INVITED REVIEW

Adaptive Immune Responses in CNS Autoimmune Disease:Mechanisms and Therapeutic Opportunities

Rhoanne C. McPherson & Stephen M. Anderton

Received: 5 December 2012 /Accepted: 13 March 2013# Springer Science+Business Media New York 2013

Abstract The processes underlying autoimmune CNS in-flammation are complex, but key roles for autoimmunelymphocytes seem inevitable, based on clinical investiga-tions in multiple sclerosis (MS) and related diseases such asneuromyelitis optica, together with the known pathogenicactivity of T cells in experimental autoimmune encephalo-myelitis (EAE) models. Despite intense investigation, thedetails of etiopathology in these diseases have been elusive.Here we describe recent advances in the rodent models thatbegin to allow a map of pathogenic and protective immunityto be drawn. This map might illuminate previous successfuland unsuccessful therapeutic strategies targeting particularpathways, whilst also providing better opportunities for thefuture, leading to tailored intervention based on understand-ing the quality of each individual’s autoimmune response.

Keywords Multiple sclerosis . EAE . Autoimmunity .

Tcells

Immune surveillance

Although the CNS was long viewed as a site of immuneprivilege, it is now understood that cells of the adaptive im-mune system are required to patrol the CNS to protect againstinfection by pathogens. Indeed, the inhibition of lymphocyteentry into the CNS can prove catastrophic owing to the re-activation of latent pathogens. In particular, several therapeutic

interventions that act in this manner have led to the develop-ment of progressive multifocal leukoencephalopathy (PML),an often fatal demyelinating condition caused by thereactivation of the JC virus within the CNS (Wenning et al.2009; Stuve et al. 2006).

Despite this necessity for lymphocytes to gain access, entryinto the CNS remains a highly regulated process, involvingboth physical and molecular barriers. The physical barriers arecomprised of the endothelial layer of the blood brain barrier(BBB), and the epithelial layer of the blood-cerebrospinalfluid barrier (BCSFB); both of which possess complex tightjunctions that limit cellular infiltration (Wolburg and Lippoldt2002; Engelhardt and Sorokin 2009). The BBB is locatedwithin the CNSmicrovasculature and separates the circulatingblood from the perivascular (Virchow-Robin) spaces of thebrain parenchyma (Bechmann et al. 2007). In contrast, theBCSFB surrounds the choroid plexus within the ventricles,the site of CSF production. The relative absence of leukocyteswithin the CNS parenchyma of the healthy brain compared tothe high numbers found in the CSF (Hickey et al. 1991;Svenningsson et al. 1995) suggests that immune cells prefer-entially enter the CNS across the BSCFB. Analysis of thecellular populations within the CSF reveals that the over-whelming majority (<90 %) are T cells, with a high CD4+/CD8+ T cell ratio compared to peripheral blood (Kivisakk etal. 2003; de Graaf et al. 2011). Moreover, the CD4+ T cellspresent within the CSF have a memory phenotype and onlyactivated, not naïve, T cells are able to enter the non-inflamedCNS (Hedlund et al. 1989; Wekerle et al. 1986). Upon acti-vation, T cells up-regulate adhesion molecules and chemokinereceptors required for their migration into tissues. The consti-tutive expression of adhesionmolecules such as VCAM-1 andICAM-1 by the epithelial cells of the choroid plexus is thoughtto enable entry of activated T cell lymphocytes, which expressthe adhesion molecule ligands VLA-4 and LFA-1, into theCNS (Steffen et al. 1996). The adhesion molecule P-selectinhas also been implicated in the migration of T cells across theBCSFB as it is similarly constitutively expressed in the

R. C. McPherson : S. M. AndertonCentre for Inflammation Research and Centre for MultipleSclerosis Research, Queen’s Medical Research Institute,University of Edinburgh, Edinburgh EH16 4TJ, UK

S. M. Anderton (*)Centre for Inflammation Research, Queen’s Medical ResearchInstitute, University of Edinburgh, 47 Little France Crescent,Edinburgh EH16 4TJ, UKe-mail: steve.anderton@ed.ac.uk

J Neuroimmune PharmacolDOI 10.1007/s11481-013-9453-9

choroid plexus, and blockade of P-selectin reduces the numberof activated effector T cells able to enter the CNS (Kivisakk etal. 2003; Carrithers et al. 2002). In addition to adhesionmolecules, chemokine-chemokine receptor interactions alsofacilitate trafficking of activated lymphocytes into target sites.The constitutive expression of the chemokine CCL20 in thechoroid plexus is thought to enable homing of CCR6+ T cellsto the BCSFB (Reboldi et al. 2009). Furthermore, T cellsdeficient in CCR6 are found to accumulate in the choroidplexus parenchyma, unable to cross into the CSF. Evidencethat activated T cells do indeed target the BCSFB as a route ofentry into the CNS stems from the observation that T cellblasts can be detected in the choroid plexus stroma within2 hours of adoptive transfer (Carrithers et al. 2002). In con-trast, the capacity of activated T cells to pass through theundisturbed BBB into the brain parenchyma is especiallylow due to the absence of adhesion molecule and chemokineexpression on the endothelial layer of the non-inflamed CNS(Piccio et al. 2002).

The permissive environment of the choroid plexus sug-gests that activated lymphocytes enter the CNS through theBCSFB in order to conduct immune surveillance. CSF isproduced by ependymal cells of the choroid plexus, andcirculates throughout the subarachnoid space (between thearachnoid & pial membranes), carrying nutrients into theCNS and removing waste products (Johanson et al. 2008).Once T cells cross into the CSF they are thought to migratethroughout the subarachnoid space where they can becomere-activated by CNS-resident antigen presenting cells (APC)displaying their cognate antigen (Kivisakk et al. 2009;Hickey and Kimura 1988). This re-activation of T cells thenpromotes activation of the endothelial layer of the BBB,initiating high level expression of cell adhesion moleculesand recruitment of T cells to the perivascular space.Migration of T cells through the BBB facilitates the estab-lishment of inflammatory foci, further recruitment of leuko-cytes and movement of T cells across the glia limitans intothe brain parenchyma (Fig. 1).

Pathogenic T helper cells and autoimmune CNSinflammation

Most of what is understood about the underlying mecha-nisms of inflammation within the CNS has been gleanedfrom animal models. In particular, the rodent model exper-imental autoimmune encephalomyelitis (EAE) has providedvaluable information about both pathogenic and protectiveadaptive immune responses within the CNS. EAE is con-sidered to model the initial inflammatory stages of MS, andhas been used in the development of many therapeuticstrategies currently in use in the clinic. The predominantcaveat being that EAE involves the experimental activation

of antigen-reactive T cells in the peripheral lymphoid or-gans, before they can migrate into the CNS to induce dis-ease, whereas the instigating factors in MS are currentlyunknown.

EAE is a demyelinating disease that usually targetsautoantigenic components of the CNS myelin sheath, mostcommonly myelin basic protein (MBP), myelin oligodendro-cyte glycoprotein (MOG), or proteolipid protein (PLP). In itssimplest form, instigation of disease requires the peripheralactivation of autoreactive T cells by immunisation with myelinantigens in adjuvant (Stromnes and Goverman 2006).Adoptive transfer models can also be achieved by in vitroactivation of myelin-reactive T cells prior to their infusion.These antigen-reactive effector T cells can migrate into theCNS as a pioneer population and become re-activated, produc-ing pro-inflammatory cytokines that activate CNS-residentmicroglia and recruit macrophages from the peripheral circu-lation and tissues (Ponomarev et al. 2005; Ajami et al. 2011).Activated microglia develop enhanced APC function andthemselves secrete pro-inflammatory cytokines such as IL-1,IL-6 and TNF-α, which acts to further disrupt the BBB andenhance the recruitment of immune cells into the CNS (Conradand Dittel 2011; Prendergast and Anderton 2009).Inflammatory macrophages induced by cytokines secreted bypathogenic CD4+ Tcells are thought to be directly responsiblefor destruction of the myelin sheath surrounding axons andcausing damage to oligodendrocytes (Epstein et al. 1983;Pender et al. 1990; Scolding and Compston 1991). The impor-tance of macrophages in EAE pathology has been illustratedby depletion studies, whereby removal of peripheral macro-phages supresses the clinical signs of disease (Huitinga et al.1990; Brosnan et al. 1981). Despite the role of activatedmacrophages in disease progression, EAE is categoricallyCD4+ T cell mediated. No other immune component cantransfer disease in isolation, but the clinical outcome is dictatedby the nature of the CD4+ T cell population used.

CD4+ effector T cells can produce different patterns ofcytokines. It is now over 25 years since Mosmann andCoffman first described two types of mouse T helper (Th)cell clone (Mosmann et al. 1986). Th1 cells were describedto produce IFN-γ in particular and not IL-4, whereas Th2cells showed the opposite profile. IFN-γ potently activatesmacrophages and so it seemed logical for EAE to be Th1-mediated. Initial evidence supported this (Ando et al. 1989;Bright et al. 1998). Th effector function is determined by thelocal cytokine milieu present at the time of initial T cellactivation by APC, with IL-12 having a key role in drivingTh1 differentiation (Hsieh et al. 1993; Macatonia et al.1995). The protection against EAE induction afforded byblockade or deficiency of the IL-12p40 subunit (Leonard etal. 1995; Bright et al. 1998; Segal et al. 1998; Becher et al.2002) further cemented the view that this was a Th1-mediated disease. However, the observation that genetic

disruption or antibody blockade of IFN-γ did not preventdisease induction (Ferber et al. 1996; Lublin et al. 1993)ruled out a key contribution of this signature Th1 cytokineto disease development. On the contrary, mice lackingIFN-γ signalling show exacerbated EAE (Ferber et al.1996; Krakowski and Owens 1996; Willenborg et al.1996). This paradox was gradually unpicked, first by thediscovery that p40 is not only a component of IL-12 (whencombined with p35), but also IL-23 (in combination withp19) (Oppmann et al. 2000). Subsequent comparison ofmice deficient in either p35 or p19 revealed that EAE wasabrogated in mice specifically deficient in IL-23 and not IL-12 (Cua et al. 2003). The requirement for IL-23 was be-lieved to involve a function for the cytokine within the CNS.

How was IL-23 influencing EAE? A following paperdescribed a novel T cell subset, distinct from Th1 thatproduced IL-17 rather than IFN-γ (Th17 cells). These cellswere believed to be induced by IL-23 and were found to bepotent inducers of EAE (Langrish et al. 2005). A distinctcharacteristic of EAE induced by these IL-23 polarised cellswas the dominant neutrophilic inflammation- a feature notordinarily observed during passive EAE induced by IL-12polarised cells or classical active EAE, where macrophagescomprise the majority of the inflammatory infiltrate (Merrillet al. 1992; Kroenke et al. 2008). In spite of these findings,inconsistencies remained as EAE induction was not abro-gated in mice deficient in IL-17 (Haak et al. 2009).Furthermore, another study also demonstrated that adoptivetransfer of Th1 cells but not Th17 cells could effectively

induce disease, and that Th17 cells could only enter theCNS in the presence of Th1 cells (O’Connor et al. 2008).Coupled with reports that both T-bet (the master regulatortranscription factor determining Th1 differentiation) and IL-23 are required for the induction of EAE (Bettelli et al.2004; Cua et al. 2003; Lovett-Racke et al. 2004; Thakkeret al. 2007), it can be concluded that EAE is not explicitly aTh1 or Th17 driven disease. Instead, whether the encepha-litogenic T cell population displays a predominantly Th1 orTh17 phenotype is thought to influence the location ofinflammatory foci and manifestation of clinical signs. Adominant Th17 phenotype has been associated with morepronounced inflammatory foci in the cerebellum and hindbrain, which is associated with “atypical” diseasecharacterised by ataxia, whereas Th1-dominated EAE pro-duces a greater inflammatory infiltrate within the spinal cordand the ascending paralysis typical of the classical diseasecourse (Stromnes et al. 2008; Rothhammer et al. 2011;Domingues et al. 2010; Wensky et al. 2005).

The debate over why different reports favour Th1 overTh17 cells, and vice versa, as key initiators of EAE can beresolved to some extent by the fact that either population canproduce GM-CSF (Mosmann et al. 1986; El-Behi et al.2011). It has been known for over a decade that micedeficient in this cytokine strongly resist EAE induction(McQualter et al. 2001), and the requirement for T cells tobe a key source has recently been re-emphasised(Ponomarev et al. 2005; Kroenke et al. 2010; Codarri et al.2011). It is thought that the production of GM-CSF may

Stroma

Choroid plexus

Epithelial cellsBCSFB

MemoryCD4+ Tcell

Subarachnoid Space(CSF)

12

Perivascularspace

Brain parenchyma

CNS-residentAPC

3

BBB

4 CD8+ T cells B cells

Pro-inflammatorycytokines

Peripheral monocytes/

macrophages

5

Fig. 1 Recruitment of CNS-reactive T cells into the CNS. 1. Memory/activated T cells bind to adhesion molecules expressed in the choroidplexus and migrate across the BCSF barrier into the CSF. 2. Once in theCSF, T cells migrate throughout the subarachnoid space. 3. T cells arereactivated by CNS-resident APC, such as macrophages and microgliapresenting auto-antigen. Activated T cells secrete pro-inflammatory

cytokines which compromise the integrity of the BBB. 4. Adhesionmolecule, and chemokine expression on the ativated endothelial layerof the BBB facilitates entry of auto-reactive CD4+, CD8+, B cells andperipheral monocytes into the perivascular spaces. 5. Infiltrating cellscontribute to the formation of inflammatory foci and can migrate into thebrain parenchyma. Adapted from (Goverman 2009)

play a role in instigating disease through the activation ofmacrophages and other APC within the CNS (Ponomarev etal. 2007; Codarri et al. 2011).

Recent studies have highlighted that T helper “lineages”are not irrevocably fixed. CD4+ cells producing IL-17 havebeen shown to trans-differentiate into IFN-γ producingcells, and elegant fate-mapping studies have shown that thisis particularly the case in the inflamed CNS during EAE(Hirota et al. 2011; Kurschus et al. 2010). Thus a model canbe drawn in which IL-17 production is triggered early afteractivation of autoantigen-responsive “Th17” cells whichalso express the IL-23 receptor, allowing best uptake ofGM-CSF production (key to pathology) (Codarri et al.2011; Yang et al. 2009), but that a collateral effect (notkey to pathology) is the down-regulation of IL-17 and up-regulation of IFN-γ (Hirota et al. 2011). This fits well withdata from cytokine gene knockout studies in that neither IL-17 nor IFN-γ are required for EAE (Willenborg et al. 1999;Haak et al. 2009), but both IL-23 and GM-CSF are (Cua etal. 2003; McQualter et al. 2001). A third cytokine known tobe required for EAE is IL-6 (Samoilova et al. 1998; Mendelet al. 1998). The role for IL-6 appears to be very early in theetiopathology of EAE, based on a series of findings. First,exogenous IL-6 can render IL-6 deficient mice susceptibleto EAE, if given around the time of immunization, but notlater (Mendel et al. 1998). Consistent with this, administra-tion of an anti-IL-6 receptor blocking antibody can preventdisease development in wild type mice, if given at the timeof priming, but cannot reverse ongoing disease (Serada et al.2008). IL-6 signalling in recently activated naïve T cellsdrives RORγt expression, which in turn drives IL-17 pro-duction and expression of the IL-23 receptor, rendering thedeveloping effector cells sensitive to the pathogenic effectsof IL-23, as above. A molecular basis for the early require-ment for IL-6 has been provided by recent observations thata) all CD4+ T cells in the CNS of mice with EAE have lostexpression of the IL-6 receptor (O’Connor et al. 2012) andb) autoantigen responsive T cells in the peripheral lymphoidorgans lose the IL-6 receptor within 7 days of immunization(Leech et al. 2013). Thus, there is no long-term requirementfor IL-6 for pathogenic T cell function in EAE. By extrap-olation, this would suggest that the likelihood of IL-6 re-ceptor blockade becoming a viable therapeutic option in MSto be low. Only time will tell (we are not aware of anyongoing trial with IL-6 blockade in MS).

In addition to directing the immune response by se-cretion of cytokines, CD4+ T cells have also demonstrat-ed cytotoxic activity towards oligodendrocytes in vitro,and cytotoxicity in response to stimulation with myelinantigens is increased in MS patients, suggesting CD4+ Tcells themselves may also directly contribute to disrup-tion of myelin integrity (Antel et al. 1994; Zaguia et al.2013; Broux et al. 2012)

CD8 T cells & B cells in CNS autoimmune inflammation

Whilst the induction of EAE is known to require CD4+ Tcells (Ben-Nun et al. 1981; Zamvil et al. 1985; Waldor et al.1985), inflammatory infiltrates within the CNS are alsofound to contain other leukocytes that can migrate into theCNS once the BBB has been compromised, such as CD8+ Tcells (Sriram et al. 1982). Indeed, the presence of CD8+ Tcells within CNS lesions of MS patients is well documented(Traugott et al. 1983a; Hauser et al. 1986; Babbe et al.2000). Due to the cytotoxic activity of CD8+ T cells it isthought that these cells can contribute to CNS autoimmunepathology by directly killing oligodendrocytes. In addition,CD8+ T cells have proven also to be encephalitogenic in amodel of EAE that employs the use of a recombinant virus(Huseby et al. 2001), and this has also been reported fromadoptive transfer models involving the transfer of myelin-reactive CD8+ T cells (Sun et al. 2003; Ford and Evavold2005), although concerns remain that these populations mayhave contained sufficient contaminating CD4+ cells to ac-count for the pathology seen. In a separate study, the transferof highly pure CD8+ cells responsive to a class I epitope ofMOG failed to induce disease (Leech et al. 2011). There isalso evidence to suggest that CD8+ T cells may play more ofa regulatory than pathogenic role in CNS autoimmune in-flammation (Koh et al. 1992; Montero et al. 2004) (Table 1).

Another lymphocyte that can contribute to autoimmunepathology in EAE is the B cell, although their exact contri-bution to disease pathogenesis is also a controversial area.Potential functions for B cells in the pathogenesis of EAEinclude enhancement of antigen presentation to T cells,secretion of pro-inflammatory cytokines and, of course,the generation of autoantibodies (Table 1). The ability toinduce disease in B cell-deficient mice and under conditionsof B cell depletion indicates that these cells are not specif-ically required for disease onset (Lyons et al. 1999;Matsushita et al. 2008; Hjelmstrom et al. 1998; Wolf et al.1996; Fillatreau et al. 2002). However, the observation thatEAE can only be induced in B cell-deficient C57BL/6 miceusing the 35–55 peptide of MOG and not the rMOG proteinsuggests some role for these cells in disease pathogenesis. Inthis context, B cells do not appear to be required as APC inthe induction of EAE using whole protein, as lymphocytesfrom rMOG-immunized B cell deficient mice are capable ofresponding to rMOG in ex vivo recall assays (Lyons et al.1999). Through studies using MOG protein from differentspecies (Oliver et al. 2003), and the reconstitution ofdisease in rMOG-immunized B cell deficient mice bytransfer of serum from rMOG immunized WT mice(Lyons et al. 2002), it has been suggested that whenusing whole protein instead of an immunodominant en-cephalitogenic T cell epitope to induce EAE, the proteinused must contain both T cell and B cell determinants

and that pathogenic antibodies may contribute to diseasepathogenesis. However, it should be stressed that therequirement for B cells for EAE development only holdstrue when using human rMOG, immunization with the“true” autoantigen, mouse rMOG, induces EAE that fullyresembles that induced by the 35–55 peptide in B celldeficient H-2b mice (Fillatreau et al. 2002).

Direct evidence that antibodies produced by B cells canplay a detrimental role in the progression of autoimmuneinflammation of the CNS has been provided by the detectionof anti-MOG antibodies bound to disintegrating myelin in amarmoset model of EAE (Genain et al. 1999), and theamelioration of disease upon treatment with B cell depletingantibodies (Barr et al. 2012; Matsushita et al. 2008).Furthermore, infusion of anti-MOG antibodies has beenshown to enhance demyelination and increase disease se-verity in a T cell-driven model of EAE (Linington et al.1988). More in-depth analysis has revealed that depletion ofB cells early in disease results in a chronic disease course,whereas depletion at a later stage accelerates resolution(Matsushita et al. 2008), thus illustrating duality in thefunction of B cells in EAE. Similarly to CD8+ T cells, ithas been postulated that B cells may be more important inthe regulation rather than pathogenesis of EAE (Wolf et al.1996; Fillatreau et al. 2002; Fillatreau et al. 2008).

Protective lymphocytes in CNS inflammation

Although pathogenic T cells can mediate the induction ofCNS autoimmune inflammation, T regulatory (Treg) cellscan contribute to the resolution of disease (Table 1).Evidence of this stems from the observation that CD4+CD25+Foxp3+ natural Treg (nTreg) accumulate in theCNS during EAE, coinciding with recovery from disease,and depletion of CD25+ cells can inhibit that recovery(McGeachy et al. 2005). Mechanisms by which Treg canmediate resolution of inflammation include the secretion of

immunosuppressive cytokines such as IL-10 and TGF-β,metabolic disruption and modulation of APC function (Liuet al. 2003; Pandiyan et al. 2007; Deaglio et al. 2007; Lianget al. 2008). The accumulation of regulatory T cells withinthe CNS results from the activation and proliferation ofthese cells within the inflamed CNS following their recruit-ment from the periphery (O’Connor et al. 2007). This sug-gests that, as a consequence of ongoing inflammation in theCNS, Treg respond in situ to enable resolution of the in-flammatory response and the subsequent recovery fromdisease. The adoptive transfer of myelin-reactive nTreg hasalso been used successfully to inhibit the induction andaccelerate the recovery of EAE (Stephens et al. 2009).Furthermore, in vitro generated Foxp3+ induced Treg(iTreg) have also been shown to effectively inhibit theinduction of EAE, highlighting the potential use of thesecells in therapeutic applications (Selvaraj and Geiger 2008;Zhang et al. 2010; O’Connor et al. 2010).

In addition to CD4+ Treg cells, a potential function ofCD8+ T cells in the regulation of CNS inflammation hasalso been proposed. EAE induced in CD8-deficient micewas reported to show increased frequency of relapses (Kohet al. 1992), and in separate studies the presence of CD8+ Tcells protected against the induction of disease (Montero etal. 2004; Jiang et al. 1992). The mode of action of these so-called CD8+ Treg remains elusive, but they may secrete theimmunosuppressive cytokine TGF-β (Chen et al. 2009).The lack of specific markers to identify regulatory CD8+T cells means that the function of these cells in controllinginflammation of the CNS remains a contentious subject.

Although Treg are crucial in the regulation of autoim-mune inflammation, it is also clear that B cells can contrib-ute to the control of pathological immune responses. B celldeficient mice exhibited an exacerbated chronic form ofEAE (Wolf et al. 1996). Consistent with this, depletion ofB cells before the induction of EAE in wild type mice, usingan anti-CD20 antibody, can exacerbate established disease(Matsushita et al. 2008). Although B cells have been shown

Table 1 Key players in the immunopathogenesis of CNS inflammation

Cell Type Pathogenic action Protective action

CD4+ T cells Secretion of pro-inflammatory cytokines such as IFN-γ, IL-17,TNF-α and GM-CSF; disrupts the integrity of the BBB, allowingrecruitment of other cell types to the inflammatory lesion, andactivates macrophages. CD4+ T cells have also demonstratedcytotoxicity towards oligodendrocytes.

Treg are recruited to the site of inflammation and dampenimmune responses by the secretion of anti-inflammatorycytokines (e.g. IL-10, TGF-β), metabolic disruption(IL-2 deprivation, and expression of CD39 & CD79) andor modulation of APC function.

CD8+ T cells Cytotoxic activity, killing of oligodendrocytes Potentially through the secretion of the anti-inflammatorycytokine TGF-β

B cells Produce anti-myelin antibodies which can opsonise the myelinsheath and oligodendrocytes, promoting macrophage-mediateddestruction. B cells can also act as APC promoting T cellactivation.

Production of the anti-inflammatory cytokine IL-10, actsto limit expansion of pathogenic lymphocytes.

to produce IL-6 and therefore may also potentially aidpathogenic T cell priming (Lampropoulou et al. 2008; Barret al. 2012), activated B cells can produce large amounts ofIL-10 (Fillatreau et al. 2002; Mizoguchi et al. 2002; Mauri etal. 2003). It is this production of IL-10 which is thought tofacilitate the resolution of EAE (Fillatreau et al. 2002;Matsushita et al. 2010). However, this effect most probablyoccurs early, before clinical disease is established, by limit-ing the size of the pathogenic T cell cohort, which is thenmore amenable to Treg mediated control within the CNS(Hoehlig et al. 2012).

EAE as a preclinical model for MS

Although the pathogenesis of EAE mirrors many of the clin-ical features seen in MS, there remain discrepancies betweenthe two diseases, which can clearly have implications for thedevelopment of therapeutic interventions. Certain therapeuticagents effective in EAE have failed to translate into the clinic;the reasons for which may include the involvement of addi-tional cells types in disease pathology. MS has long beenconsidered a CD4+ T cell mediated disease due to the simi-larities in pathology with EAE, the presence of MBP-reactiveT cell clones in brains of MS patients and the genetic associ-ation with the MHC II allele HLA-DRB1*15 (Haines et al.1998; Barcellos et al. 2003; Allen et al. 1994). Whilst immu-nohistochemical analyses of lesions inMS and EAE reveal thepresence of both CD4+ and CD8+ T cells (Traugott et al.1983a, b), in MS lesions it is CD8+ T cells, rather thanCD4+ T cells, that predominate (Booss et al. 1983; Hauseret al. 1986; Bradl et al. 2005). In addition, CD8+ Tcells in MSlesions show oligoclonal expansion (Babbe et al. 2000), andCNS-reactive CD8+ T cells have been identified in MS pa-tients (Crawford et al. 2004). Furthermore, genetic associa-tions with MHC I alleles have also been identified (Rubio etal. 2002; Boon et al. 2001). These observations and reportsthat CD8+ T cells might be pathogenic in certain models ofEAE (Huseby et al. 2001; Sun et al. 2003; Ford and Evavold2005) suggest that specifically targeting only CD4+ T cellsmay not prove clinically efficacious, although the exact role ofCD8+ T cells in the pathogenesis or protection from diseaseremains unclear.

In addition, MS is a highly heterogeneous disease withvaried clinical symptoms, and therefore probably manydifferences in disease pathogenesis between individuals.As a generalization, an initial relapsing-remitting (RRMS)course later advances to a secondary progressive phase(SPMS), where cumulative neurodegeneration without re-mission are hallmark features (Venken et al. 2010; Spain etal. 2009). The paradigm has been that relapses in RRMS areassociated with more pronounced inflammation, whereasremission reflects a reduction in inflammation (Bruck

2005). Multiple relapses lead to an accumulation in neuro-logical damage and axonal loss, and once this reaches acritical level there is a change in the disease phenotype fromoverriding inflammation to predominant neurodegeneration,leading to SPMS (Trapp et al. 1999; Spain et al. 2009).However, evidence for the persistence of inflammation intoSPMS is clear (Serafini et al. 2004; Kutzelnigg et al. 2005;Frischer et al. 2009). In contrast, primary progressive (PP)MS is characterised by progressive axonal degeneration inthe absence of any overt inflammation, and therefore isprimarily thought of as a neurodegenerative disease(Matthews 2004; Trapp and Nave 2008).

Those mouse EAE models that involve relapsing-remitting, or chronic/relapsing disease (for example inSJL, or Biozzi/ABH mice) do not yet lend themselves toadvanced interrogation of pathogenic T cell developmentand function (TCR transgenic T cell transfer systems arenot well-advanced in these models). Those models that dohave TCR transgenics available tend to result in eitherchronic or monophasic, rather than relapsing, diseasecourses. However, new models are being developed, someof which involve the spontaneous development of pathology(Pollinger et al. 2009; Krishnamoorthy et al. 2009). Withthis range of models available, further cellular and molecu-lar studies in EAE will no doubt continue to provide newtherapeutic opportunities

The tried and the tested; therapeutic intervention in MS

Until relatively recently, the mainstay of MS treatment wasnon-specific immunomodulation. Steroids have long beenused to treat exacerbations (Then Bergh et al. 2006; Durelliet al. 1986). However, this has no beneficial effect on therate of relapses (Ciccone et al. 2008; Zivadinov et al. 2001).Nevertheless, continued study of the effects of steroids isvalid. For example, methylprednisolone has recently beenreported to reduce levels of Th1, Th2 and Th17 cytokines,as well as IL-6 and IL-23 in RRMS, whilst increasingpotentially immunosuppressive IL-10, TGF-b and IL-27(Muls et al. 2012). The subsequent advent of disease mod-ifying therapies (DMTs) has provided the potential benefitsof decreased relapse rates and inhibition disability progres-sion (Table 2). The array of therapeutic interventions thathas been tested in EAE for the potential use in the treatmentof MS is large, whilst the translation of these to the clinichas been low.

Modulation of T cell priming/function

Interferon-β therapeutics (Avonex®, Rebif®, Betaferon®,Extavia®) include both interferon-β-1a and interferon-β-1b, and have been reported to reduce the frequency of

relapses in RRMS (Paty and Li 1993; Jacobs et al. 1996;Kappos et al. 2007). The mode of action is not fully under-stood, but studies in EAE have suggested potential mecha-nisms including the inhibition of antigen presentation, T cellproliferation, and effector cytokine production (Markowitz2007; Noronha et al. 1993; Barna et al. 1989) as well as anincrease in production of anti-inflammatory cytokines suchas IL-10, and TGF-β (Yasuda et al. 1999). IFN-β mostlikely affects cells of the myeloid lineage, rather than havedirect effects on adaptive immune cells (Brendecke andPrinz 2012). EAE is to a large extent dependent on afunctional NLRP3 inflammasome. Mice that lack this havea milder form of disease, but this cannot be reduced byadministration of IFN-β. This provides a molecular basisfor the action of IFN-β via inhibition of the NLRP3 signal-ling pathway in myeloid cells (Inoue et al. 2012).

Despite these beneficial effects, flu-like side-effects arecommon and not all patients respond to this treatment (Rioet al. 2006; Horakova et al. 2012). Response to IFN-βtherapy may be dependent upon the phenotype of disease,with a study describing IFN-β therapy as beneficial in Th1-mediated EAE but not in Th17-biased EAE (Axtell et al.2010). Whether this observation will truly translate to allowthe development of a predictive biomarker for IFN-β re-sponsiveness, based on the quality of the autoaggressive Tcell response remains uncertain.

Glatiramer acetate (GA)(Copaxone®) is a synthetic aminoacid polymer containing the four most common amino acidsfound in MBP. Although this was designed as an MBP “mim-ic”, it shares no defined sequence homology with theautoantigen and so it remains something of a puzzle as towhy this compound was able to suppress EAE. Despite thisobservation being made over four decades ago (Teitelbaum etal. 1971), again mode of action is not absolutely defined.Nevertheless, GA is widely used in RRMS and is reportedto reduce the relapse rate and delay the onset of disability(Johnson et al. 1995; Simpson et al. 2002). GAwas reported toswitch off T cell responses to the MBP (82–100) epitope(Aharoni et al. 1999) and to induce changes in T cell

phenotype from Th1/Th17 to Th2/Th3/Treg (Aharoni et al.1997; Begum-Haque et al. 2008). The main mode of action is,however, thought to be non-specific, acting through effects onAPC rather than T cells (Weber et al. 2004, Vieira et al. 2003).Despite the effectiveness of GA in treating EAE, its effective-ness in MS is a matter of debate. A Cochrane review haspreviously stated no beneficial effect compared to placebo(Munari et al. 2004; Munari and Filippini 2004; Comi et al.2005)(Table 3), despite several trials comparing the effective-ness of GA to IFN-β therapeutics in RR-MS that demonstrat-ed no differences (Flechter et al. 2002; Mikol et al. 2008;O’Connor et al. 2009).

Mitoxantrone (Novantrone®), an immunosuppressive cy-totoxic drug that functions to inhibit the proliferation of Tand B cells (Fidler et al. 1986; Wang et al. 1986), is used asa second line therapy to treat aggressive forms of MS thatare refractory to treatment with interferon-β and GA.Treatment with mitoxantrone can decrease the number ofdemyelinating MS lesions and inhibit the progression ofdisability (Edan et al. 1997; Hartung et al. 2002), but long-lasting immunosuppression is a concern.

BG-12 (dimethyl fumarate), a fumaric acid ester reportedto induce apoptosis of T cells and promote Th2 cytokineproduction (Treumer et al. 2003; de Jong et al. 1996;Ghoreschi et al. 2011), seems close to approval as an oraldrug for MS having performed well in a phase 3 clinicaltrial; reportedly reducing relapse rate, number of lesions onMRI and the progression of disability (Gold et al. 2012).

Modulating lymphocyte trafficking

Natalizumab (Tysabri®) is the first monoclonal antibody to belicenced for use in MS, and was developed in EAE (Yednocket al. 1992; Steinman 2005). Natalizumab blocks the adhesionmolecule α-4 integrin (a component of VLA-4), inhibiting thebinding of lymphocytes to endothelial cell walls via VCAM-1and therefore blocking the progress of these cells across theBBB into the CNS (Hutchinson 2007; Rice et al. 2005). Inclinical trials natalizumab, has been shown to reduce the

Table 2 Current therapies used in the treatment in MS; mechanisms & disadvantages

Current therapeutics Mechanism Disadvantages

Interferon-β Potential inhibition of antigen presentation, T cell proliferationand pro-inflammatory cytokine production. Also blocks theNLRP3 inflammasome.

Non-specific immunosuppression

Glatiramer actetate Potential immune deviation from pro-inflammatory CD4+ T cellsresponses to regulatory

Non-specific immunosuppression

Mitoxantrone Inhibits proliferation of T & B cells Non-specific immunosuppression and cardiacadverse effects

Natalizumab Blockade of α-4 integrin; inhibiting lymphocyte entry into the CNS Non-specific effects and prevents normalimmune surveillance of the CNS

Fingolimod Inhibits the egress of lymphocytes from lymph nodes, thereforepreventing trafficking to the CNS

Non-specific effects and inhibits normalimmune surveillance of the CNS

relapse rate and the development of gadolinium-enhancinglesions in RRMS (Polman et al. 2006; Rudick et al. 2006).The drug was briefly withdrawn from the market followingdeaths of several patients who developed PML due toreactivation of JC virus (Kleinschmidt-DeMasters and Tyler2005; Langer-Gould et al. 2005). This highlighted the impor-tance of maintaining normal immune surveillance in the CNSduring therapeutic intervention. Although natalizumab is nowused widely, JC virus is a relatively common infection(Taguchi et al. 1982), and therefore the development of PMLremains a risk. Serology allows patients to be tested forPML prior to use of natalizumab, as well as continuedmonitoring for sero-conversion. The rebound of clinicaldisease following withdrawal of the drug highlights thatnatalizumab does not tackle the underlying autoaggressiveimmune response. It also shows that this response is notentirely self-contained and perpetuated within the CNSitself. Rather, continued repopulation (with as yet,undefined) immune cells from the periphery seems neces-sary to maintain the disease. Recent studies in EAE havealso reported that, although blockade of α-4 integrin caneffectively prevent the entry of autoaggressive Th1 cellsinto the spinal cord, it does not inhibit Th17 cell entry intothe brain parenchyma (Rothhammer et al. 2011) (Table 3).Instead, this was dependent of LFA-1/ICAM-1 interactions.This echoes the reported differential sensitivity of these two Tcell types to IFN-β treatment as described above, and againsuggests that immune profiling might be able to identify thosepatients most likely to respond to therapy.

Figolimod (Gilenya®) was recently approved as an oraltherapy for MS. Fingolimod affects immune cell traffickingupstream of the effects of natalizumab, by inhibiting theegress of lymphocytes from lymph nodes by blocking thesphingosine 1-phosphate receptor, S1P1 (Brinkmann et al.2010). This was shown to be efficacious in both the preven-tion and treatment of EAE (Fujino et al. 2003; Webb et al.2004). Despite demonstrating clinical efficacy in MS(Kappos et al. 2010), a formal review of fingolimod hasbeen conducted recently due to the occurrence of severalsudden or unexplained fatalities (Lindsey et al. 2012).

Lost in translation

Despite the obvious beneficial uses of EAE in the develop-ment of therapeutics for MS, some approaches that provedeffective at inhibiting EAE have been ineffective or evendetrimental in MS (Table 3). As EAE is undoubtedly aCD4+ mediated disease, it was not surprising that depletionof CD4+ T cells proved beneficial in preventing and ame-liorating disease in this model (Waldor et al. 1985; Sriramand Roberts 1986). However, translation of anti-CD4 de-pleting antibodies into MS showed minimal effects (vanOosten et al. 1997), whereas subsequent studies using anti-CD52 (Campath-1H), a monoclonal antibody that depletesall lymphocytes, demonstrated a reduction in MRI markersof disease and accumulation of disability (Moreau et al.1994; Coles et al. 2008). This highlights the potential im-portance of CD8+ T cells and/or B cells in the pathogenesis

Table 3 Comparison of the tried and tested therapeutics in EAE and MS

Mode of action Therapeutic agent Effective in EAE? Effective in MS?

Modulation of T cell function Interferon-β, Glatirameracetate

IFN-β ameliorates Th1 drivenEAE but exacerbates Th17-driven EAE. Glatiramer acetateprotects against the inductionof EAE

Reported reduction in relapse rateand delay in onset of disabilityin RR-MS, but efficacy is currentlyunder debate.

Inhibition of T cell trafficking Fingolimod, Natalizumab Blockade of α-4 integrin inhibitsentry of Th1 cells into the CNSbut not Th17 cells. Fingolimodis effective at both inhibiting andtreating EAE.

Reduction in relapse rate and therisk of disability progression inRR-MS, but complications existdue to the inhibition of immunesurveillance in the CNS

Depletion of immune cells Monoclonal anti-CD4 antibody,Campath-1H, Rixtuximab

CD4+ T cell depletion is effectiveat preventing and treating EAE.Campath-1H was not tested inEAE. Whereas, Rituximab doesnot prevent the induction of EAEbut has been shown to ameliorateongoing EAE (Matsushita et al.2008; Barr et al. 2012)

No benefit of CD4+ T cell depletionin MS, but reduction in diseaseactivity and accumulation ofdisability in RR-MS after depletionof all lymphocytes (Campath-1H).Campath-1H has an associated riskof secondary autoimmune disease(Jones et al. 2009; Cossburn et al. 2011).Rituximab has been shown to reducerelapse rates in RR-MS (Bar-Or etal. 2008; Hauser et al. 2008)

Manipulation ofinflammatory cytokines

anti-p40, anti-TNF-α Both anti-p40 and anti-TNF-α areeffective at inhibiting EAE

anti-p40 had no effect in MS and anti-TNF-α exacerbated disease.

of MS compared to EAE. Indeed, both Campath-1H(Alemtuzumab®) and the B cell-depleting anti-CD20 anti-body Rituximab (Rituxan®) are currently in clinical trialsfor the treatment of MS. Campath-1H was not tested inEAE, since mice do not express CD52, the human targetof the antibody.

Manipulation of pro-inflammatory cytokines thought tobe important in disease pathogenesis or regulation has againproved effective in EAE but not in MS. Several studies havedemonstrated a potential regulatory role for IFN-γ, wherebyblockade of IFN-γ has been shown to exacerbate EAE, andconcurrently administration of IFN-γ can suppress EAE(Billiau et al. 1988; Duong et al. 1992; Voorthuis et al.1990). Furthermore, it has been observed that treatment withIFN-β can induce IFN-γ production by CD4+ T cells undernon-polarising conditions, and that the production of IL-10by these cells requires the presence of IFN-γ (Axtell et al.2010). Despite these findings, a clinical trial involving theadministration of IFN-γ in MS patients in order to test itstoxicity induced relapses and exacerbated disease (Panitchet al. 1987), which may be somewhat unsurprising given thewell-defined pro-inflammatory nature of this cytokine.

Although studies antagonising the action of pro-inflammatory cytokines demonstrated that blockade ofTNF-α, or the p40 subunit of both IL-12 and IL-23 inhibitsEAE (Selmaj et al. 1991; Baker et al. 1994; Leonard et al.1995), in clinical trials anti-TNF-α exacerbated MS andanti-p40 had no effect on disease (van Oosten et al. 1996;The Lenercept Multiple Sclerosis Study Group 1999;Leonard et al. 1995; Segal et al. 2008). Therefore, a paradoxexists as to why blockade of or treatment with cytokines caninhibit EAE but have no effect or even exacerbate MS.Whether the failure of anti-p40 reflected a poor ability ofthe antibody to cross the BBB remains uncertain, but otherbiologic options are beginning to appear. Antibodies thatonly target IL-23 are now available and anti-IL-17 is undertest in MS and clinically isolated syndrome. The discoverythat GM-CSF is a critical cytokine in the development ofEAE, (discussed above) suggests that targeting GM-CSF,which has already proven effective at inhibiting EAE (El-Behi et al. 2011; McQualter et al. 2001) is a worthwhilestrategy to test.

Smarter therapeutics for tomorrow?

As with all therapies that systemically target even individualcomponents of the immune response, the current therapeuticregimes for MS remain unsatisfactory from the immunolo-gist’s perspective. Insufficient immune surveillance canleave the patient susceptible to (sometimes life threatening)infections and the risk of neoplasia is a long-term concern.As such, there remains a pressing need to develop methods

of specifically targeting the discrete pathologic processes atplay, whilst leaving normal/desired immune function intact.Is this Holy Grail within sight?

The discovery of Treg and their ability to suppress inflam-matory responses in the CNS prompts the investigation intothe use of these cells in the treatment of MS. Indeed, adoptivetransfer of either nTreg or iTreg have proven to be potentinhibitors of EAE (Selvaraj and Geiger 2008; Stephens et al.2009; O’Connor et al. 2010). Furthermore, it has been dem-onstrated that antigen-reactive Treg are more effective atinhibiting immune responses than polyclonal Treg (Tarbellet al. 2004). This inherent characteristic provides a mechanismthat allows trafficking of the Treg to the site of inflammationas determined by the target antigen, and therefore negates thepotential for non-specific immunosuppression. Despite thebeneficial effects of Treg, concerns have been raised over theirtherapeutic use due to their ability to lose Foxp3 expression &produce pro-inflammatory cytokines under certain conditions(Xu et al. 2007; Yang et al. 2008). However, a study byO’Connor et al., has demonstrated that although in vitrogenerated Treg can lose Foxp3 expression and produceIFN-γ, these cells are only weakly pathogenic and retain theirsuppressive capacity (O’Connor et al. 2010). Together, thesestudies highlight the importance for further scrutiny of Tregand how best to exploit their suppressive function therapeuti-cally without contributing to pathogenicity. The benefits ofusing therapeutic Treg administration in MS seem clear con-sidering evidence that suggests Treg are functionally impairedin MS patients (Viglietta et al. 2004; Venken et al. 2008). Thisdeficiency in Treg function implies an inability to controlinflammation and could explain why multiple relapses occurin MS, therefore, the administration of Treg could help re-establish homeostasis. At present, we are unaware of any trialof Treg therapy in MS. However, trials of this approach havecommenced in graft-versus-host-disease, kidney transplanta-tion and type I diabetes.

An obvious means of targeting only those autoantigen-responsive T cells that are driving pathology is the use theautoantigen. Induction of immune tolerance using antigen-based therapy is well established in experimental models ofautoimmune and allergic disease (Larche and Wraith 2005;Hochweller et al. 2006). Such approaches have been devel-oped in EAE using peptide antigens (Gaur et al. 1992;Critchfield et al. 1994; Liu andWraith 1995) and are currentlyin clinical trials for MS (Wraith 2009). The administration oftarget immunodominant peptides in a tolerogenic form haslong been known to induce tolerance in antigen-reactive Tcells (Gaur et al. 1992; Hoyne et al. 1993). This can occurvia deletion, the induction of anergy or regulation of theautoreactive Tcells (Miller et al. 2007; Hochweller et al. 2006).

In mice, antigen-based therapies are straightforward because,most often, the disease is induced by active immunization andso we know the T cell epitopes recognized by the pathogenic T

cells. Although a series of epitopes recognized within humanMBP, PLP and MOG that are recognized by T cells from MSpatients have been identified (Burns et al. 1983; Zhang et al.1994; Ota et al. 1990; Sun et al. 1991), it is still uncertain whichare really driving disease. Also, it seems likely that these mightvary between patients, even those sharing MHC alleles. Thus,the current peptide-based approaches are using combinations ofmultiple peptides. An alternative for the future will be to tailorthe antigenic peptides used according to the HLA type andimmune response of the individual. The need to understand infine detail all the autoantigenic epitopes recognized by patho-genic T cells might not be necessary, if we can find a robustmeans of instating dominant immunological tolerance. That is,the induction of a regulatory function within a discrete antigen-responsive population, that is then capable of counteracting thepathogenic effects of other T cells. Proof of principle for theinduction of bystander suppression using individual myelinpeptides was provided in EAE some 15 years ago (AndertonandWraith 1998). This may not require the resulting regulatorycells to express Foxp3, but IL-10 production does seem to be akey requirement (Burkhart et al. 1999; Sundstedt et al. 1997;Nicolson et al. 2006; Sundstedt et al. 2003), at least in somemodels. That said, the in vivo induction/expansion of antigen-responsive Foxp3 cells following antigen administration hasbeen reported in other systems (Kretschmer et al. 2006;Tarbell et al. 2004). Reliably harnessing the undoubted powersof regulatory Tcells (whatever their precise nature) represents anongoing, but extremely exciting challenge.

Acknowledgments Work in the authors’ laboratory is supported bygrants from the UK Medical Research Council and the Wellcome Trust.

Conflict of Interest The authors have no financial conflicts of inter-est to declare.

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