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Intrathecal immune reset in multiple sclerosis: Exploring a new concept

Medical Hypotheses, 3, 82, pages 300 - 309

Abstract

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

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

Abbreviations: Ab - antibody, AI - antibody index, AICD - activation-induced cytidine deaminase, ASC - antibody secreting cells, BBB - blood–brain barrier, CDR - complement determining region, CIS - clinically isolated syndrome, CNS - central nervous 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 - oligoclonal bands, PML - progressive multifocal leukoencephalopathy, PP - primary progressive MS, rAb - recombinant antibody, RR - relapsing–remitting MS, SP - secondary progressive MS, TLO - tertiary lymphoid organ.

Introduction

Multiple sclerosis (MS) is the most frequent chronic inflammatory and demyelinating disorder of the central nervous system (CNS) in young adults and remains the second cause of disability in young people. Although most patients during the early phase of the disease suffer from a relapsing–remitting form of MS (RR-MS) characterized by acute relapses usually followed by a complete remission, the majority will develop a secondary progressive form (SP-MS). The impairment is mostly independent from the initial RR phase and is mainly driven by the late SP phase. Although treatments directed against the RR phase may have a slight preventive effect on the SP phase, none of them has unfortunately been shown to halt the ongoing secondary phase. Considering that most of the debilitating burden is driven by the progressive phase, building-up a therapeutic strategy dedicated to this phase remains a challenging goal.

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

We briefly review the main actors in this intrathecal response leading to slow axonal burning. Then we hypothesize the exploration of a new potential therapeutic avenue in progressive MS. In other words, whereas it has long been considered that the inability of therapies to penetrate the blood–brain-barrier (BBB) is a crucial obstacle, we propose to take advantage of this to safely reset the intrathecal immune system in order to halt the slow axonal burning underlying secondary MS. We review the literature in support of the rationale for treating MS with intrathecal drugs dedicated to safely clearing the local immune response.

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

All the available treatments are directed against the inflammatory component of the RR phase but they fail to actively delay or prevent the onset of SP-MS, to cure it, or to prevent any kind of steady impairment. Furthermore, in primary progressive MS (PP-MS), no treatment has ever proved to be efficient [2] . Another clue to the limited efficacy of drugs in the early phase is the brain atrophy rate, which is a key point when considering the dynamic of impairment. This rate remains essentially constant and high throughout the course of MS, from clinically isolated syndrome (CIS) to PP-MS[3] and [4]. Interestingly, even in the RR-MS phase treated by the most active treatments for preventing relapses, i.e. alemtuzumab or autologous stem cell transplantation, the brain atrophy rate decreases but always fails to normalize and remains high[5], [6], [7], and [8].

Spreading of subpial cortical lesions drives the late disability

Although demyelination of the cortex and deep gray matter nuclei has long been known, the extent of cortical demyelination remained grossly underestimated until recent immunohistochemical methods demonstrating their presence in the very early stages of the disease [9] . Lesion burden increases with time to become more prominent than white matter lesions in the secondary phase (review in[10] and [11], and the cortical lesion load may even be a key event in the transition from RR-MS to SP-MS[12] and [13]. Cortical pial lesions (type III) extend from the pial surface to the superficial cortical layers [14] and represent approximately half of the cortical lesions [15] , affecting 60% of the cortical ribbon of the brain, cerebellum and hippocampus[16], [17], and [18]. They harbor distinct features: constant depth of demyelination waning at cortical layer 5 [15] and a large extension over the multiple gyri [15] . Cortical lesions are different from white matter lesions. They are devoid of inflammatory cells and macrophages[15] and [19], have sparse deposition of complement and immunoglobulins (Ig) (in [20] ) and lack detectable serum-derived proteins, suggesting that an immune response underlying the cortical pathology occurs in the meninges[11] and [21]. Regional gray matter demyelination and atrophy are not driven by underlying white matter lesion load[22] and [23]. Progressive MS is associated with cortical demyelination and diffuse normal appearing white matter injury, which invariably occurs on a background of meningeal, perivascular and parenchymal inflammation[16] and [24].

Cortical inflammatory lesions are highly correlated with cortical atrophy and disability within each MS subgroup[25] and [26]. In a large five-year follow-up study, change in cortical matter fraction, new cortical lesions and clinical impairment (expanded disability score, EDSS) were highly correlated [25] . Age at onset of wheelchair use and death are correlated with extent of grey matter damage [10] . On the other hand, benign MS is characterized by an initially low cortical lesion charge (of about a third in RR-MS patients) and significantly lower new cortical lesions [25] , whereas the number of shadow plaques in the white matter is not different in benign MS [10] . In a long-term cohort, benign MS demonstrated higher normal gray matter volume than non-benign MS that were close to those of controls [26] . On the contrary, patients with a high level of cortical lesions at baseline showed greater progression of both clinical and grey matter atrophy at 5 years [25] . Several studies have found cognitive skills and motor impairment to be correlated with cortical atrophy[26], [27], and [28]. Moreover, cortical pathology evolves at similar rates in all MS subtypes, with a higher baseline cortical lesion load in SP-MS due to the longer disease duration [25] . In conclusion, whereas the cortex is mostly spared in benign MS, a high load of subpial cortical lesions drives the brain atrophy and the clinical burden in non-benign MS.

Intrathecal synthesis is robust over time and treatments

Intrathecal synthesis occurs as a very early disease event and the proportion of patients with OCB tends to increase over time [29] . In longitudinal CSF studies, OCB pattern is robust and OCB have never been seen to go away with time [30] although changes in band intensity and acquisition of new bands [31] may occur. Regarding the clonal repertoire of CSF Ig, clonal rearrangements are conserved over time and a higher number of clones is found in patients with the longest disease duration, suggesting a continuous clonal expansion over time[32] and [33]. Antibody index[34], [35], and [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] and [38]that is resistant to high-dosage steroid infusions[30], [39], and [40]. Even if steroids transiently decrease the IgG index in most but not all patients, the decrease in range of CSF IgG synthesis is low and the CSF total protein concentration remains unaffected [39] . Weekly intramuscular or intrathecal β-IFN[37] and [41], azathioprine [42] , natalizumab [43] , rituximab[44], [45], and [46]or daclizumab [47] have essentially no effect upon intrathecal secretion.

In conclusion, intrathecal secretion and OCB pattern are early-occurring events in the course of MS, which, once acquired, persist essentially unchanged throughout life, whatever the various therapies available, and then remain stable or gradually worsen over time.

Intrathecal synthesis confers a worse prognostic value

Demonstration of CSF OCB at the index event is a highly independent predictor of clinical recurrence[48], [49], [50], and [51], especially if OCB target lipid antigens [52] . The presence of an MRZ pattern (intrathecal reaction againstMeasles,Rubella andZoster viruses) in CIS predicts progression to definite MS [53] . The number of OCB and the IgM index are thought to positively correlate with the course of the disease[54] and [55]. Mean EDSS is higher in patients with OCB, especially in the event of IgM OCB[56] and [57], and EDSS correlates with IgG1 index and CSF free light chains[58] and [59].

In numerous studies, CSF IgM have been associated with a poorer clinical long-term outcome[57], [60], [61], [62], and [63], a lower brain volume [64] , and a decrease in brain parenchyma fraction over time [65] .

Absence of intrathecal secretion in some patients reflects technical limitations but not absence of intrathecal inflammation

About 3% of MS patients in cohorts lack intrathecal IgG synthesis [35] . This might just be due to the low sensitivity of test since MRZ reaction, high CSF IgA synthesis, high IgA index[66] and [67], oligoclonal free κ light-chains [68] , clonalVHand complement determining region (CDR) rearrangements[69] and [70]are observed, suggesting that OCB tests are insufficiently sensitive in OCB-negative patients. Moreover, OCB negativity at baseline tends to become positive for half of patients in whom lumbar puncture is repeated [71] . Cumulated data suggest that MS patients apparently devoid of intrathecal secretion may in fact have a milder secretion below the sensitivity threshold of the common tests. However, this smoldering intrathecal reaction may have clinical consequences since many studies have confirmed that OCB-negative patients are more prone to have lower EDSS, a benign form, and a delayed and lower risk of impairment milestones[54], [71], [72], [73], [74], [75], and [76].

Intrathecally secreted Ig target the brain

CSF Ig constitute a private repertoire, which may harbor toxic properties [77] . Patterns of demyelinating lesions with Ig and complement deposition in and around macrophages have been described in brain fragments mainly obtained from biopsies, in which fixation is immediate[78], [79], and [80]. Application of CSF to cultured cells (rat cerebellar granule neurons) significantly labels the axonal surface with IgM dots [64] and purified antibodies against MOG from MS serum bind to intact myelin in rat [81] . Various recombinant antibodies (rAb) synthesized from CD138+ CSF B-cells of MS patients stain most of the glia components[82] and [83]and many specific antibodies have been shown to react against cultured oligodendrocytes or human CNS tissue (in [84] ). In animal models, anti-MOG antibodies purified from human MS serum strongly enhanced lesions without increasing inflammation[81] and [85]. In a model of focal demyelination using implantation of various hybridoma cells in rat spinal cord, demyelination strongly depended on the target of the hybridoma complement and involved [86] . The latter experiment is reminiscent of the infiltration by plasma cells inside old burned-out demyelinating plaques [87] . Functional electrophysiological modifications have also been observed in older experiments[88] and [89]. In conclusion, the hypothesis that CSF Ig could play an active role in brain dysfunction, both at functional and 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 CSF although some authors recovered rare higher IgG2 or IgG3 indices[90] and [91]. Over-representation of a single VH type family with heavy chain sequences in MS plaques, periplaque white matter and CSF, but not blood, has been demonstrated [92] , mostly with VH4 and VH2. This bias precedes the onset of OCB and is typical of MS, contrary to other CNS disorders [92] . A robust demonstration of clonal expansion of a single ancestor gene was made by the analysis of the clonal diversification from an ancestor gene accumulating substitutions [93] , a diversification mostly confined to the intrathecal compartment [94] . The main targets of somatic hypermutations in CDR are within RGYW/WRCY motifs [70] , which are targeted by the activation-induced cytidine deaminase (AICD) specifically expressed in lymphoid organs.

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

CSF is a fostering milieu immortalizing inflammation

The CSF is bathed by many cytokines involved in the traffic and survival of inflammatory cells (e.g. BAFF, CXCL13), so a self-sustained intrathecal inflammation is fostered. BAFF is a key chemokine for the maturation and survival of B cells and is constitutively produced in the CNS by astrocytes [95] . CSF BAFF dosage was higher in a subset of patients with ⩾6 OCB [96] . One of the receptors of BAFF is BCMA, which enhances the long-term survival of plasma cells. Up-regulation of BAFF expression in active and inactive MS lesions, where BAFF reaches levels of concentration similar to those in lymphatic tissue [95] , may provide a fostering environment to long-lived B-cells [97] .

The chemokine CXCL13, which is mostly secreted by follicular dendritic cells [95] , is a key regulator of B-cell recruitment and is selectively a chemoattractant for B lymphocytes and B-helper T cells via its exclusive receptor CXCR5 [98] , which is expressed in 20–30% of blood and CSF CD4+ T cells (45) and by virtually all the CSF B cells [45] . CXCL13−/− mice fail to develop lymph nodes and CXCL13 is essential for establishing and maintaining the lymphoid tissue architecture. Likewise, the homing of B-cells to lymph nodes is CXCR5-dependent (in [99] ). Immunostaining of CXCL13 revealed this cytokine in lymphoid follicles of the meninges, [100] in blood vessels in chronic active lesions [101] and in the endothelium of primary CNS lymphomas [95] . Patients with OCB had high levels of CXCL13 [98] . The rare CIS cases associating high CXCL13 levels without OCB should be compared with the finding that CXCL13 secretion in CSF anticipates the OCB in neuroborreliosis [102] . The mean level is higher if the MRZ pattern is present, demonstrating an association of CXCL13 with a polyspecific intrathecal B-cell response in MS. Moreover, CXCL13 levels strongly correlated with both CSF plasma cells count, B cell count [103] and IgG index [98] . Steroids and natalizumab also affect CSF CXCL13 levels [103] .

Meningeal tertiary lymphoid organs (TLO) are identified in all MS subtypes

Meningeal CD20+ are elevated in all MS subtypes but Ig+ plasma cells are higher in PP-MS and SP-MS patients [104] . Axonal damage (APP) correlates well with each parameter of inflammation: B cells, T cells, plasma cell, HLA-D macrophages or microglia [104] . Neurite loss correlates with meningeal infiltration B and T cells, and neuronal loss predominates in the superficial layers [105] . In SP-MS and PP-MS series, 30–41% had meningeal aggregates of inflammatory cells reminiscent of tertiary lymphoid organs[20] and [105]. All these aggregates were ectopic follicles with germinal centers based on the presence of a reticulum of CD35+ and CXCL13+ stromal/dendritic follicular cells, proliferating CD20+/Ki67+ B cells, Ig + plasma/plasmablast cells and CD138+ plasma cells [20] . However, a fundamental difference in structure with lymphoid follicles was the lack of mantle zone, the presence of CD27+ memory B cells, and infiltration by numerous CD4+ and CD8+ T lymphocytes [106] . Plasma cell distribution is variable: either diffuse around most parenchymal and meningeal blood vessels in one patient [100] or scattered inside the parenchyma of chronic inactive lesions [100] . CXCL13 immunoreactivity is confined to dendritiform cells inside intra-meningeal B-cell follicles [100] . Proliferating cells in B-cell follicles, mostly CD20+ cells, are observed at rates varying from 0% to 43% [100] . CCL21 and adhesion molecule peripheral node addressin (PNAd), which selectively binds to naïve T and B lymphocytes and allows their homing to secondary lymphoid organs, are absent [100] , suggesting that the homing 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 more intense subpial demyelination[20] and [21]; a lower density of neurites both in normal appearing gray matter and in type III lesions [20] ; a higher degree of atrophy and cell loss in the superficial cortex (up to 65%); a profound loss of superficial neurons, astrocytes and oligodendrocytes (up to 70%); and a large increase in microglia numbers in superficial layers (up to 70%) with diffuse microglial activation in the presence of TLO [106] . A topographical and quantitative relationship is frequently observed between parenchymal cortical macrophages–microglia and meningeal infiltrates[14], [19], and [21]. Moreover, a study revealed a 90% probability that at least moderate meningeal inflammation was topographically associated with subpial lesions [9] . Examination of acute subpial lesions confirmed a destructive inflammation with ongoing neuro-degeneration and loss of oligodendrocytes [9] .

TLO identification and count are underestimated owing to technical limitations

Since brain TLO have recently been described, stringent universal criteria are lacking, which may explain their wide variation in frequency. Moreover, TLO are minute structures in low numbers and are highly likely be missed by the sampling process (i.e. 8 blocks per patient with a mean of 6 ± 3 follicles in the positive block sections) [20] . Considering the focal nature of tracked meningeal lesions and the usually low sample number, negative results should not be considered as definite proof of the absence of meningeal lesions in these patients, since a lower number of TLO may explain the sample negativity. Lastly, the convexity of the gyrus may undergo a mechanical abrasion of the meningeal surface during the fixation procedure, which may explain the predominance of TLO in the deep sulci [9] . B-cell aggregates are always adjacent to subpial lesions but the contrary is not true[21] and [106], which may suggest that B-cell aggregates located in different planes may have been missed. A definitive ascertainment of TLO in an autopsied brain would require the reinforcement of sampling density to be sure of the stereological relationship between cortical lesions and TLO.

TLO correlates with a worse clinical follow-up

Clues for a correlation between meningeal infiltration and shorter mean clinical duration were given in an old series [87] . The proportion of ectopic follicles decreases with the age of progression onset [20] . Both in SP-MS and PP-MS patients, age at clinical onset, age to be wheelchair-bound, and age at death were all lower in TLO patients[20], [21], [105], and [106]. The difference was even more striking in women with TLO who died nearly 20 years earlier than 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+ T cells, CD20+ B-cells and Ig+ cells) or the degenerative parameters (APP, synaptophysin), suggesting that the halt in neurodegeneration 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 reflects an underlying benign form of MS [104] . In conclusion, the prominent cortical lesions suggest a non-targeted mechanism responsible for cell injury [106] . A superficial diffusible factor (e.g. IFNγ, TNFα), for example by indirect activation of microglia inducing inflammation-driven neurodegeneration [106] , would better explain the type III subpial cortical lesions (see above), which —an exception in MS pathology – are not centered by blood vessels [11] .

Intrathecal TLOs are able to mount a local mature immune response

The T cell clonal response was demonstrated to be both ‘private’ to brain regions and ‘public’ since shared throughout the brain in all MS patients [108] , and clonal expansion persists for years [109] . Private B cell clonotypes are widely distributed throughout the brain and CSF but are absent in peripheral blood[94] and [110]. A high load of mutations in CDR reflects germinal center maturation[69], [70], and [83]. This selection process of high activity antibodies requires a positive selective pressure based on the presentation of antigen by follicular dendritic cells in germinal centers. Moreover, the persistence of clonal rearrangements over many years —far exceeding the lifetime of B cells – confirms the local proliferation of B cell clones[32] and [34].

Moreover, in an animal model, a locally restricted intrathecal response was demonstrated against a brain delivered antigen (ovalbumin) [111] . Using a PLP178–191induced model of relapsing experimental autoimmune encephalomyelitis (EAE)—an animal model of RR-MS, it was demonstrated that the spreading to PLP139–151is detected inside the CNS days before being detectable in the peripheral lymphoid compartments, and that PLP139–151transgenic T cell activation is exclusive to the CNS [112] . Only CNS dendritic cells (with the exception of microglia and macrophages) were able to induce naïve T cell proliferation with endogenously processed peptide [112] . This experimental set favors the hypothesis that naïve T cells gain access to inflamed CNS where they 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/complement activation has been found in gray matter type III cortical plaques, and that (b) in vitro, death of oligodendrocytes may be independent from secretion of Ig or some cytokines, Lisak et al. [113] speculated that molecules other than B-cells could be relevant to explain cortical demyelination. In vitro study of supernatants obtained from B cell cultures of MS patients led to higher levels of oligodendrocyte death than did supernatants from control patients [113] . Mixed glial culture and microglia exposed to poly(I:C) induce a microglial activation with secretion of TNFα restricted to microglia, in turn inducing the lethality of oligodendrocytes dependent on the TNFα/TNFR1 pathway [114] . Complementary experiments demonstrated that numerous TLR agonists triggered TNFα production by microglia, resulting in the reduced viability of oligodendrocytes [114] . TNFα and IFNγ induce apoptosis of human oligodendroglial cell lines in vitro, either individually in a dose-dependent fashion or by synergy [115] . Although in vivo proof —for example, from transplanted TLO to CNS – is still lacking, the abovementioned arguments suggest that cortical TLOs may secrete a combination of cytokines that are locally noxious to cortical cells.

Each cell component of TLO may play a role in the maintenance of a noxious intrathecal response: B-cells are able to proliferate and differentiate to immortal plasma cells; T cells are continuously maturing in interaction with B cells; dendritic cells provide support for respective maturation and homing. Consequently, targeting resident TLO as a whole appears promising[112], [116], and [117]. However, one may foresee how the systemic targeting of lymphoid tissue may be deleterious owing to an unwanted generalized immune suppression. We propose to take advantage of the intrathecal compartmentation of inflammation to blunt it without targeting systemic lymphoid tissue.

None of the available MS drugs deplete intrathecal Ig secretion

As demonstrated before, intrathecal Ig secretion provides a valuable (although underestimated) approximation of persistent intrathecal inflammation. We now discuss the efficiency of each available drug with regard to intrathecal immunosuppression.

Irradiation

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

Steroids

Various protocols of steroid infusions —ranging from iv to intrathecal injections of various steroids – have been described in the literature but without any sustained clinical success upon impairment. In series examining CSF, a disassociation is observed between a transient decrease in IgG synthesis and the preservation of OCB[118], [119], [120], and [121].

Interferon beta

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

Other non-specific immunosuppressive agents

We found no data on CSF parameters following conventional treatment with methotrexate (iv or intrathecal), cyclophosphamide, mitoxantrone or mycophenolate mofetil.

Autologous stem cell transplantation

Stem cell transplantation often fails to halt clinical progression of MS[122], [123], and [124]and acute demyelinating lesions may persist [125] . Although no control group was provided, the latter results are in line with the expected natural history of the progressive group. Brain atrophy is neither halted nor prevented even in the absence of new inflammatory lesions[6] and [7]. At pathological level, axons stained by APP —which is a marker of acute axonal degeneration – were identified in all cases both inside plaques and in normal-appearing white matter [126] , confirming a persistent ongoing diffuse degeneration.

This failure to suppress disease progression correlates with the remaining active intrathecal inflammation. Receiver intrathecal inflammatory cells persisted after bone marrow transplantation although all the blood cells were of donor origin [127] . Pretreatment OCB mostly persisted or became positive[7], [128], and [129]. Importantly, the failure to cure OCB was linked to the persistence of plasma cells rather to a longer half-life of Ig in CSF. Stem cell transplantation failure testifies to the full autonomy of intrathecal inflammation even after a peripheral autoimmune reset.

Natalizumab

Natalizumab prevented leukocyte transmigration to CNS from the first injection, while CSF white blood cells, CD19+, CD4+ and CD8+ T cells, CD138+ plasma cells and CD209+ MHC class II dendritic cells were lowered to the same count as that of controls [130] . The number of doses needed to deplete dendritic cells from perivascular spaces of deep white matter is unknown, as well is the maximal amount of depletion that may be expected [131] . The delayed onset of progressive multifocal leukoencephalopathy (PML) after the first year of natalizumab therapy suggests that long-term uninterrupted use of natalizumab eventually leads to a reduction in dendritic cells at levels unable to prevent PML onset [131] . The reduced number of CD209+ dendritic cells strongly suggests that these cells egress from the blood [131] . Although all the patients had OCB before natalizumab, 16% were controlled negative and the proportion of intrathecal synthesized fraction (Reibergram) in the normal range increased from 20% to 45% [132] . This suggests that intrathecal secretion is merely repressed rather than suppressed by natalizumab.

FTY720

Longitudinal CSF analysis during the treatment of MS patients showed that while the cell count decreased, the IgG index and OCB persisted [133] .

Rituximab

Rituximab is a monoclonal antibody targeting all the B cells expressing high levels of CD20 (CD20bright), whereas a minor population of B cells expressing a lower concentration of CD19 (CD19dim) may be resistant to rituximab, all the more at low concentration, and may expand during reconstitution [134] . Plasma cells, which do not express CD20 but secrete high levels of Ig, are fully resistant. The rituximab concentration reached in the CSF does not exceed 0.2% of its concentration in serum in oncological settings [135] . After rituximab infusion in blood, CSF floating cells were dramatically decreased for several months[44], [45], and [136], far exceeding the half-life of rituximab. CSF CXCL13 and CCL19 were decreased at week 24 compared to baseline whereas other cytokines remained unchanged [45] . In serum, the IgG and IgM against MOG and MBP, which were found in some patients, slightly decreased after infusion. However, CSF IgG level, IgG index and OCB number remained essentially unchanged[45] and [46]. In fact, this failure to lower intrathecal Ig secretion was predictable owing to the absence of effect of blood-infused rituximab on serum IgG and IgA levels, contrary to a minor effect upon IgM levels[46] and [137].

Rituximab fails to clinically improve PP-MS [137] . In CSF, a minor depletion of CD19+ B-cells was achieved in some patients [134] . For several months after rituximab therapy, CSF B cells were mostly CD19dimpost-switch B cells (IgM-IgD-CD38-) displaying a low light-scatter profile (indicative of a resting state) and only a minority were plasma cells [134] . From 14 to 20 months, CD19brightrepopulate and CD19dimexpand 5- to 9-fold and recover their activated state [134] . The most important point is the lack of CD19+ CSF B-cell depletion after rituximab treatment [134] . Two explanations have been proposed: (1) the critical CSF rituximab concentration may not be reached; (2) CSF B cells are mostly advanced memory or plasma cells, which are naturally prone to resist rituximab owing todimexpression of CD19/20 [134] . Giving rituximab by the intrathecal route should overcome these two limitations, and should achieve clearance of CSF CD20dimB cells. Considering the safety of intrathecal rituximab [138] , there is an urgent need to undertake clinical trials. Moreover, intrathecal rituximab may necessitate co-administration of intravenous rituximab to destroy CD20 lymphocytes, which may replenish the intrathecal compartment early, or to block their entry to the CNS with natalizumab. If this combination were to prove safe, could CSF CD20+ cell depletion be a sufficient condition to expect a cure from the progressive disease? In particular, can intrathecal plasma cells (which secrete potentially toxic Ig and are naturally resistant to rituximab) and TLOs (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 disorders. In chronic active antibody-mediated rejection, rituximab decreases B-cells from TLO of explanted rejected kidneys but fails to eliminate them [139] . TLO from rituximab-treated patients secrete more Ig anti-donor alloantibodies —although a third less than explanted grafts without rituximab – and express higher levels of BAFF [139] . In rheumatoid arthritis, clinical response correlates with synovial B-cell depletion [140] , while synovial B-cells, unlike circulating cells, and although profoundly depleted (up to −90%) are not eliminated by rituximab [141] . Moreover, synovial immunoglobulin synthesis, although not suppressed, was lowered by −50% in correlation with clinical response, but the synovial expression of BAFF, APRIL and SDF1 was not altered [140] .

Supposing an optimal entrance of rituximab into the intrathecal compartment, the elective targeting of CD20 B cells would spare CSF plasma cells (CD20- or nested B-cells) and could be expected to have little impact on intrathecal IgG secretion, just as blood IgG secretion remains relatively unchanged after rituximab blood infusion, even after several rounds of treatments. Data acquired in a mouse model demonstrate that plasma cells are long-lived and that depletion of mature and memory B-cells by anti-CD20 neither dramatically affects plasma cell numbers nor the pre-existing 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 from ASC, rendering ASC partially sensitive to rituximab depletion [142] . This strategy of ASC depletion holds promise for applications in other tissues, for example TLO.

Since TLO are considered to be the major target in CSF, the targeting of peripheral secondary lymphoid organs might be a viable working model. Human tonsils grafted in immunodeficient mice were highly enriched in long-lived B cells, plasma cells and T cells [143] . Finally, levels of circulating human IgG were 5-fold lower than controls after rituximab and 100-fold lower after alemtuzumab [143] . Both IgM levels and IgM secreting cells increased 4 weeks after rituximab, reflecting the generation of IgM secreting cells in the graft from a precursor B-cell population that survived rituximab treatment [143] . In conclusion, rituximab alone cannot be expected to reach OCB depletion even if an optimal access to CNS is obtained, so other mAbs need to be considered.

How might a ring-fenced intrathecal response become a strong therapeutic advantage – conceptualization of intrathecal immune reset

As reviewed above, impairment in MS is driven by the progressive phase, which is associated with a persistent intrathecal immune response. This response is largely spared by immunosuppressive treatments given systemically. This fact has largely been interpreted in the literature in two ways. It was first suggested that progressive MS disease activity could not be diminished through immune mechanisms. As demonstrated above, this assertion cannot be validated until the intrathecal compartment is efficiently targeted by immunosuppressive treatments. Secondly, the intrathecal immune response, i.e. OCB, is not considered to be sufficient alone to trigger and feed the protracted CNS tissue damage and MS relapses [144] . There are several lines of evidence for the direct or indirect toxicity of immune cells owing to their byproducts (i.e. intrathecal antibodies, cytokines). As proposed by Tourtellotte, ‘one of the goals of an effective treatment in MS may be the eradication of CNS IgG synthesis [40] .

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

Intrathecal injection by lumbar puncture has long been used to deliver various drugs to the meningeal and CNS compartment. Animal and human experimentation has shown that CSF-delivered drugs 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 be sufficient to achieve this goal. As a consequence, CSF drug drainage to blood would not reach sufficiently high concentrations to attain the threshold for systemic action, thus avoiding the side effect of immune suppression and secondary immune reaction. Ideally, intrathecal immune reset could be achieved with a single drug administration. Since the peripheral immune system and immune cell transmigration across the BBB are both preserved, the reconstitution of a normal intrathecal defense should be short, a matter of days or weeks, in line with the immune reconstitution syndrome delay after plasma exchange of natalizumab in PML. The PML risk is driven by the persistent depletion of brain T cells and dendritic cells [146] , which should not be a safety concern with this technique. Moreover, considering that the goal of our strategy, i.e. immunosuppression, is the same as that already driving peripheral immune suppression with routine drugs (see above), elective targeting of the immunity nested in the intrathecal compartment, which is normally devoid of this reaction, should not increase our assessment of the risk involved.

There are several plausible scenarios. If persistent intrathecal inflammation drives the clinical progression, this therapy could hopefully halt the course of MS. If intrathecal inflammation also drives relapses, this could be very promising for an early cure for MS. In a different scenario, a process (e.g. degeneration) might drive intrathecal inflammation, which could then be reconstituted from the periphery after being cured. Even in this worse scenario, the therapy might cause the neo-synthesized intrathecal autoreactive clones to be less aggressive than those preceding the intrathecal reset.

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

Several strategies should be considered to achieve intrathecal immune depletion. In bone marrow transplantation, immune suppression is usually obtained by the combination of cytotoxic drugs that cannot be used by the intrathecal route owing to obvious risks of side-effects. Moreover, a pitfall of HSCT is the inability to vanquish most of the humoral immunity [147] owing to the chemoresistance of plasma cells. For these reasons, an ideal intrathecal treatment 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 their accuracy and tolerability. The considerable feedback obtained from the use of intrathecal rituximab in lymphoma is a promising starting point for finding new mAbs for intrathecal administration. Although intrathecal rituximab trials may be set up, we do not believe that rituximab alone would achieve an intrathecal immune depletion, mostly because plasma cells are naturally resistant (see above). In fact, a combination of mAbs might be able to destroy all the electively targeted white cells, including B cells, plasma cells, T cells and dendritic cells acting as APC. Candidate antibodies should be able to destroy targeted cells in the intrathecal context (low availability of complement factors). Multiple candidates have already been reported to be able to extensively target B and T lymphocytes (e.g. rituximab, alemtuzumab), but none of them is fully polyvalent, especially upon plasma cells and dendritic cells.

Plasma cells should also be targeted

Plasma cells are notoriously hard to eradicate owing to their innate resistance to radiation and to most of the currently immunosuppressive drugs. Long-lived plasma cells play a key role in the maintenance of antibody response in lupus, Sjögren’s syndrome and rheumatoid arthritis [148] . In myasthenia, MuSK antibodies are eliminated by targeting CD20 with rituximab thanks to their production by short-lived plasma cells continuously regenerated by plasmablasts, whereas AChR are almost unchanged owing to their dependence on long-lived plasma cells [148] . In vitro, exposure of human thymus from myasthenic patients to bortezomib at 60-fold lower concentrations than those reached in vivo induced a selective apoptosis of plasma cells in a few hours [148] . In vivo, off-label use of bortezomib (in single or dual cycles) in autoimmune patients often leads to a lowering in the target autoAb level, a less pronounced decrease in blood total Ig and Ig against measles and tetanus toxoid (review in [148] ). Although bortezomib failed to completely cure the autoAb in a few autoimmune patients, its use might be promising in autoimmune disorders [148] . However, the major concern with bortezomib is drug-induced hyperalgic neuropathy, which is cumulative and may no longer occur under carfilzomib 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 cytotoxicity against CD38. The latter is able to kill plasma cells effectively inside the preserving bone marrow micro-environment and at extremely low 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-blocking activity in vitro, leading to an effect upon circulating B-cells similar to that of rituximab and also reaching cycling and non-cycling plasma cells, leading to a decrease in IgM and IgG levels [152] . This effect is evident upon subsets of B-cells that are relatively resistant to anti-CD20 depletion (i.e. germinal centers) [152] . Many other drugs targeting the micro-environment of survival niches are under development (see [153] ).

Dendritic cells should also be targeted. FLT3 is a tyrosine kinase restrictively expressed in CD34+ dendritic cells. In MS brain, FLT3 is detectable in perivascular CD209+ dendritic cells, in some CD209-CD68+ macrophages/microglia and in normal grey matter [154] . Lestaurtinib, which inhibits FLT3, induces apoptosis of dendritic cells and decreases the severity of EAE [155] . Efalizumab is a humanized monoclonal antibody directed against the alpha-subunit of the integrin LFA-1 (CD11a) and used in psoriasis [156] . Efalizumab blocks and down-regulates CD11a on lymphocytes, thereby inhibiting their migration into the target tissue especially through the BBB, as demonstrated in vitro for peripheral blood monocular cells [156] . Unfortunately, excepting rituximab, none of these drugs has ever been used in intrathecal administration, so preclinical studies are required.

Conclusion

Several lines of evidence suggest that an intrathecal injection of one or several mAb might have a considerable effect on progressive MS. We propose the following plan: (1) Selecting a combination of candidate mAbs able to achieve a stepwise intrathecal immune suppression combining the targeting of B/T-cells and plasma cells, and eventually targeting dendritic cells in order to obtain aplasia with complete destruction of TLO in vitro; (2) Testing the intrathecal 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 patients with close CSF monitoring. In the event of biological success and if the procedure was entirely safe, long-term clinical and MRI follow-up could be undertaken. Such a study can already be undertaken with rituximab alone, since safety data are available from previous use in meningeal lymphomas [138].

Since impairment in the secondary phase is very slow and takes years to progress, the primary outcome should be the rate of brain atrophy, with clinical outcome only as a secondary outcome. As previously suggested, intrathecal secretion could be considered as: (1) either a possible actor in brain toxicity and atrophy via autoreactive Ig; (2) or as a simple marker of an ongoing immortalized intrathecal inflammation. We suggest that the close monitoring of intrathecal inflammation (intrathecal secretion, OCB, floating cells, cytokines) should be the main target of further trials in progressive MS. The long-term normalization of CSF parameters could become a key treatment issue.

Conflict of interest

No conflict of interest.

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Footnotes

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