathology can be attributed to studies showing a deposition of antibodies and complement in acute MS lesions (Lucchinetti et al., 2000) and histopathological studies confirming an antibody mediated demyelination (Lucchinetti et al., 1996; Storch et al., 1998) . Furthermore, clonally expanded B lymphocytes were discovered in chronic MS plaques and in the CSF of MS patients (Qin et al., 1998; Colombo et al., 2000; Owens et al., 2003). Latest studies highlight the role of B cells in the disease pathogenesis, as B cell depletion with the chimeric anti-CD20 monoclonal antibody rituximab had an impact on reduced inflammatory brain lesions in MS patients (Hauser et al., 2008). Nevertheless, there is a lack of unique biomarkers for MS, although numerous studies have focused on the presence of autoantibodies against potential antigens of the myelin sheath (Figure 3) and infectious

One promising potential candidate as target antigen for MS is the myelin oligodendrocyte glycoprotein (MOG), a CNS specific antigen, which has been studied for several decades now. This transmembrane protein is localized on the outer membrane of myelin sheaths and

cytokines and antibodies.

agents in serum and CSF of patients (Reindl et al., 2006).

oligodendrocytes (Figure 3) (Brunner et al., 1989).

**2.1 Anti-MOG antibodies and MS** 

Anti-MOG antibodies are pathogenic *in vitro* (Kerlero de Rosbo et al., 1990) and *in vivo*, as immunization with the MOG protein induces severe experimental autoimmune encephalomyelitis (EAE), which is commonly used as animal model for MS (Linington et al., 1993; Amor et al., 1994; Adelmann et al., 1995; Genain et al., 1995; Weissert et al., 1998). Immunization of MOG in adjuvant or adoptive transfer of activated myelin-specific T cells results in a MS like pathology in EAE (Goverman, 2009). Anti-MOG antibodies are proposed to augment disease severity by enhancing T cell and macrophage initiated demyelination (Zhou et al., 2006). In addition to their relevance in animal models, anti-MOG autoantibodies were discovered in active MS brain lesions (Genain et al., 1999), yet their presence in the CSF and serum of MS patients remains controversial and there are ongoing studies trying to solve these conflicting outcomes (Reindl et al., 2006). Following a publication of our group showing that increased serum anti-MOG and anti-MBP IgM antibodies in patients with clinically isolated syndrome (CIS), the most common first manifestation of MS, predict early conversion to clinically definite MS (Berger et al., 2003), numerous other studies were performed with correlations ranging from highly significant (Greeve et al., 2007; Tomassini et al., 2007), significant in a subanalysis (Rauer et al., 2006; Kuhle et al., 2007a) or not significant at all (Rauer et al., 2006; Kuhle et al., 2007a; Kuhle et al., 2007b). Possible explanations for this controversy could be linked to discrepancies in antibody assays, study designs and the investigated populations (Bar-Or & Antel, 2008). Former studies used mainly Western Blot, ELISA and liquid phase assays for the analysis of anti-MOG antibodies, which detect primarily linear epitopes of the MOG protein or partially refolded MOG. These assays were performed using bacterially expressed fragments of the MOG protein (MOG1-125). However, binding of pathogenic serum anti-MOG antibodies might require native MOG with its posttranslational modifications. As the commonly used Western Blot and ELISA techniques led to inconsistent results, new assays were developed detecting conformational epitopes of MOG. Therefore, cell-based assays were used reflecting the correct formation and glycosylation of native MOG in the human CNS. Increased levels of autoantibodies to native MOG were observed in MS patients during relapse and in SP-MS, compared to patients in remission and controls when performing an ELISA coated with native MOG expressed by eukaryotic cells (Gaertner et al., 2004). A higher frequency of antibodies to native MOG was detected using cell lines expressing full

Relevance of Autoantibodies for the Classification and Pathogenesis of Neurological Diseases 169

infrequently detected in patients with ADEM (Stuve et al., 2005; Franciotta et al., 2008), yet a biomarker with high specificity is warranted. Recently, ADEM has attracted rising interest due to the discovery of anti-MOG autoantibodies in a subset of patients (O'Connor et al., 2007). In their study, O`Connor and colleges used a tetramer radioimmunoassay and detected serum antibodies directed to folded MOG tetramer in patients with ADEM in higher concentrations compared to adult MS cases (O'Connor et al., 2007). This observation was confirmed by several other publications (Brilot et al., 2009; Di Pauli et al., 2011; Lalive et al., 2011). Serum antibodies to native MOG were most commonly reported in pediatric ADEM patients. Although, a recent study of our group detected native MOG autoantibodies predominantly in children, we additionally observed few adult anti-MOG antibody positive ADEM cases (Di Pauli et al., 2011). Furthermore, we performed longitudinal analysis of anti-MOG antibodies and showed that a decrease of anti-MOG antibodies in ADEM patients was associated with a more favorable clinical outcome (Di Pauli et al., 2011). Anti-MOG IgG was detected in the CSF of high titer seropositive patients, suggesting a rather peripheral production of antibodies directed to MOG (Di Pauli et al., 2011). Even though anti-MOG antibodies might support the diagnosis of ADEM in a subset of patients, additional biomarkers are warranted for the remaining large proportion of anti-MOG antibody negative ADEM patients. Furthermore, the relevance of ADEM specific high titer anti-MOG

NMO is a rare devastating inflammatory demyelinating disease of the CNS. In former times it was believed to be a severe variant of MS, the most common neurological disease in young adults. In contrast to MS, it has several unique features (Table 1). NMO is characterized by the occurrence of optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM) extending over three or more vertebral segments (Wingerchuk et al., 1999; Cree, 2008), which can lead to blindness and paraplegia within several years of disease onset (Wingerchuk et al., 1999; Wingerchuk & Weinshenker, 2003). Furthermore, NMO commonly follows a more aggressive disease course compared to MS and has a high rate of morbidity and mortality in patients who receive no special treatment (Wingerchuk & Weinshenker, 2003). Especially at disease onset, the diagnosis can be complicated by a long lasting time interval between the occurrence of LETM and ON. Whereas OCB are detected in the CSF of approximately 90% of MS patients (Kabat et al., 1948), they are rarely or transiently present in patients with NMO (0-37%) (Wingerchuk et al., 1999). In addition, the diagnosis of NMO can be supported by the detection of CSF pleocytosis (>50 x106 white blood cell count /L) during acute relapses (Zaffaroni, 2004), which is not indicative for MS. Originally, NMO was described in 1894 by Eugene Devic and Gault as acute monophasic disorder with simultaneous occurrence of ON and LETM (Minagar et al., 2002). Due to a tremendous increase in the scientific interest for this disease, many aspects from the original view of NMO have changed. Nowadays, NMO is characterized as a mainly relapsing disease (80-90%), with a minority of patients suffering from a monophasic course (Wingerchuk et al., 2007; Sellner et al., 2010). Whereas NMO was initially described by a lack of brain MRI lesions, MS-atypical brain lesions are found in some NMO patients primarily at sites of high AQP4 expression (Pittock et al., 2006). However, a negative brain MRI at disease onset is not indicative for MS (Jarius et al., 2008b). The explosive rise in the field of NMO research was mainly due to the discovery of NMO-IgG autoantibodies, mostly IgG1,

antibodies for disease pathogenesis should be further analyzed.

**3. Anti-AQP4 autoantibodies in NMO** 

length human MOG (Lalive et al., 2006; Zhou et al., 2006). Although both studies used cell based assays, the frequency of anti-MOG antibodies within the MS disease course varied in both studies. Lalive and colleges reported increased titers of serum anti-MOG antibodies in patients with CIS, RR-MS and to a smaller extend in SP-MS, but not in healthy controls or PP-MS patients (Lalive et al., 2006). This is in contrast to a study of Zhou, observing the highest frequency of pathogenic autoantibodies to MOG in PP-MS patients using a flow cytometry-based assay. Moreover, Zhou and colleges demonstrated a pathogenic role of human anti-MOG antibodies *in vitro* and *in vivo* following injection into susceptible rat models (Zhou et al., 2006). Summarizing, the relevance of anti-MOG antibodies in MS remains controversial. Latest findings using a novel tetramer radioimmunoassay indicated the presence of conformation dependent anti-MOG antibodies is a subset of pediatric patients with acute disseminated encephalomyelitis (ADEM) and pediatric MS, but rarely in adult onset MS (O'Connor et al., 2007).

#### **2.2 High titer anti-MOG antibodies in ADEM patients**

ADEM is a rarely occurring inflammatory demyelinating disease of the CNS, brain and spinal cord, with an unknown relationship to MS. In patients with ADEM, acute or subacute multifocal large bilateral white matter lesions, frequently involving deep grey matter regions are accompanied by the occurrence of encephalopathy (Mikaeloff et al., 2004; Krupp et al., 2007). Although guidelines have been published to support the diagnosis of ADEM, diagnosis can be complicated, as exact diagnostic criteria are missing. Thus, the incidence remains to be investigated. Some publications suggest a prevalence rate of 0.8 per 100,000 affected patients per year (Leake et al., 2004). Whereas some reports describe no gender predisposition (Dale et al., 2000; Leake et al., 2004), most studies indicate a slight male preponderance in ADEM patients (Pavone et al., 2010). Although the majority of patients with ADEM follow a monophasic disease course, recently recurrent or multiphasic forms have been described with a lower incidence rate (Rust, 2000; Hynson et al., 2001). ADEM commonly occurs after a vaccination (post-vaccination encephalomyelitis) or infection (postinfection encephalomyelitis). In a study of Tenembaum, analyzing 84 pediatric ADEM patients, neurological disturbances occurred in 74% of patients following vaccination or infection (Tenembaum et al., 2002). ADEM is more often described in pediatric patients and juveniles (Leake et al., 2004), however, adult cases have also been reported (Schwarz et al., 2001). In contrast to the persistent disease course of MS, 57-89% of ADEM patients show complete recovery (Dale et al., 2000; Tenembaum et al., 2002). Furthermore, acute treatment with corticosteroids, immunoglobulins and plasma exchange often results in amelioration of ADEM patients, for which reason a biomarker is of high relevance in order to stratify MS and ADEM. Primarily at disease onset, ADEM can be misdiagnosed as CIS (Mikaeloff et al., 2007). The International Consensus criteria of 2007 can serve as guidelines for diagnosing CIS or ADEM (Krupp et al., 2007). Recently, a retrospective study was published analyzing the role of MRI in 28 children with MS and 20 ADEM patients (Callen et al., 2009). Herby, Callen et al. demonstrated a lower age of onset for ADEM patients compared to pediatric MS. This study invented new MRI diagnostic criteria to help differentiating RR-MS from monophasic ADEM at disease onset, yielding a high sensitivity (81%) and specificity (95%) (Callen et al., 2009). Contrary to MS which is typically associated with the development of new lesions, ADEM lesions usually resolve or show residual findings (Kesselring et al., 1990). Therefore, a follow-up MRI within a time period not shorter than 6 months is helpful for diagnosis (Kesselring et al., 1990). Analysis of CSF can support the diagnosis, as OCB are infrequently detected in patients with ADEM (Stuve et al., 2005; Franciotta et al., 2008), yet a biomarker with high specificity is warranted. Recently, ADEM has attracted rising interest due to the discovery of anti-MOG autoantibodies in a subset of patients (O'Connor et al., 2007). In their study, O`Connor and colleges used a tetramer radioimmunoassay and detected serum antibodies directed to folded MOG tetramer in patients with ADEM in higher concentrations compared to adult MS cases (O'Connor et al., 2007). This observation was confirmed by several other publications (Brilot et al., 2009; Di Pauli et al., 2011; Lalive et al., 2011). Serum antibodies to native MOG were most commonly reported in pediatric ADEM patients. Although, a recent study of our group detected native MOG autoantibodies predominantly in children, we additionally observed few adult anti-MOG antibody positive ADEM cases (Di Pauli et al., 2011). Furthermore, we performed longitudinal analysis of anti-MOG antibodies and showed that a decrease of anti-MOG antibodies in ADEM patients was associated with a more favorable clinical outcome (Di Pauli et al., 2011). Anti-MOG IgG was detected in the CSF of high titer seropositive patients, suggesting a rather peripheral production of antibodies directed to MOG (Di Pauli et al., 2011). Even though anti-MOG antibodies might support the diagnosis of ADEM in a subset of patients, additional biomarkers are warranted for the remaining large proportion of anti-MOG antibody negative ADEM patients. Furthermore, the relevance of ADEM specific high titer anti-MOG antibodies for disease pathogenesis should be further analyzed.
