Medical Hypotheses 82 (2014) 300–309

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Medical Hypotheses

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Intrathecal immune reset in multiple sclerosis: Exploring a new concept

0306-9877/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mehy.2013.12.015

Abbreviations: Ab, antibody; AI, antibody index; AICD, activation-inducedcytidine deaminase; ASC, antibody secreting cells; BBB, blood–brain barrier; CDR,complement determining region; CIS, clinically isolated syndrome; CNS, centralnervous system; CSF, cerebrospinal fluid; EDSS, expanded disability score; HSCT,hematopoietic stem cell transplantation; Ig, immunoglobulins; MRZ pattern,intrathecal reaction against measles rubella and zoster viruses; OCB, oligoclonalbands; PML, progressive multifocal leukoencephalopathy; PP, primary progressiveMS; rAb, recombinant antibody; RR, relapsing–remitting MS; SP, secondaryprogressive MS; TLO, tertiary lymphoid organ.⇑ Tel.: +33 (0) 559924848.

E-mail address: mickael_bonnan@yahoo.fr

Mickael Bonnan ⇑Service de Neurologie, Hôpital F. Mitterrand, 4 bd Hauterive, 64046 Pau, France

a r t i c l e i n f o

Article history:Received 8 October 2013Accepted 19 December 2013

a b s t r a c t

Multiple sclerosis impairment is mainly driven by the progressive phase, whose pathology remains elu-sive. No drug has yet been able to halt this phase so therapeutic management remains challenging. It wasrecently demonstrated that late disability correlates with the spreading of cortical subpial lesions, andtertiary lymphoid organs (TLO) were identified in close apposition with these lesions. TLO are of crucialimportance since they are able to mount a complete local immune response, as observed in the intrathe-cal compartment from the moment MS is diagnosed (i.e. oligoclonal bands). This article examines theconsequences of this intrathecal response: giving a worst clinical prognostic value and bearing argumentsfor possible direct brain toxicity, intrathecal secretion should be targeted by drugs abating both B-lym-phocytes and plasma cells. Another consequence is that intrathecal secretion has value as a surrogatemarker of the persistence of an ongoing intrathecal immune reaction after treatment. Although it is stillunsure which mechanism or byproduct secreted by TLO triggers cortical lesions, we propose to target TLOcomponents as a new therapeutic avenue in progressive MS.

Whereas it was long considered that the inability of therapies to penetrate the blood–brain-barrier wasa crucial obstacle, our proposed strategy will take advantage of the properties of the BBB to safely resetthe intrathecal immune system in order to halt the slow axonal burning underlying secondary MS. Wereview the literature in support of the rationale for treating MS with intrathecal drugs dedicated to clear-ing the local immune response. Since many targets are involved, achieving this goal may require a com-bination of monoclonal antibodies targeting each cell sub-type. Hope might be rekindled with a one-shotintrathecal multi-drug treatment in progressive MS.

� 2013 Elsevier Ltd. All rights reserved.

Introduction

Multiple sclerosis (MS) is the most frequent chronic inflamma-tory and demyelinating disorder of the central nervous system(CNS) in young adults and remains the second cause of disabilityin young people. Although most patients during the early phaseof the disease suffer from a relapsing–remitting form of MS (RR-MS) characterized by acute relapses usually followed by a com-plete remission, the majority will develop a secondary progressiveform (SP-MS). The impairment is mostly independent from the ini-tial RR phase and is mainly driven by the late SP phase. Althoughtreatments directed against the RR phase may have a slight pre-

ventive effect on the SP phase, none of them has unfortunatelybeen shown to halt the ongoing secondary phase. Considering thatmost of the debilitating burden is driven by the progressive phase,building-up a therapeutic strategy dedicated to this phase remainsa challenging goal.

Even if the exact pathophysiology of SP-MS remains to be com-pletely clarified, the hallmark of this phase is the restriction of theimmune response down to the intrathecal compartment, leading toprogressive extensive cortical lesions and clinical impairment [1].Interestingly, this partition occurs so early in the disorder thatIgG intrathecal synthesis and oligoclonal bands are even part ofthe diagnostic criteria.

We briefly review the main actors in this intrathecal responseleading to slow axonal burning. Then we hypothesize the explora-tion of a new potential therapeutic avenue in progressive MS. Inother words, whereas it has long been considered that the inabilityof therapies to penetrate the blood–brain-barrier (BBB) is a crucialobstacle, we propose to take advantage of this to safely reset theintrathecal immune system in order to halt the slow axonal burn-ing underlying secondary MS. We review the literature in supportof the rationale for treating MS with intrathecal drugs dedicated tosafely clearing the local immune response.

M. Bonnan / Medical Hypotheses 82 (2014) 300–309 301

Drugs for relapsing–remitting (RR) phase fail to preventatrophy and conversion to SP-MS: treatment of progressive MSis required

All the available treatments are directed against the inflamma-tory component of the RR phase but they fail to actively delay orprevent the onset of SP-MS, to cure it, or to prevent any kind ofsteady impairment. Furthermore, in primary progressive MS (PP-MS), no treatment has ever proved to be efficient [2]. Another clueto the limited efficacy of drugs in the early phase is the brain atro-phy rate, which is a key point when considering the dynamic ofimpairment. This rate remains essentially constant and highthroughout the course of MS, from clinically isolated syndrome(CIS) to PP-MS [3,4]. Interestingly, even in the RR-MS phase treatedby the most active treatments for preventing relapses, i.e. ale-mtuzumab or autologous stem cell transplantation, the brain atro-phy rate decreases but always fails to normalize and remains high[5–8].

Spreading of subpial cortical lesions drives the late disability

Although demyelination of the cortex and deep gray matter nu-clei has long been known, the extent of cortical demyelination re-mained grossly underestimated until recent immunohistochemicalmethods demonstrating their presence in the very early stages ofthe disease [9]. Lesion burden increases with time to become moreprominent than white matter lesions in the secondary phase (re-view in [10,11], and the cortical lesion load may even be a keyevent in the transition from RR-MS to SP-MS [12,13]. Cortical piallesions (type III) extend from the pial surface to the superficial cor-tical layers [14] and represent approximately half of the cortical le-sions [15], affecting 60% of the cortical ribbon of the brain,cerebellum and hippocampus [16–18]. They harbor distinct fea-tures: constant depth of demyelination waning at cortical layer 5[15] and a large extension over the multiple gyri [15]. Cortical le-sions are different from white matter lesions. They are devoid ofinflammatory cells and macrophages [15,19], have sparse deposi-tion of complement and immunoglobulins (Ig) (in [20]) and lackdetectable serum-derived proteins, suggesting that an immune re-sponse underlying the cortical pathology occurs in the meninges[11,21]. Regional gray matter demyelination and atrophy are notdriven by underlying white matter lesion load [22,23]. ProgressiveMS is associated with cortical demyelination and diffuse normalappearing white matter injury, which invariably occurs on a back-ground of meningeal, perivascular and parenchymal inflammation[16,24].

Cortical inflammatory lesions are highly correlated with corticalatrophy and disability within each MS subgroup [25,26]. In a largefive-year follow-up study, change in cortical matter fraction, newcortical lesions and clinical impairment (expanded disability score,EDSS) were highly correlated [25]. Age at onset of wheelchair useand death are correlated with extent of grey matter damage [10].On the other hand, benign MS is characterized by an initially lowcortical lesion charge (of about a third in RR-MS patients) and sig-nificantly lower new cortical lesions [25], whereas the number ofshadow plaques in the white matter is not different in benign MS[10]. In a long-term cohort, benign MS demonstrated higher nor-mal gray matter volume than non-benign MS that were close tothose of controls [26]. On the contrary, patients with a high levelof cortical lesions at baseline showed greater progression of bothclinical and grey matter atrophy at 5 years [25]. Several studieshave found cognitive skills and motor impairment to be correlatedwith cortical atrophy [26–28]. Moreover, cortical pathologyevolves at similar rates in all MS subtypes, with a higher baselinecortical lesion load in SP-MS due to the longer disease duration

[25]. In conclusion, whereas the cortex is mostly spared in benignMS, a high load of subpial cortical lesions drives the brain atrophyand the clinical burden in non-benign MS.

Intrathecal synthesis is robust over time and treatments

Intrathecal synthesis occurs as a very early disease event andthe proportion of patients with OCB tends to increase over time[29]. In longitudinal CSF studies, OCB pattern is robust and OCBhave never been seen to go away with time [30] although changesin band intensity and acquisition of new bands [31] may occur.Regarding the clonal repertoire of CSF Ig, clonal rearrangementsare conserved over time and a higher number of clones is foundin patients with the longest disease duration, suggesting a contin-uous clonal expansion over time [32,33]. Antibody index [34–36]and peptidic targets of the OCB IgG are constant over time [31].

Each patient has a unique pattern (‘OCB fingerprint’) of CSF OCB[37,38] that is resistant to high-dosage steroid infusions [30,39,40].Even if steroids transiently decrease the IgG index in most but notall patients, the decrease in range of CSF IgG synthesis is low andthe CSF total protein concentration remains unaffected [39].Weekly intramuscular or intrathecal b-IFN [37,41], azathioprine[42], natalizumab [43], rituximab [44–46] or daclizumab [47] haveessentially no effect upon intrathecal secretion.

In conclusion, intrathecal secretion and OCB pattern are early-occurring events in the course of MS, which, once acquired, persistessentially unchanged throughout life, whatever the various ther-apies available, and then remain stable or gradually worsen overtime.

Intrathecal synthesis confers a worse prognostic value

Demonstration of CSF OCB at the index event is a highly inde-pendent predictor of clinical recurrence [48–51], especially ifOCB target lipid antigens [52]. The presence of an MRZ pattern(intrathecal reaction against Measles, Rubella and Zoster viruses)in CIS predicts progression to definite MS [53]. The number ofOCB and the IgM index are thought to positively correlate withthe course of the disease [54,55]. Mean EDSS is higher in patientswith OCB, especially in the event of IgM OCB [56,57], and EDSS cor-relates with IgG1 index and CSF free light chains [58,59].

In numerous studies, CSF IgM have been associated with apoorer clinical long-term outcome [57,60–63], a lower brain vol-ume [64], and a decrease in brain parenchyma fraction over time[65].

Absence of intrathecal secretion in some patients reflectstechnical limitations but not absence of intrathecalinflammation

About 3% of MS patients in cohorts lack intrathecal IgG synthe-sis [35]. This might just be due to the low sensitivity of test sinceMRZ reaction, high CSF IgA synthesis, high IgA index [66,67], oli-goclonal free j light-chains [68], clonal VH and complement deter-mining region (CDR) rearrangements [69,70] are observed,suggesting that OCB tests are insufficiently sensitive in OCB-nega-tive patients. Moreover, OCB negativity at baseline tends to be-come positive for half of patients in whom lumbar puncture isrepeated [71]. Cumulated data suggest that MS patients apparentlydevoid of intrathecal secretion may in fact have a milder secretionbelow the sensitivity threshold of the common tests. However, thissmoldering intrathecal reaction may have clinical consequencessince many studies have confirmed that OCB-negative patientsare more prone to have lower EDSS, a benign form, and a delayedand lower risk of impairment milestones [54,71–76].

302 M. Bonnan / Medical Hypotheses 82 (2014) 300–309

Intrathecally secreted Ig target the brain

CSF Ig constitute a private repertoire, which may harbor toxicproperties [77]. Patterns of demyelinating lesions with Ig and com-plement deposition in and around macrophages have been de-scribed in brain fragments mainly obtained from biopsies, inwhich fixation is immediate [78–80]. Application of CSF to culturedcells (rat cerebellar granule neurons) significantly labels the axonalsurface with IgM dots [64] and purified antibodies against MOGfrom MS serum bind to intact myelin in rat [81]. Various recombi-nant antibodies (rAb) synthesized from CD138+ CSF B-cells of MSpatients stain most of the glia components [82,83] and many spe-cific antibodies have been shown to react against cultured oligo-dendrocytes or human CNS tissue (in [84]). In animal models,anti-MOG antibodies purified from human MS serum strongly en-hanced lesions without increasing inflammation [81,85]. In a mod-el of focal demyelination using implantation of various hybridomacells in rat spinal cord, demyelination strongly depended on thetarget of the hybridoma complement and involved [86]. The latterexperiment is reminiscent of the infiltration by plasma cells insideold burned-out demyelinating plaques [87]. Functional electro-physiological modifications have also been observed in olderexperiments [88,89]. In conclusion, the hypothesis that CSF Igcould play an active role in brain dysfunction, both at functionaland anatomical levels, is largely justified.

Somatic hypermutations and VH bias suggest local antigen-driven maturation

The IgG subclass IgG1 is significantly elevated in most MS CSFalthough some authors recovered rare higher IgG2 or IgG3 indices[90,91]. Over-representation of a single VH type family with heavychain sequences in MS plaques, periplaque white matter and CSF,but not blood, has been demonstrated [92], mostly with VH4 andVH2. This bias precedes the onset of OCB and is typical of MS, con-trary to other CNS disorders [92]. A robust demonstration of clonalexpansion of a single ancestor gene was made by the analysis ofthe clonal diversification from an ancestor gene accumulating sub-stitutions [93], a diversification mostly confined to the intrathecalcompartment [94]. The main targets of somatic hypermutations inCDR are within RGYW/WRCY motifs [70], which are targeted bythe activation-induced cytidine deaminase (AICD) specifically ex-pressed in lymphoid organs.

In summary, B-cells in CSF provide the cardinal features of anantigen-driven humoral immune response: clonal expansion andsomatically hypermutated VH family sequences [92], suggestingthat these lineage cells have been expanded by antigen and haveundergone a germinal center reaction [95].

CSF is a fostering milieu immortalizing inflammation

The CSF is bathed by many cytokines involved in the traffic andsurvival of inflammatory cells (e.g. BAFF, CXCL13), so a self-sus-tained intrathecal inflammation is fostered. BAFF is a key chemo-kine for the maturation and survival of B cells and isconstitutively produced in the CNS by astrocytes [95]. CSF BAFFdosage was higher in a subset of patients with P6 OCB [96]. Oneof the receptors of BAFF is BCMA, which enhances the long-termsurvival of plasma cells. Up-regulation of BAFF expression in activeand inactive MS lesions, where BAFF reaches levels of concentra-tion similar to those in lymphatic tissue [95], may provide a foster-ing environment to long-lived B-cells [97].

The chemokine CXCL13, which is mostly secreted by folliculardendritic cells [95], is a key regulator of B-cell recruitment and isselectively a chemoattractant for B lymphocytes and B-helper T

cells via its exclusive receptor CXCR5 [98], which is expressed in20–30% of blood and CSF CD4+ T cells (45) and by virtually allthe CSF B cells [45]. CXCL13�/� mice fail to develop lymph nodesand CXCL13 is essential for establishing and maintaining the lym-phoid tissue architecture. Likewise, the homing of B-cells to lymphnodes is CXCR5-dependent (in [99]). Immunostaining of CXCL13revealed this cytokine in lymphoid follicles of the meninges,[100] in blood vessels in chronic active lesions [101] and in theendothelium of primary CNS lymphomas [95]. Patients with OCBhad high levels of CXCL13 [98]. The rare CIS cases associating highCXCL13 levels without OCB should be compared with the findingthat CXCL13 secretion in CSF anticipates the OCB in neuroborrelio-sis [102]. The mean level is higher if the MRZ pattern is present,demonstrating an association of CXCL13 with a polyspecific intra-thecal B-cell response in MS. Moreover, CXCL13 levels strongly cor-related with both CSF plasma cells count, B cell count [103] and IgGindex [98]. Steroids and natalizumab also affect CSF CXCL13 levels[103].

Meningeal tertiary lymphoid organs (TLO) are identified in allMS subtypes

Meningeal CD20+ are elevated in all MS subtypes but Ig+ plas-ma cells are higher in PP-MS and SP-MS patients [104]. Axonaldamage (APP) correlates well with each parameter of inflamma-tion: B cells, T cells, plasma cell, HLA-D macrophages or microglia[104]. Neurite loss correlates with meningeal infiltration B and Tcells, and neuronal loss predominates in the superficial layers[105]. In SP-MS and PP-MS series, 30–41% had meningeal aggre-gates of inflammatory cells reminiscent of tertiary lymphoid or-gans [20,105]. All these aggregates were ectopic follicles withgerminal centers based on the presence of a reticulum of CD35+and CXCL13+ stromal/dendritic follicular cells, proliferatingCD20+/Ki67+ B cells, Ig + plasma/plasmablast cells and CD138+plasma cells [20]. However, a fundamental difference in structurewith lymphoid follicles was the lack of mantle zone, the presenceof CD27+ memory B cells, and infiltration by numerous CD4+ andCD8+ T lymphocytes [106]. Plasma cell distribution is variable:either diffuse around most parenchymal and meningeal blood ves-sels in one patient [100] or scattered inside the parenchyma ofchronic inactive lesions [100]. CXCL13 immunoreactivity is con-fined to dendritiform cells inside intra-meningeal B-cell follicles[100]. Proliferating cells in B-cell follicles, mostly CD20+ cells, areobserved at rates varying from 0% to 43% [100]. CCL21 and adhe-sion molecule peripheral node addressin (PNAd), which selectivelybinds to naïve T and B lymphocytes and allows their homing tosecondary lymphoid organs, are absent [100], suggesting that thehoming is dependent on various markers specific to brain tissues(see [107]).

Spatial correlation of meningeal TLO and cortical lesions

TLO in patients are associated with the following: a 6-fold moreintense subpial demyelination [20,21]; a lower density of neuritesboth in normal appearing gray matter and in type III lesions [20]; ahigher degree of atrophy and cell loss in the superficial cortex (upto 65%); a profound loss of superficial neurons, astrocytes and oli-godendrocytes (up to 70%); and a large increase in microglia num-bers in superficial layers (up to 70%) with diffuse microglialactivation in the presence of TLO [106]. A topographical and quan-titative relationship is frequently observed between parenchymalcortical macrophages–microglia and meningeal infiltrates[14,19,21]. Moreover, a study revealed a 90% probability that atleast moderate meningeal inflammation was topographically asso-ciated with subpial lesions [9]. Examination of acute subpial le-

M. Bonnan / Medical Hypotheses 82 (2014) 300–309 303

sions confirmed a destructive inflammation with ongoing neuro-degeneration and loss of oligodendrocytes [9].

TLO identification and count are underestimated owing totechnical limitations

Since brain TLO have recently been described, stringent univer-sal criteria are lacking, which may explain their wide variation infrequency. Moreover, TLO are minute structures in low numbersand are highly likely be missed by the sampling process (i.e. 8blocks per patient with a mean of 6 ± 3 follicles in the positiveblock sections) [20]. Considering the focal nature of tracked men-ingeal lesions and the usually low sample number, negative resultsshould not be considered as definite proof of the absence of men-ingeal lesions in these patients, since a lower number of TLO mayexplain the sample negativity. Lastly, the convexity of the gyrusmay undergo a mechanical abrasion of the meningeal surface dur-ing the fixation procedure, which may explain the predominance ofTLO in the deep sulci [9]. B-cell aggregates are always adjacent tosubpial lesions but the contrary is not true [21,106], which maysuggest that B-cell aggregates located in different planes may havebeen missed. A definitive ascertainment of TLO in an autopsiedbrain would require the reinforcement of sampling density to besure of the stereological relationship between cortical lesions andTLO.

TLO correlates with a worse clinical follow-up

Clues for a correlation between meningeal infiltration andshorter mean clinical duration were given in an old series [87].The proportion of ectopic follicles decreases with the age of pro-gression onset [20]. Both in SP-MS and PP-MS patients, age at clin-ical onset, age to be wheelchair-bound, and age at death were alllower in TLO patients [20,21,105,106]. The difference was evenmore striking in women with TLO who died nearly 20 years earlierthan those without TLO [20].

A negative correlation of inflammation with age is observed[104]. Moreover compared with pathologically active patients,pathologically inactive patients showed no difference with age-matched controls in any of the inflammatory parameters (CD3+ Tcells, CD20+ B-cells and Ig+ cells) or the degenerative parameters(APP, synaptophysin), suggesting that the halt in neurodegenera-tion parallels the vanishing of the inflammatory reaction [104].Since the neurological disability of these inactive patients was high(mean EDSS 8.5), this lower inflammatory burden unlikely reflectsan underlying benign form of MS [104]. In conclusion, the promi-nent cortical lesions suggest a non-targeted mechanism responsi-ble for cell injury [106]. A superficial diffusible factor (e.g. IFNc,TNFa), for example by indirect activation of microglia inducinginflammation-driven neurodegeneration [106], would better ex-plain the type III subpial cortical lesions (see above), which —anexception in MS pathology – are not centered by blood vessels [11].

Intrathecal TLOs are able to mount a local mature immuneresponse

The T cell clonal response was demonstrated to be both ‘private’to brain regions and ‘public’ since shared throughout the brain inall MS patients [108], and clonal expansion persists for years[109]. Private B cell clonotypes are widely distributed throughoutthe brain and CSF but are absent in peripheral blood [94,110]. Ahigh load of mutations in CDR reflects germinal center maturation[69,70,83]. This selection process of high activity antibodies re-quires a positive selective pressure based on the presentation ofantigen by follicular dendritic cells in germinal centers. Moreover,

the persistence of clonal rearrangements over many years —farexceeding the lifetime of B cells – confirms the local proliferationof B cell clones [32,34].

Moreover, in an animal model, a locally restricted intrathecalresponse was demonstrated against a brain delivered antigen(ovalbumin) [111]. Using a PLP178–191 induced model of relapsingexperimental autoimmune encephalomyelitis (EAE)—an animalmodel of RR-MS, it was demonstrated that the spreading toPLP139–151 is detected inside the CNS days before being detectablein the peripheral lymphoid compartments, and that PLP139–151

transgenic T cell activation is exclusive to the CNS [112]. OnlyCNS dendritic cells (with the exception of microglia and macro-phages) were able to induce naïve T cell proliferation with endog-enously processed peptide [112]. This experimental set favors thehypothesis that naïve T cells gain access to inflamed CNS wherethey undergo an epitope spreading driven by CNS dendritic cells[112].

Intrathecal TLOs are also noxious owing to antibody-independent toxicity

Considering that: (a) pathologically, no Ig deposition/comple-ment activation has been found in gray matter type III cortical pla-ques, and that (b) in vitro, death of oligodendrocytes may beindependent from secretion of Ig or some cytokines, Lisak et al.[113] speculated that molecules other than B-cells could be rele-vant to explain cortical demyelination. In vitro study of superna-tants obtained from B cell cultures of MS patients led to higherlevels of oligodendrocyte death than did supernatants from controlpatients [113]. Mixed glial culture and microglia exposed topoly(I:C) induce a microglial activation with secretion of TNFa re-stricted to microglia, in turn inducing the lethality of oligodendro-cytes dependent on the TNFa/TNFR1 pathway [114].Complementary experiments demonstrated that numerous TLRagonists triggered TNFa production by microglia, resulting in thereduced viability of oligodendrocytes [114]. TNFa and IFNc induceapoptosis of human oligodendroglial cell lines in vitro, either indi-vidually in a dose-dependent fashion or by synergy [115]. Althoughin vivo proof —for example, from transplanted TLO to CNS – is stilllacking, the abovementioned arguments suggest that cortical TLOsmay secrete a combination of cytokines that are locally noxious tocortical cells.

Each cell component of TLO may play a role in the maintenanceof a noxious intrathecal response: B-cells are able to proliferate anddifferentiate to immortal plasma cells; T cells are continuouslymaturing in interaction with B cells; dendritic cells provide sup-port for respective maturation and homing. Consequently, target-ing resident TLO as a whole appears promising [112,116,117].However, one may foresee how the systemic targeting of lymphoidtissue may be deleterious owing to an unwanted generalized im-mune suppression. We propose to take advantage of the intrathe-cal compartmentation of inflammation to blunt it withouttargeting systemic lymphoid tissue.

None of the available MS drugs deplete intrathecal Ig secretion

As demonstrated before, intrathecal Ig secretion provides avaluable (although underestimated) approximation of persistentintrathecal inflammation. We now discuss the efficiency of eachavailable drug with regard to intrathecal immunosuppression.

Irradiation

Plasma cells and IgG synthesis are insensitive to irradiation[118].

304 M. Bonnan / Medical Hypotheses 82 (2014) 300–309

Steroids

Various protocols of steroid infusions —ranging from iv to intra-thecal injections of various steroids – have been described in theliterature but without any sustained clinical success upon impair-ment. In series examining CSF, a disassociation is observed be-tween a transient decrease in IgG synthesis and the preservationof OCB [118–121].

Interferon beta

Essentially no change occurred in CSF drawn at 104 weeks con-cerning IgG index and OCB [41].

Other non-specific immunosuppressive agents

We found no data on CSF parameters following conventionaltreatment with methotrexate (iv or intrathecal), cyclophospha-mide, mitoxantrone or mycophenolate mofetil.

Autologous stem cell transplantation

Stem cell transplantation often fails to halt clinical progressionof MS [122–124] and acute demyelinating lesions may persist[125]. Although no control group was provided, the latter resultsare in line with the expected natural history of the progressivegroup. Brain atrophy is neither halted nor prevented even in theabsence of new inflammatory lesions [6,7]. At pathological level,axons stained by APP —which is a marker of acute axonal degener-ation – were identified in all cases both inside plaques and in nor-mal-appearing white matter [126], confirming a persistent ongoingdiffuse degeneration.

This failure to suppress disease progression correlates with theremaining active intrathecal inflammation. Receiver intrathecalinflammatory cells persisted after bone marrow transplantationalthough all the blood cells were of donor origin [127]. Pretreat-ment OCB mostly persisted or became positive [7,128,129]. Impor-tantly, the failure to cure OCB was linked to the persistence ofplasma cells rather to a longer half-life of Ig in CSF. Stem cell trans-plantation failure testifies to the full autonomy of intrathecalinflammation even after a peripheral autoimmune reset.

Natalizumab

Natalizumab prevented leukocyte transmigration to CNS fromthe first injection, while CSF white blood cells, CD19+, CD4+ andCD8+ T cells, CD138+ plasma cells and CD209+ MHC class II den-dritic cells were lowered to the same count as that of controls[130]. The number of doses needed to deplete dendritic cells fromperivascular spaces of deep white matter is unknown, as well is themaximal amount of depletion that may be expected [131]. The de-layed onset of progressive multifocal leukoencephalopathy (PML)after the first year of natalizumab therapy suggests that long-termuninterrupted use of natalizumab eventually leads to a reductionin dendritic cells at levels unable to prevent PML onset [131].The reduced number of CD209+ dendritic cells strongly suggeststhat these cells egress from the blood [131]. Although all the pa-tients had OCB before natalizumab, 16% were controlled negativeand the proportion of intrathecal synthesized fraction (Reiber-gram) in the normal range increased from 20% to 45% [132]. Thissuggests that intrathecal secretion is merely repressed rather thansuppressed by natalizumab.

FTY720

Longitudinal CSF analysis during the treatment of MS patientsshowed that while the cell count decreased, the IgG index andOCB persisted [133].

Rituximab

Rituximab is a monoclonal antibody targeting all the B cellsexpressing high levels of CD20 (CD20bright), whereas a minor pop-ulation of B cells expressing a lower concentration of CD19(CD19dim) may be resistant to rituximab, all the more at low con-centration, and may expand during reconstitution [134]. Plasmacells, which do not express CD20 but secrete high levels of Ig, arefully resistant. The rituximab concentration reached in the CSFdoes not exceed 0.2% of its concentration in serum in oncologicalsettings [135]. After rituximab infusion in blood, CSF floating cellswere dramatically decreased for several months [44,45,136], farexceeding the half-life of rituximab. CSF CXCL13 and CCL19 weredecreased at week 24 compared to baseline whereas other cyto-kines remained unchanged [45]. In serum, the IgG and IgM againstMOG and MBP, which were found in some patients, slightly de-creased after infusion. However, CSF IgG level, IgG index and OCBnumber remained essentially unchanged [45,46]. In fact, this fail-ure to lower intrathecal Ig secretion was predictable owing tothe absence of effect of blood-infused rituximab on serum IgGand IgA levels, contrary to a minor effect upon IgM levels [46,137].

Rituximab fails to clinically improve PP-MS [137]. In CSF, a min-or depletion of CD19+ B-cells was achieved in some patients [134].For several months after rituximab therapy, CSF B cells weremostly CD19dim post-switch B cells (IgM-IgD-CD38-) displaying alow light-scatter profile (indicative of a resting state) and only aminority were plasma cells [134]. From 14 to 20 months, CD19bright

repopulate and CD19dim expand 5- to 9-fold and recover their acti-vated state [134]. The most important point is the lack of CD19+CSF B-cell depletion after rituximab treatment [134]. Two explana-tions have been proposed: (1) the critical CSF rituximab concentra-tion may not be reached; (2) CSF B cells are mostly advancedmemory or plasma cells, which are naturally prone to resist ritux-imab owing to dim expression of CD19/20 [134]. Giving rituximabby the intrathecal route should overcome these two limitations,and should achieve clearance of CSF CD20dim B cells. Consideringthe safety of intrathecal rituximab [138], there is an urgent needto undertake clinical trials. Moreover, intrathecal rituximab maynecessitate co-administration of intravenous rituximab to destroyCD20 lymphocytes, which may replenish the intrathecal compart-ment early, or to block their entry to the CNS with natalizumab. Ifthis combination were to prove safe, could CSF CD20+ cell deple-tion be a sufficient condition to expect a cure from the progressivedisease? In particular, can intrathecal plasma cells (which secretepotentially toxic Ig and are naturally resistant to rituximab) andTLOs (which support their survival) be completely neglected?

Rituximab may fail to cure TLO— Time to broaden mAb choices

It appears crucial to reanalyze the data obtained in other disor-ders. In chronic active antibody-mediated rejection, rituximab de-creases B-cells from TLO of explanted rejected kidneys but fails toeliminate them [139]. TLO from rituximab-treated patients secretemore Ig anti-donor alloantibodies —although a third less thanexplanted grafts without rituximab – and express higher levels ofBAFF [139]. In rheumatoid arthritis, clinical response correlateswith synovial B-cell depletion [140], while synovial B-cells, unlikecirculating cells, and although profoundly depleted (up to �90%)are not eliminated by rituximab [141]. Moreover, synovial immu-

M. Bonnan / Medical Hypotheses 82 (2014) 300–309 305

noglobulin synthesis, although not suppressed, was lowered by�50% in correlation with clinical response, but the synovial expres-sion of BAFF, APRIL and SDF1 was not altered [140].

Supposing an optimal entrance of rituximab into the intrathecalcompartment, the elective targeting of CD20 B cells would spareCSF plasma cells (CD20- or nested B-cells) and could be expectedto have little impact on intrathecal IgG secretion, just as bloodIgG secretion remains relatively unchanged after rituximab bloodinfusion, even after several rounds of treatments. Data acquiredin a mouse model demonstrate that plasma cells are long-livedand that depletion of mature and memory B-cells by anti-CD20neither dramatically affects plasma cell numbers nor the pre-exist-ing Ab levels [142]. These long-lived ASC (antibody secreting cells)were mostly concentrated in bone marrow [142]. Interestingly,antibody blocking of LFA1/VLA4 purges bone marrow niches fromASC, rendering ASC partially sensitive to rituximab depletion [142].This strategy of ASC depletion holds promise for applications inother tissues, for example TLO.

Since TLO are considered to be the major target in CSF, the tar-geting of peripheral secondary lymphoid organs might be a viableworking model. Human tonsils grafted in immunodeficient micewere highly enriched in long-lived B cells, plasma cells and T cells[143]. Finally, levels of circulating human IgG were 5-fold lowerthan controls after rituximab and 100-fold lower after ale-mtuzumab [143]. Both IgM levels and IgM secreting cells increased4 weeks after rituximab, reflecting the generation of IgM secretingcells in the graft from a precursor B-cell population that survivedrituximab treatment [143]. In conclusion, rituximab alone cannotbe expected to reach OCB depletion even if an optimal access toCNS is obtained, so other mAbs need to be considered.

How might a ring-fenced intrathecal response become a strongtherapeutic advantage – conceptualization of intrathecalimmune reset

As reviewed above, impairment in MS is driven by the progres-sive phase, which is associated with a persistent intrathecal im-mune response. This response is largely spared byimmunosuppressive treatments given systemically. This fact haslargely been interpreted in the literature in two ways. It was firstsuggested that progressive MS disease activity could not be dimin-ished through immune mechanisms. As demonstrated above, thisassertion cannot be validated until the intrathecal compartmentis efficiently targeted by immunosuppressive treatments. Sec-ondly, the intrathecal immune response, i.e. OCB, is not consideredto be sufficient alone to trigger and feed the protracted CNS tissuedamage and MS relapses [144]. There are several lines of evidencefor the direct or indirect toxicity of immune cells owing to theirbyproducts (i.e. intrathecal antibodies, cytokines). As proposed byTourtellotte, ‘one of the goals of an effective treatment in MS maybe the eradication of CNS IgG synthesis’ [40].

In many autoimmune disorders (e.g. lupus), the completedepletion of peripheral autoimmunity followed by bone marrowtransplantation often cures the underlying disease by ‘resetting’the immune system. Unlike in peripheral autoimmune disorders,bone marrow transplantation fails to reset intrathecal immunityand fails to cure MS. We now propose that intrathecal immunit,should be directly targeted via the intrathecal route, with drugsaimed at eliminating simultaneously all the actors of intrathecalimmunity, thereby achieving an immune intrathecal reset.

Intrathecal injection by lumbar puncture has long been used todeliver various drugs to the meningeal and CNS compartment. Ani-mal and human experimentation has shown that CSF-delivereddrugs reach all surfaces bathed by the CSF as far as the deep sulci[145]. There are many advantages in this strategy. The intrathecal

compartment has a low volume so low amounts of drugs should besufficient to achieve this goal. As a consequence, CSF drug drainageto blood would not reach sufficiently high concentrations to attainthe threshold for systemic action, thus avoiding the side effect ofimmune suppression and secondary immune reaction. Ideally,intrathecal immune reset could be achieved with a single drugadministration. Since the peripheral immune system and immunecell transmigration across the BBB are both preserved, the reconsti-tution of a normal intrathecal defense should be short, a matter ofdays or weeks, in line with the immune reconstitution syndromedelay after plasma exchange of natalizumab in PML. The PML riskis driven by the persistent depletion of brain T cells and dendriticcells [146], which should not be a safety concern with this tech-nique. Moreover, considering that the goal of our strategy, i.e.immunosuppression, is the same as that already driving peripheralimmune suppression with routine drugs (see above), elective tar-geting of the immunity nested in the intrathecal compartment,which is normally devoid of this reaction, should not increaseour assessment of the risk involved.

There are several plausible scenarios. If persistent intrathecalinflammation drives the clinical progression, this therapy couldhopefully halt the course of MS. If intrathecal inflammation alsodrives relapses, this could be very promising for an early cure forMS. In a different scenario, a process (e.g. degeneration) mightdrive intrathecal inflammation, which could then be reconstitutedfrom the periphery after being cured. Even in this worse scenario,the therapy might cause the neo-synthesized intrathecal autoreac-tive clones to be less aggressive than those preceding the intrathe-cal reset.

Cooking a monoclonal Abs soup: multiple drugs to targetmultiple hits from TLO

Several strategies should be considered to achieve intrathecalimmune depletion. In bone marrow transplantation, immune sup-pression is usually obtained by the combination of cytotoxic drugsthat cannot be used by the intrathecal route owing to obvious risksof side-effects. Moreover, a pitfall of HSCT is the inability to van-quish most of the humoral immunity [147] owing to the chemore-sistance of plasma cells. For these reasons, an ideal intrathecaltreatment should obey some fundamental rules:

(1) absence of brain and nerve cytotoxicity;(2) simultaneous targeting of each immune cell subtype (B cells,

plasma cells, T cells and dendritic cells);(3) achieve a complete/near complete immune depletion after a

single pulse;(4) limited systemic effect after CSF drainage.

Monoclonal antibodies are suitable candidates owing to theiraccuracy and tolerability. The considerable feedback obtained fromthe use of intrathecal rituximab in lymphoma is a promising start-ing point for finding new mAbs for intrathecal administration.Although intrathecal rituximab trials may be set up, we do not be-lieve that rituximab alone would achieve an intrathecal immunedepletion, mostly because plasma cells are naturally resistant(see above). In fact, a combination of mAbs might be able to de-stroy all the electively targeted white cells, including B cells, plas-ma cells, T cells and dendritic cells acting as APC. Candidateantibodies should be able to destroy targeted cells in the intrathe-cal context (low availability of complement factors). Multiple can-didates have already been reported to be able to extensively targetB and T lymphocytes (e.g. rituximab, alemtuzumab), but none ofthem is fully polyvalent, especially upon plasma cells and dendriticcells.

306 M. Bonnan / Medical Hypotheses 82 (2014) 300–309

Plasma cells should also be targeted

Plasma cells are notoriously hard to eradicate owing to their in-nate resistance to radiation and to most of the currently immuno-suppressive drugs. Long-lived plasma cells play a key role in themaintenance of antibody response in lupus, Sjögren’s syndromeand rheumatoid arthritis [148]. In myasthenia, MuSK antibodiesare eliminated by targeting CD20 with rituximab thanks to theirproduction by short-lived plasma cells continuously regeneratedby plasmablasts, whereas AChR are almost unchanged owing totheir dependence on long-lived plasma cells [148]. In vitro, expo-sure of human thymus from myasthenic patients to bortezomibat 60-fold lower concentrations than those reached in vivo induceda selective apoptosis of plasma cells in a few hours [148]. In vivo,off-label use of bortezomib (in single or dual cycles) in autoim-mune patients often leads to a lowering in the target autoAb level,a less pronounced decrease in blood total Ig and Ig against measlesand tetanus toxoid (review in [148]). Although bortezomib failedto completely cure the autoAb in a few autoimmune patients, itsuse might be promising in autoimmune disorders [148]. However,the major concern with bortezomib is drug-induced hyperalgicneuropathy, which is cumulative and may no longer occur undercarfilzomib therapy or under marizonimb (salinosporamide A),which is the only proteasome inhibitor known to cross the BBB[149].

Daratumumab is a human mAb inducing both a complement-dependent and an antibody-dependent cellular cytotoxicityagainst CD38. The latter is able to kill plasma cells effectively insidethe preserving bone marrow micro-environment and at extremelylow concentrations [150]. Mention should be made of anti-229,which is largely expressed by leukocytes and plasma cells [151],and of anti-BR3 which has both an ADCC and a BAFF-blockingactivity in vitro, leading to an effect upon circulating B-cells similarto that of rituximab and also reaching cycling and non-cyclingplasma cells, leading to a decrease in IgM and IgG levels [152]. Thiseffect is evident upon subsets of B-cells that are relatively resistantto anti-CD20 depletion (i.e. germinal centers) [152]. Many otherdrugs targeting the micro-environment of survival niches are un-der development (see [153]).

Dendritic cells should also be targeted. FLT3 is a tyrosine kinaserestrictively expressed in CD34+ dendritic cells. In MS brain, FLT3is detectable in perivascular CD209+ dendritic cells, in someCD209-CD68+ macrophages/microglia and in normal grey matter[154]. Lestaurtinib, which inhibits FLT3, induces apoptosis of den-dritic cells and decreases the severity of EAE [155]. Efalizumab is ahumanized monoclonal antibody directed against the alpha-sub-unit of the integrin LFA-1 (CD11a) and used in psoriasis [156]. Efa-lizumab blocks and down-regulates CD11a on lymphocytes,thereby inhibiting their migration into the target tissue especiallythrough the BBB, as demonstrated in vitro for peripheral bloodmonocular cells [156]. Unfortunately, excepting rituximab, noneof these drugs has ever been used in intrathecal administration,so preclinical studies are required.

Conclusion

Several lines of evidence suggest that an intrathecal injection ofone or several mAb might have a considerable effect on progressiveMS. We propose the following plan: (1) Selecting a combination ofcandidate mAbs able to achieve a stepwise intrathecal immunesuppression combining the targeting of B/T-cells and plasma cells,and eventually targeting dendritic cells in order to obtain aplasiawith complete destruction of TLO in vitro; (2) Testing the intrathe-cal tolerance and kinetics of these mAbs in normal animals; (3)Testing the combination in animal models of progressive MS; (4)

testing intrathecal injection of a combination in progressive MS pa-tients with close CSF monitoring. In the event of biological successand if the procedure was entirely safe, long-term clinical and MRIfollow-up could be undertaken. Such a study can already be under-taken with rituximab alone, since safety data are available fromprevious use in meningeal lymphomas [138].

Since impairment in the secondary phase is very slow and takesyears to progress, the primary outcome should be the rate of brainatrophy, with clinical outcome only as a secondary outcome. Aspreviously suggested, intrathecal secretion could be consideredas: (1) either a possible actor in brain toxicity and atrophy via auto-reactive Ig; (2) or as a simple marker of an ongoing immortalizedintrathecal inflammation. We suggest that the close monitoringof intrathecal inflammation (intrathecal secretion, OCB, floatingcells, cytokines) should be the main target of further trials in pro-gressive MS. The long-term normalization of CSF parameters couldbecome a key treatment issue.

Conflict of interest

No conflict of interest.

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