Multiple sclerosis: Neuroimmune crosstalk and therapeutic targeting

SUMMARY

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system afflicting nearly three million individuals worldwide. Neuroimmune interactions between glial, neural, and immune cells play important roles in MS pathology and offer potential targets for therapeutic intervention. Here, we review underlying risk factors, mechanisms of MS pathogenesis, available disease modifying therapies, and examine the value of emerging technologies, which may address unmet clinical needs and identify novel therapeutic targets.

INTRODUCTION

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by neuroinflammation and neurodegeneration. In 2020, the MS atlas reported that one individual with an average age of 32 years is diagnosed with MS every 5 min around the world, adding to about 2.8 million individuals currently living with the disease. Strikingly, MS worldwide prevalence has risen by 30% since 2013.¹ This increase may be partially attributed to improved reporting and diagnosis, but much of its underlying factors remain unknown. As for MS therapies, despite an improvement in efficacy and options, they remain mostly limited to relapsing-remitting MS. In this review, we summarize the current understanding of MS predisposing factors, mechanisms of disease pathology, and therapeutic interventions.

DISEASE PRESENTATION AND PATHOLOGY

Historical records describe a disease reminiscent of MS as early as the 14^th century.² In 1868, Jean-Martin Charcot named this disease sclérose en plaques, later adapted to multiple sclerosis.³ Charcot and collaborators established that the pathological hallmarks of MS include lesions in CNS white and gray matter areas with variable degrees of demyelination, perivascular immune cell infiltration, reactive gliosis, and/or neurodegeneration.³ Axonal transection and blood-brain barrier (BBB) disturbances were later described as additional features of these lesions.⁴

MS diagnosis and monitoring

MS is diagnosed based on clinical, biochemical, and radiological features that indicate a dissemination in time and space of CNS lesions.⁵ These features include symptoms of motor, sensory, and/or cognitive dysfunction; the presence of oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF)-antibodies indicative of CNS inflammation-; and the detection of CNS lesions by magnetic resonance imaging (MRI) using T2-weigthed scans to provide information about lesion load; or the contrast agent gadolinium to detect active inflammatory lesions. MRI advances have improved the monitoring of CNS damage and, consequently, disease activity, progression, and response to therapy.^6,7 Moreover, recent developments in positron emission tomography (PET) imaging have enabled the examination of microglial and astrocyte responses linked to MS progression.^8,9

A persistent challenge in MS diagnosis and management is the need for blood biomarkers. However, recent large population studies and the identification of relevant confounding factors may enable the clinical use of serum neurofilament light chain (sNfL), a marker of neuronal damage sNfL, for disease monitoring.^10,11 Other biomarkers such as serum glial fibrillary acid protein (GFAP),¹² antibodies,^13,14 and metabolites¹⁵ may provide additional tools to monitor disease progression and response to therapy.

MS disease forms

Relapsing remitting MS (RRMS) is the most common clinical form of MS diagnosed in 85%–90% of patients between the ages 20–40, characterized by symptomatic attacks (relapses) followed by periods of recovery (remissions). Clinically isolated syndrome (CIS) refers to a first episode of neurologic symptoms followed by complete or partial recovery. The incidental detection of MS-like CNS lesions in the absence of symptoms often leads to a diagnosis of radiologically isolated syndrome, considered a pre- or sub-clinical form of MS.¹⁶

RRMS often transitions into secondary progressive MS (SPMS), characterized by the progressive and irreversible accumulation of neurologic disability. Furthermore, 10%–15% of patients display a progressive disease course from diagnosis (primary-progressive MS, PPMS). However, early sub-clinical symptoms may interfere with the differentiation of SPMS and PPMS. In addition, relapses may hide disease progression, which may be present even during early MS stages.¹⁷ Thus, it has been suggested that specific disease outcomes, such as progression independent of relapse activity, might be more clinically useful.¹⁸ While RRMS is 2–4 times more frequent in females than in males, PPMS shows increased prevalence in males,¹ suggesting that sex-specific factors (e.g., hormones) influence disease onset and progression.¹⁹

MS pathology

CNS lesions at their earliest stage are called active lesions, featuring myelin loss, myelin debris, immune cell infiltration (particularly in the perivascular space), BBB disruption, reactive gliosis, and axonal damage. Active lesions are mostly linked to early disease in RRMS patients.²⁰ Inactive lesions depict areas of old demyelinating injury with minimal glia and immune cell presence and activity; they become more abundant in patients with extended disease duration.²⁰

Mixed active/inactive lesions–also known as chronic active or smoldering lesions–are also demyelinated but show a border of activated microglia/macrophages with foamy or ramified phagocytic phenotypes, axonal damage, subtle BBB disruption, and some immune cell infiltration adjacent to a largely hypocellular center with minimal microglia/macrophage presence and moderate T cell infiltration.²⁰ Interestingly, active/inactive lesions have been associated with MS progression and are more frequent in older patients.⁵ These and other findings suggest a role for age as a determinant of disease progression, probably because of deficits in CNS repair mechanisms and/or pathways that regulate peripheral and compartmentalized CNS inflammation. These findings also suggest that active/inactive lesions may be useful to monitor the response of therapies aimed to arrest MS progression.

Please note that CNS pathology in MS is not restricted to focal lesions. Diffuse injury, atrophy, BBB dysregulation, perivascular astrogliosis, and/or non-demyelinating inflammation are detected in both white and gray matter.²⁰ Moreover, inflammatory immune cell infiltrates and ectopic lymphoid follicles are detected in the meninges of MS patients, in association with severe cortical pathology, neurodegeneration, and a progressive disease course.^21,22 Thus, multiple CNS regions and tissue microenvironments are involved in MS pathology, even within the same patient, reflecting the contribution of multiple mechanisms to disease pathology.

MS PREDISPOSING FACTORS

MS develops as a result of a dynamic interplay between genetic, environmental, and lifestyle factors²³ (Figure 1). These factors modulate pathogenic processes even before MS diagnosis, in a prodromal phase that may display depression, anxiety, gastrointestinal issues, and elevated sNfL levels indicative of neuroaxonal degeneration.²⁴

Figure 1. Genetic and environmental factors that shape MS pathogenesis.

(A) Genetic studies linked MS susceptibility to autosomal and X chromosome genes. HLA variants were shown to promote pathogenic cross-talk between B and T lymphocytes. Autosomal variations are comparatively understudied, but increasing evidence suggest their involvement in dysregulated lymphocyte and glial responses. The impact of X-linked gene variants remains to be determined.

(B) The gut microbiome is altered in MS. Specific bacterial species have been shown to promote pathogenic T and B cell responses while limiting Tregs.

(C) EBV infection has been postulated to be a trigger of CNS autoimmunity in MS by boosting the production of pathogenic factors by B cells, by increasing the APC function of B cells, and by triggering cross-reactive responses with CNS antigens by molecular mimicry.

(D) MS-promoting responses of immune and glial cells are influenced by environmental factors (e.g., exposure to chemicals) and lifestyle choices (e.g., diet).

Genetics

The lifetime risk of developing MS in first-degree relatives and monozygotic twins of affected individuals was calculated to be more than 7-fold and 100-fold higher than in the general population, respectively, suggesting that genetic factors control MS susceptibility. Initial studies linked MS susceptibility to the MHC locus, paving the way for the identification of additional genetic factors²⁵ (Table 1). The HLA gene cluster in the polymorphic MHC locus is considered a major genetic MS risk factor, with notable variants in class II HLA-DRB1 (e.g., HLA-DRB1*15:01) and HLA-DPB1 genes and class I HLA-A and HLA-B genes.²⁵ In addition, a recent large study identified 233 genetic variants linked to MS susceptibility, 200 of which are located outside the MHC region, and one in the X chromosome²⁶ (Figure 1A). Several of these non-MHC MS susceptibility genes play immune functions (e.g., IL2RA, IL7RA, and CD58) and are enriched in adaptive and innate immune cells (e.g., T cells and microglia).²⁶ Interestingly, known gene variants only explain ~48% of MS genetic predisposition,²⁶ suggesting that additional predisposing genetic factors are yet to be identified. Ongoing studies aim to elucidate the mechanisms through which genetic variants linked to MS contribute to disease pathogenesis and progression. For example, the HLA-DR15 haplotype was shown to promote the activation of autoreactive T cells by antigen-presenting B cells,^27,28 while MS risk variants linked to NF-κB signaling²⁹ and MERTK³⁰ were linked to astrocyte and microglial responses linked to MS progression.

Table 1.

Representative genes linked to MS

Chromosome	Key Susceptibility Loci	Function(s)	Expression	Mechanism	p-value of Genome Wide Significancea
6	HLA Class II and I regions (E.g. HLA-DRB1, HLA-DPB1, HLA-A, HLA-B)	Adaptive immune regulation and function	Ubiquitous	Antigen presentation	1.62E-1916 - 4.95E-08
16	CLEC16A (C-type lectin domain containing 16)	Regulator of mitophagy	Ubiquitous	Antigen presentation; inflammatory response	3.70E-71 - 4.09E-09
1	CD58 (lymphocyte function-associated antigen 3, LFA3)	CD2 ligand; cell adhesion molecule; costimulatory molecule	Macrophages; dendritic cells	T cell activation	4.55E-70
10	IL2RA (IL-2 receptor alpha or CD25)	High affinity receptor for cytokine IL-2	T and B cells	T cell activation	2.96E-65 - 1.76E-15
12	TNFRSF1A (TNF receptor superfamily member 1A, CD120a)	TNFα receptor	Ubiquitous	Inflammatory response	2.24E-47 - 4.13E-10
3	CD86 (B7-2)	Ligand for CD28 and CTLA-4; costimulatory molecule	Macrophages; dendritic cells; B cells	T cell activation	6.98E-43 - 8.32E-10
1	EVI5 (ecotropic viral integration Site 5)	Regulator of cell cycle and cell division	Ubiquitous	Unclear	2.95E-30
11	CD6	ALCAM ligand; costimulatory receptor	T cells	T cell activation; cell trafficking	2.00E-29
5	IL7R (IL-7 receptor, CD127)	IL-7 receptor	Ubiquitous	T cell proliferation	1.58E-28
17	STAT3 (signal transducer and activator of transcription 3)	Transcription factor	Ubiquitous	T cell polarization	2.32E-28
11	CXCR5 (C-X-C motif chemokine receptor 5, CD185)	CXCL13 receptor	B cells	Cell trafficking	4.53E-26 - 2.10E-19
1	Vascular cell adhesion molecule 1 (VCAM1)	VLA-4 ligand; cell trafficking molecule	Ubiquitous	Cell trafficking	1.11E-22
20	CYP24A1 (Cytochrome P450 24A1)	Mitochondrial enzyme	Ubiquitous	Vitamin D metabolism	1.92E-19 - 3.13E-10
20	CD40	Receptor for CD40L; costimulatory molecule	Macrophages; dendritic cells; B cells	T cell activation	5.28E-19
16	IRF8 (interferon regulatory factor 8)	Transcription factor	Macrophages; dendritic cells; B cells	Transcription control	2.83E-17
3	EOMES (Eomesodermin, TBR2)	Transcription factor	T lymphocytes and NK cells	T cell response	1.55E-16
6	ATXN1 (ataxin 1)	DNA binding protein	Ubiquitous	B-cell modulation	1.62E-13
2	MERTK (MER proto-oncogene tyrosine kinase)	Kinase	Microglia	Phagocytosis	1.11E-11
X	VGLL1 (vestigial like family member 1)	Transcription cofactor	Unclear	Unclear	6.86E-09

^ap values obtained from the International Multiple Sclerosis Genetics Consortium.²⁶

Microbiome

The microbiome has gained major attention in MS.^31,32 In particular, the intestinal microbiome has been shown to promote the development of CNS-reactive pathogenic T cells³³ and modulate the activity of CNS-resident cells.^15,34,35 Interestingly, the transplantation of intestinal microbiota from MS patients worsened experimental autoimmune encephalomyelitis (EAE), a pre-clinical MS model.^36,37 Indeed, analyses of the MS intestinal microbiome revealed alterations linked to pro- and anti-inflammatory responses (Figure 1B). Notably, the genera Akkermansia was found to be elevated in RRMS and progressive MS and promote T helper 1 responses.^36-38 However, other studies linked Akkermansia to lower MS clinical disability³⁹ and showed that the administration of specific Akkermansia strains ameliorate EAE.^39,40 These findings suggest strain-specific effects of Akkermansia on mechanisms of MS pathogenesis.

Associations between the MS intestinal microbiome and regulatory T cells (Tregs) have also been described. The genera Parabacteroides, which is reduced in MS patients, was shown to stimulate IL-10⁺ Tregs.³⁶ Moreover, transplantation of microbiota from MS patients reduces IL-10 production in EAE.³⁷ Conversely, it was recently shown that IgA-producing plasma cells induced in the gut migrate to the CNS and suppress inflammation via IL-10 production.⁴¹ IgA-producing plasma cells reactive with the intestinal microbiome were recently detected in active MS lesions.⁴² Collectively, these studies highlight the role of the microbiome in the control of MS pathology. Future studies should translate these findings into therapeutic interventions, while extending these investigations to the microbiome in other tissues. For example, it was recently shown that the lung microbiome regulates CNS inflammation in rat EAE,⁴³ but overall, little is known about lung microbiome perturbations in MS.

Microbial infections

Charcot’s associate, Pierre Marie, noted that MS is often brought about during an infection.⁴⁴ Epstein-Barr virus (EBV) infection is associated with MS, as highlighted by the recent analysis of a large cohort of blood samples obtained before MS diagnosis, which established that the risk of MS is increased 32-fold after infection with EBV but not with other viruses.⁴⁵ The mechanisms through which EBV may contribute to MS development are many, including the triggering of CNS autoimmunity because of dysregulated T cell activation and/or molecular mimicry with EBV antigens (Figure 1C). Because of its tropism for B cells, EBV may boost their ability to activate pathogenic T cells. In addition, EBV-reactive T cells can cross-react with CNS antigens,^46,47 and EBV was also found to induce a self-reactive B cell immune response targeting the oligodendrocyte and astrocyte antigen GlialCAM.⁴⁸ However, these findings do not imply that EBV causes MS, but rather that immune mechanisms linked to EBV infection might cooperate with other environmental and/or genetic factors to trigger disease initiation in susceptible individuals. Novel vaccines may enable preventive interventions targeting EBV in MS and other diseases,⁴⁹ with the major caveat that vaccination should be performed at an early age, posing an important challenge for clinical trial design and overall feasibility.

Other factors

Race, ethnicity, latitude, diet, obesity, smoking, and environmental exposures (e.g., sunlight, pollutants) are also thought to contribute to MS development.^23,50 Studies of the mechanisms involved in the environmental regulation of MS showed, for example, that the nutrient methionine promotes CNS inflammation via the epigenetic reprogramming of CD4⁺ T lymphocytes,⁵¹ and that the herbicide linuron boosts astrocyte pathogenic activities driven by the transcription factor XBP1.⁵² Thus, combined epidemiologic and mechanistic studies may identify both novel MS predisposing factors and mechanisms of disease pathogenesis.

MECHANISMS OF DISEASE PATHOLOGY

Extensive analyses of patient samples, interventional clinical trials and pre-clinical model studies established a central role for peripheral leukocytes (T cells, B cells, myeloid cells) in MS pathology. Their aberrant responses damage oligodendrocytes and neurons, while activating CNS-resident cells such as microglia and astrocytes. Thus, MS pathology can be linked to four interdependent processes controlled by genetic and environmental factors: (1) activation of self-reactive lymphocytes, (2) CNS immune invasion, (3) immunoregulation, and (4) progressive neurodegeneration. Targeting these diverse processes with combinatorial approaches is needed for the effective therapeutic management of MS.

Activation of self-reactive lymphocytes

Decades of basic, translational, and clinical research established MS as an autoimmune disease. Seminal reports on the induction of EAE by immunization with CNS antigens supported a role for T cell autoimmunity in MS pathogenesis, while providing useful pre-clinical models. It should be kept in mind that different EAE models recapitulate specific aspects of MS, including an isolated acute CNS autoimmune attack induced in the C57BL/6 mouse strain by immunization with myelin oligodendrocyte glycoprotein (MOG)_35-55, a relapsing-remitting course induced in SJL mice by immunization with proteolipid protein (PLP)_139-151, or secondary progressive disease induced in non-obese diabetic (NOD) mice by immunization with MOG_35-55. Other EAE models are based on the adoptive transfer of myelin-specific T cells, or the transgenic expression of myelin-specific T cell receptor, which results in spontaneous EAE development. Indeed, no EAE model captures all the inflammatory and neurodegenerative processes linked to MS. One additional limitation is that most EAE models are driven by CD4⁺ T cells targeting one or a few CNS antigens, and may not recapitulate the complex autoimmune responses driven by CD4⁺ cells, CD8⁺ T cells and B cells in MS.

Self-reactive lymphocytes

CD8⁺ and CD4⁺ T cells reactive with myelin and other antigens are detected in active MS lesions.⁵³ Important insights on the role of T cells in MS were provided by reports of EAE induction in naive animals by the adoptive transfer of myelin-reactive T cells.^54,55 Although CD8⁺ T cells are dominant in MS lesions, more is known about CD4⁺ T cell specificity and regulation in MS, reflecting technical limitations for the analysis of human CD8⁺ T cells and the paucity of pre-clinical models for their investigation. However, recent developments have re-invigorated the study of CD8⁺ T cells in EAE.^56,57

CD4⁺ T helper (T_H) cells are classified based on the expression of cytokines, transcription factors and effector functions. IFNγ⁺ T_H1 cells controlled by the transcription factor Tbet, and IL-17⁺ T_H17 cells controlled by the transcription factor RORγt play pivotal roles in MS and EAE pathology⁵⁸ (Figure 2A). Importantly, these T_H subtypes include functionally different subsets controlled by additional transcriptional regulators. For example, non-pathogenic T_H17 cells show a limited ability to induce tissue pathology and express transcriptional modules linked to immune regulation, which include Il10 and Ahr.^59,60 Conversely, pathogenic T_H17 cells promote tissue destruction and express IL-23-driven transcriptional programs which include Csf2, Ifng and Tbx21 among other genes. Interestingly, increased T_H1 and T_H17 cell responses in MS have been linked to acute relapses and other disease outcomes.^61,62 Further analyses of T_H subsets reported increased pathogenic IFNγ⁺ IL-17⁺ T_H17 cells in MS relapses, and increased IL-10⁺ IL-17⁺ T_H17 cells in stable RRMS patients.⁶³ When analyzing these findings in the context of disease monitoring and treatment, it should be kept in mind that these T_H17 subsets may be interconvertible in vivo, as suggested by the AHR-induced conversion of pathogenic T_H17 cells into IL-10⁺ anti-inflammatory T cells,⁶⁴ and the IL-23-driven conversion of intestinal non-pathogenic TH17 cells into EAE-promoting pathogenic CXCR6⁺ T_H17 cells.⁶⁵ Thus, the analysis of a few cytokines or transcription factors may fail to assign T_H cells to a specific subtype and more in-depth analyses may be needed, for example, single-cell epigenetic-based lineage tracing.⁶⁶

Figure 2. Immune mechanisms and CNS invasion in MS.

(A) APCs (DCs, macrophages, B cells) and T cells first interact in the periphery, leading to activation and dysregulation of peripheral immunity and the secretion of inflammatory molecules.

(B) Peripheral immune cells invade the CNS through the BLMB, BCSFB, and BBB where they can interact with local APC subsets.

(C) Impaired Treg suppressive activity contributes to MS pathogenesis.

The adoptive transfer of CNS-specific T_H1 or T_H17 cells induces EAE, but different T_H subtypes induce pathology in different CNS locations, suggesting differences in homing and tissue interactions.⁶⁷ Indeed, the mechanisms used by T_H1 and T_H17 cells to promote CNS pathology are diverse, involving crosstalk with peripheral immune cells and CNS-resident cells. For example, T_H1 cells stimulate macrophages and microglia and hinder oligodendrocyte precursor cell survival and differentiation.^68-70 T_H17 cells disrupt the BBB, promote inflammatory phagocyte infiltration, modulate glial functions, and interact with oligodendrocytes and neurons to promote axonal damage and neurodegeneration.^70-72

Interactions between myeloid and CD4⁺ T cells are important contributors to MS pathogenesis (Figure 2A). Pathogenic T cells in MS and EAE produce granulocyte-macrophage colony-stimulating factor (GM-CSF),^73-77 which triggers the release of Ly6C^hi classical monocytes from the bone marrow and boosts their pathogenic activities,⁷⁸ highlighting the role of CD4⁺ T cell-monocyte interactions in CNS pathology. Within the CNS, GM-CSF promotes microglial activation,⁷⁹ and in combination with IFNγ, it differentiates Ly6C^hi monocytes into inflammatory macrophages.⁶⁸ In addition, GM-CSF boosts the differentiation of disease-promoting astrocyte subsets,⁷² while interfering with anti-inflammatory astrocyte functions.³⁵ Interestingly, chronic inflammation induces GM-CSF production by astrocytes,^15,52,74,80 suggesting a positive feedback loop that perpetuates pathogenic myeloid cell and astrocyte responses in progressive MS.

CD8⁺ T cells constitute the predominant T cell population in MS lesions⁵⁷ (Figure 2A). CD8⁺ T cells bearing tissue-resident memory phenotypes were detected in mixed active/inactive lesions and shown to be clonally expanded in the CSF of MS patients.^81,82 Interestingly, tissue resident CD8⁺ T cells were recently identified as putative drivers of compartmentalized autoimmune CNS damage.^83,84 Thus, novel experimental approaches^85-87 and pre-clinical models should enable a better characterization of CD8⁺ T cell responses and their dysregulation in MS.

B cells contribute to MS pathology through multiple mechanisms (Figure 2A). OCBs are a classic feature of MS, but the contribution of antibodies to disease pathology is still debated. Interestingly, the accumulation of B cells in meningeal ectopic follicles is linked to cortical pathology in MS,²² probably reflecting the contribution of B-cell produced GM-CSF, TNFα, IL-6, IL-12, IL-15 and other soluble factors in the promotion of inflammation⁸⁸ and neurotoxicity.⁸⁹ In addition, B cells play important roles as antigen-presenting cells (APCs).⁹⁰ Indeed, the beneficial effects of B-cell targeting antibodies in MS have been linked to the suppression of T cell activation.⁹⁰ However, it is still not clear whether these B cell targeted therapies interfere with T cell activation in CNS ectopic follicles, and whether additional therapeutic targets should be identified to target specific B cell populations.

Antigen presentation

The peripheral priming of naive T cells and their reactivation in the CNS are essential steps in EAE and MS pathology (Figures 2A and 2B). The analysis of clinical samples identified antigens recognized by the autoimmune T cell response in MS, including myelin basic protein (MBP), MOG, and PLP, among others (Table 2).^27,48,53 The breadth of the autoimmune response distinguishes MS from other more focalized CNS demyelinating diseases, such as neuromyelitis optica (characterized by antibodies to aquaporin 4), MOG antibody disease, and acute disseminated encephalomyelitis (both characterized by antibodies to MOG)⁹¹ (Table 2).

Table 2.

Representative MS autoantigens

Autoantigen	Expression	Function	Reacting Cell Types
Myelin basic protein (MBP)a	Oligodendrocytes	Myelin-associated	T and B cells
Myelin oligodendrocyte glycoprotein (MOG)b	Oligodendrocytes	Myelin-associated	T and B cells
Proteolipid protein (PLP)	Oligodendrocytes	Myelin-associated	T and B cells
Myelin-associated glycoprotein (MAG)	Oligodendrocytes	Myelin-associated	T and B cells
Myelin-associated oligodendrocyte basic protein (MOBP)	Oligodendrocytes	Myelin-associated	T cells
2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase)	Oligodendrocytes	Myelin-associated	T and B cells
Transaldolase	Oligodendrocytes	Enzyme	T and B cells
Transketolase	Oligodendrocytes	Enzyme	B cells
KIR4.1	Oligodendrocyte; astrocytes	Potassium channel	B cells
GlialCAM (HEPACAM)a	Oligodendrocytes; astrocytes	Cell adhesion	B cells
Reticulon 4 (NOGO-A)	Oligodendrocytes; neurons	Inhibitor of neurite outgrowth	B cells
Contactin-2	Neurons	Cell adhesion	T cells
Neurofilament light chain	Neurons	Cytoskeleton	B cells
Tubulin	Neurons	Cytoskeleton	B cells
Neurofascin	Neurons	Cell adhesion	B cells
RAS guanyl-releasing protein 2 (RASGRP2)	Neurons	Nucleotide exchange factor	T cells
β-synuclein (SNCB)	Neurons	Synaptic protein	T cells
Prokineticin 2 (PROK2)	Neurons	Circadian clock	T cells
Reticulon 3 (RTN3)	Neurons	Protein transporter	T cells
Synaptosome associated protein 91 (CALM)	Neurons	Vesicle transport	T cells
Anoctamin 2 (TMEM16B)a	Neurons; astrocytes	Chlorine channel	B cells
S100β	Astrocytes	Ca2+ binding protein	T cells
Aquaporin 4c	Astrocytes	Water channel	B cells
Fatty acid. binding protein 7 (FABP7)	Astrocytes	Hydrophobic ligand transporter	T cells
GDP-l-fucose synthase (GFUS)	Ubiquitous	Enzyme	T cells
αB-crystallin (CRYAB)	Ubiquitous	Small heat shock protein	T and B cells

^aautoantigens cross-reactive with EBV antigens.

^balso in MOG antibody disease and acute disseminated encephalomyelitis.

^calso in neuromyelitis optica spectrum disorders.

The specific autoantigens targeted by the immune response changes during the course of MS as a result of mechanisms of intra- and inter-molecular epitope spreading.⁵⁸ Indeed, the analysis of pediatric MS samples with antigen microarrays established epitope spreading as an early pathogenic event, probably driven by self-immunization against antigens released from tissue damaged by the autoimmune response.⁹² In addition, infections and the commensal microbiome also influence the specificity of the MS autoimmune response via molecular mimicry,^48,53 further promoting patient-to-patient heterogeneity. However, autoimmune targets linked to specific MS pathogenic processes have been identified,^13,93,94 suggesting that novel antigen identification methods^85-87 may enable patient staging and monitoring, and guide the development of antigen-specific immunomodulatory therapies.

Pre-clinical studies and the analysis of patient samples detected the drainage of antigens and dendritic cells drain from the CNS into cervical lymph nodes (CLNs), where they initiate T cell responses.⁹⁵ Reports on meningeal lymphatic vessels draining antigens and immune cells from the CSF and the meninges into the CLNs further support these findings.^96,97 However, a recent publication reported a central role for APCs in the leptomeninges, but not in the dura or CLNs, in T cell activation in EAE and MS.⁹⁸ Thus, future studies should evaluate the relative contribution of these anatomic locations in the acute and chronic activation of CNS-reactive T cells in MS, and in other neurologic diseases.

Strikingly, anatomically distant sites participate in the control of CNS-reactive T cells. For example, in EAE, T cells home to different CNS locations as a function of their priming in inguinal or mesenteric lymph nodes.⁹⁹ Indeed, commensal bacteria in mucosal compartments such as the gut, skin and lungs modulate effector or regulatory activities in myelin-reactive T cells.^{33,65,100-102} These findings highlight potential roles of the microbiome in the control of autoimmune responses through the production of factors that modulate T cell activation and polarization, and via the expression of cross-reactive microbial antigens which may activate self-reactive T cells.

APCs

Dendritic cells (DCs), macrophages, and B cells are considered major APCs involved in T cell activation in MS and EAE. Surprisingly, recent data suggest that microglia do not play a central APC role, at least in acute EAE models.^103,104

Peripheral monocytes that cross the BBB differentiate into perivascular DCs that promote T_H1 and T_H17 responses.^105,106 DCs containing myelin debris are detected in the perivascular space of active MS lesions and in the meninges near infiltrating T lymphocytes.^106,107 In EAE, conventional type 2 DCs (cDC2s) dwell in the meninges and reactivate pathogenic T cells.^103,105,108 DCs are also detected in the choroid plexus in EAE and MS but their capacity to reactivate encephalitogenic T cells remains to be determined.^103,109 Indeed, some of these T cell-APC interactions may dampen T cell autoimmunity, rather than boost it.¹¹⁰

A macrophage subset capable of antigen presentation to T cells was identified on the abluminal side of BBB blood vessels and also in active and smoldering MS lesions.^111,112 Macrophages were identified as the largest meningeal APC subset at peak EAE, surpassing DCs by ~4-fold and promoting CNS T cell infiltration.¹⁰² In MS, leptomeningeal macrophages were associated with cortical lesions and with meningeal lymphoid-like structures linked to worse disease outcome.²² More recently, MHC-II⁺ macrophages in dural sinuses were shown to present CNS antigens to T cells during homeostatic CNS surveillance, in addition to cDC2s.¹⁰⁸ DCs and macrophages are also present in the choroid plexus. Thus, the relative contribution of dural and choroidal APCs to MS pathogenesis remains to be defined.

Mechanistic studies on B-cell-targeting disease modifying therapies (DMTs) underscore the role of B cells as APCs in MS.^88,90 Indeed, B cells induce the proliferation of CNS-recruited T_H1 and T_H17 cells in MS.²⁷ Moreover, B cells are detected in the perivascular space and in meningeal lymphoid-like structures adjacent to subpial cortical lesions.^22,113 Collectively, these findings highlight the potential of DCs, macrophages, and B cells as targets for the therapeutic induction of antigen-specific tolerance. However, reports on T cell activation by type 3 innate lymphoid cells and neutrophils in autoimmune neuroinflammation suggest that additional APCs may also play important roles in the control of autoimmune T cells in MS^114,115; but little is known about their contribution to different disease stages and therapeutic potential.

CNS immune invasion

CNS T cell infiltration involves three major CNS barriers (Figure 2B); a fourth meningeal layer named the subarachnoid lymphatic-like membrane has been recently identified in the mouse and human subarachnoid space,¹¹⁶ but its role in EAE and MS remains to be determined: (1) The BBB is the main gateway to the CNS parenchyma.¹¹⁷ (2) The blood-leptomenin-geal barrier (BLMB) provides access to subpial CNS regions and, to some extent, the CSF.¹¹⁸ More recently, immune cell trafficking from the skull bone marrow into the meninges was described, potentially through channels whose roles in disease pathogenesis are not clearly defined.^119,120 (3) The choroid plexus represents the main portal linking the peripheral blood with the CSF.¹²¹

Migration through these CNS barriers involves cell-cell interactions mediated by cell adhesion molecules, chemokines, and their receptors. These interactions are mediated by classical molecules such as ICAM-1/LFA-1 and VCAM-1/VLA-4, and more recently identified ones such as DICAM, MCAM, ALCAM, and JAML.¹²² Importantly, these molecules are potential therapeutic targets to block the access of peripheral lymphocytes to the CNS, as exemplified by the VLA-4 blocking antibody natalizumab. However, unspecific therapeutic approaches, such as natalizumab, also interfere with physiologic mechanisms of CNS immune surveillance that limit the reactivation of common viruses, and thus could enable the development of progressive multifocal leukoencephalopathy.¹²³

Immunoregulation

Multiple mechanisms prevent tissue damage caused by dysregulated immune responses. CD4⁺ FoxP3⁺ Tregs and IL-10-producing type 1 regulatory (Tr1) cells have been extensively studied in MS and EAE.^50,124-127 In EAE, Tregs have been shown to suppress the development of autoimmune neuroinflammation and promote remyelination ^128,129 while Tr1 cells have been assigned a more dominant role in the resolution of CNS inflammation, for example through their ability to suppress microglial and astrocyte responses via IL-10 production.^130,131 Indeed, the induction of IL-10⁺ Tr1 cells by nasal anti-CD3 is undergoing clinical testing in MS. Similarly, IL-4 is reportedly neuroprotective in EAE,¹³² suggesting its potential for MS treatment.

Several observations suggest Treg dysfunction in MS, including reports of Treg decreased abundance, suppressive function, stability, migratory capacity and/or the relative resistance of effector T cells.¹²⁴ In addition, it has been suggested that epitope spreading gives rise to pathogenic T cell clones that evade regulation by antigen-specific Tregs and may be associated with MS relapses.⁵⁸ Interestingly, genetic variants linked to MS map to genes associated with immunoregulation, such as IL2RA and CTLA4.¹³³

CD8⁺ T cells endowed with regulatory function have also been described.^134-136 A decrease in their frequency and function has been reported in MS patients during relapses and to some extent in progressive MS patients.^137,138 However, less is known about the regulatory mechanisms used by CD8⁺ regulatory T cells and their clinical relevance, in part due to the lack of specific markers to unequivocally define, characterize and study them. The recent characterization of CD8⁺ regulatory T cells characterized by the expression of inhibitory killer immunoglobulin-like receptors (KIRs) is likely to open new avenues for their investigation in MS.¹³⁹

B cells also limit CNS inflammation, while promoting oligodendrogenesis and remyelination,^140-142 but it is still unclear whether defects in B cell-driven immune regulation are linked to specific MS stages. Nevertheless, IL-10⁺ B cells controlled by the intestinal microbiome were recently reported to migrate to the CNS and suppress MS pathogenesis.⁴¹ The characterization of these and other immunoregulatory mechanisms may guide the development of antigen-specific therapies for MS and other autoimmune diseases.

CNS INFLAMMATION AND MS PROGRESSION

A pressing challenge in MS research is the identification of mechanisms of disease progression and therapeutic targets to arrest it. Indeed, most therapies targeting peripheral immune cells show limited efficacy in progressive MS, suggesting that compartmentalized CNS inflammation drives disease progression.^143,144 Astrocytes, microglia, and oligodendrocytes play largely non-overlapping roles in the control of neurotransmission, phagocytosis, inflammation, neurodegeneration, and myelination.^145-147 Importantly, each of these glial cell types is comprised of specialized cell subsets.^72,148-151 Hence, the development of therapies for progressive MS requires the identification of relevant cell subsets and targets for their specific modulation.

Astrocytes are the most abundant glial cell in the CNS, providing trophic and metabolic support for neurons, while recycling neurotransmitters from the extracellular space.¹⁴⁵ Microglia are resident macrophages that seed the CNS early during neurodevelopment and play key roles in the removal of cell debris and, together with astrocytes, the modulation of inflammation.¹⁴⁶ Finally, oligodendrocytes myelinate CNS neuronal axons to ensure proper conduction of action potentials, while providing additional support for neuron metabolism. Oligodendrocytes are also the main target of the autoimmune response in MS.¹⁵² Recent studies have highlighted not only the heterogeneity of these cell types across different CNS regions and activation states, but also their functional interactions.

Neuroimmune cross-talk within the CNS in MS

Astrocytes, oligodendrocytes, microglia, and neurons control development, inflammation, metabolism, and neuronal activity through complex cell-cell interactions (Figure 3).^145-147 Studies in MS patients and animal models identified vulnerabilities in defined neuron subsets (Figure 3A). An analysis of cortical lesions identified a population of layer 2-layer 3 cortical neurons that degenerate in MS,¹⁴⁹ which displayed the activation of pathways linked to cell stress and disrupted metabolism, including CLU, NEFL, PPIA, and long non-coding RNAs. These findings suggest that stress-associated programs in neurons are linked to disease pathology, although it remains to be determined whether these programs promote or limit pathology. Moreover, studies in a cortical lesion animal model suggest that the activation of neuronal activity precedes neurodegeneration.¹⁵³ For instance, elevated calcium signaling in dendritic spines precedes their elimination by macrophages or microglia. Consistent with these data, neural network dysfunction is detected in EAE (even in its remission phase) and in MS patients.^154,155 These findings suggest that perturbations in neuronal networks drive neurodegeneration-promoting programs. However, it is still not clear to what extent spinal cord neurodegeneration in pre-clinical models recapitulates findings in the cortex of MS patients.

Figure 3. Cell-cell interactions in the CNS.

(A) Mechanisms driving cortical pathology identified in pre-clinical models and clinical samples. Remyelination deficits have been linked to microglia-oligodendrocyte cross-talk mediated via a type I interferon response. Additional mechanisms linked to neuron dysfunction include long non-coding RNAs, mitochondrial and endoplasmic reticulum-associated signaling pathways, and synaptic pruning controlled by calcium signaling.

(B) Mechanisms that mediate cross-talk among CNS-resident and CNS-recruited immune cells. C3 has been implicated in the microglial pruning of neuronal synapses (top-left). Astrocyte-produced chemokines promote the recruitment of pro-inflammatory CCR2⁺ monocytes via CCL2 (bottom-left). Ligand-receptor pairs implicated in the cross-talk among CNS-resident astrocytes and microglia promote regulatory or pro-inflammatory signals (top-right). Bidirectional modulation of astrocyte-T cell responses can boost or limit MS pathogenesis via epigenetic modifications, apoptosis, and pro-inflammatory cytokine signaling (bottom-right).

CNS-infiltrating immune cells and resident glial cells promote neurodegeneration and contribute to MS progression¹⁵⁶ (Figure 3B). Indeed, pro-inflammatory monocyte recruitment to the CNS is correlated with EAE severity¹⁵⁷; astrocytes promote monocyte recruitment to the CNS.¹⁵⁸ In addition, inflammation-induced perturbations in astrocyte metabolism interfere with neuronal metabolic support.^159,160 Interestingly, dysfunctional redox metabolism activates the unfolded protein response (UPR),¹⁶¹ which can boost pathology-promoting astrocyte responses in multiple contexts.^52,162,163 The activation of the UPR-associated XBP1 transcription factor increases pro-inflammatory cytokine and chemokine production by astrocytes.⁵² Conversely, the specific ablation of XBP1 or its upstream activators IRE1a or SigmaR1 in astrocytes, ameliorates EAE, decreasing microglial activation and CNS infiltration by pro-inflammatory monocytes.^52,163 Collectively, these data suggest that the therapeutic modulation of CNS-resident cells and their interactions may identify novel therapeutic targets for progressive MS.

Astrocytes and cell-cell interactions are attractive candidates for therapeutic intervention in MS and other neurologic diseases,¹⁴⁵ but specific astrocyte responses and subsets have to be identified first. For example, the ablation of reactive astrocytes during the early phase of NOD EAE worsens the disease,⁸⁰ but astrocyte ablation in the chronic progressive phase leads to disease amelioration, suggesting the need to target specific astrocyte pathogenic activities and/or functional subsets.⁸⁰ Indeed, these pathogenic roles of astrocytes were driven by the dysregulation of sphingolipid metabolism through a signaling pathway involving cPLA2 and the MAVS protein, classically involved in anti-viral immune responses.¹⁵⁹ cPLA2-MAVS signaling boosted pro-inflammatory NF-κB signaling and decreased production of lactate, which supports neuronal metabolism. Interestingly, MAVS⁺ astrocytes also express complement C3, a marker of an astrocyte subset that secretes neurotoxic lipids.^164,165 C3 may also contribute to MS pathology by participating in microglial synaptic pruning.¹⁶⁶

Additional microglial and astrocyte functions have also been linked to disease pathology in single-cell analyses of MS CNS samples.¹¹¹ These studies agree with scRNA-seq analyses of microglia in MS patients¹⁴⁸ and reports of microbiome-regulated communication between specific astrocyte and microglial subsets.³⁴ Importantly, MS-associated microglial subpopulations bear transcriptional signatures similar to those found in other neurologic diseases.¹⁶⁷

Finally, it should be kept in mind that some glial subsets actively limit CNS pathology. For example, microglial subsets have been found to support myelin integrity,¹⁶⁸ limit neurodegeneration driven by oxidized phosphatidylcholine¹⁶⁹ and actively engulf pathogenic T_H17 cells.¹⁷⁰ Similarly, a subset of TRAIL⁺ astrocytes has been shown to induce apoptosis in pathogenic T cells.³⁵ Collectively, these data highlight the diversity of glial responses relevant to MS pathology, and the need to develop approaches for the monitoring and therapeutic modulation of specific glial subsets.

Interrogation of CNS-resident cell subsets in MS

The extent to which disease-associated microglial and astrocyte subsets interact to coordinate and regulate their responses is still not fully understood. However, new tools have enabled the study of glial subset interactions in health and disease. For instance, advanced sequencing methods have provided new insights. One example is PIC-seq, a scRNA-seq-based tool for the analysisof interacting cells isolated based on defined surface markers.¹⁷¹ PIC-seq was initially deployed to study DC-T cell biology, but it has been recently used to study CNS neuroimmune interactions.¹⁷²

Likewise, novel barcoding approaches have enabled the comprehensive investigation of cell-cell interactions in the CNS. For example, RABID-seq labels interacting cells in vivo using a virus-delivered RNA-encoded barcode that can be detected by scRNA-seq.¹⁶⁰ RABID-seq recently uncovered multiple interactions and communication mechanisms between astrocytes and microglia in the context of CNS inflammation, identifying Sema4D-PlexinB1/2 and EphrinB3-EphB3 signaling as candidate therapeutic targets for MS.

These and other approaches for the study of cell-cell interactions in vivo are complemented by novel platforms for the comprehensive investigation of mechanisms of cell communication in forward genetic screens, classic approaches to first screen phenotypes following systematic or random genetic perturbations and then identify the perturbed gene responsible. In particular, the need to enable forward genetic screens of cell-cell interaction mechanisms led to the development of SPEAC-seq (Systematic Perturbation of Encapsulated Associated Cells followed by sequencing). SPEAC-seq is based on the co-encapsulation in droplets of two cells: cell 1 harbors a random single guide RNA derived from a genome-wide CRISPR library while cell 2 expresses a reporter that indicates the effect that gene inactivation in cell 1 has on cell 2.¹⁷³ Combining SPEAC-seq with an in vivo Perturb-seq screen, we recently identified microglia-produced amphiregulin as a suppressor of EAE-promoting astrocyte responses. The microglial production of amphiregulin was induced by ST2 signaling triggered by IL-33 released from astrocytes during EAE. This regulatory loop was also functional in human astrocytes and microglia. These and other tools for the study of cell-cell interactions, their perturbations, and in-depth characterization of the subsets involved are likely to identify novel mechanisms of disease pathogenesis and therapeutic targets for progressive MS.

Similarly, microglial subsets inhibit remyelination through their interactions with oligodendrocytes,¹⁷⁴ such as via interferon alpha and beta receptor 2 (Figure 3A). It is possible that these microglial subsets cross-talk with oligodendrocyte subsets previously identified in EAE and MS, which express CDH20, MHC-II and other molecules that facilitate immune cell interactions.^150,151 Although the specific role of MHC-II expressed by this oligodendrocyte subset remains to be further investigated, the modulation of the interaction between oligodendrocytes and immune cells may enable effective remyelination therapies for MS.¹⁵² Indeed, secreted factors including IGF-1, antibodies, and axon guidance cues, regardless of their cellular source, control remyelination in pre-clinical MS models and may guide the development of new therapies.⁵

Finally, an important concept is the distinction between bona fide cell populations and transcriptional activation states. For example, multiple astrocyte subsets have been identified in EAE and MS,^{72,111,149,150} but it is still unknown if they constitute populations with defined functions.¹⁴⁵ Analogously, microglial subsets have been identified in MS and other disorders based on scRNA-seq transcriptional signatures,^148,167 but recent studies suggest that some of these subsets include myeloid cells of different origins, with opposing roles in CNS pathology.¹⁷⁵ Follow up studies on the origin, regulation and function of glial subsets identified in scRNA-seq analyses is needed. In this context, FIND-seq (Focused Interrogation of cells by Nucleic acid Detection and Sequencing) integrates nucleic acid cytometry and microfluidics to enable in-depth analyses of cell subsets of interest based on nucleic acid markers (RNA or DNA). In combination with genetic and perturbation approaches, FIND-seq may reveal the role of these subsets in the pathology of MS and other neurologic diseases.

THERAPEUTIC INTERVENTIONS

The development of disease modifying therapies (DMTs) for MS has been spectacular over the last three decades. Following IFN-β-1b approval in 1993, over 15 DMTs have been approved¹⁷⁶ (Table 3). Most DMTs target the peripheral immune system, with mechanisms of action broadly defined as immunomodulatory, anti-trafficking or immunodepleting.

Table 3.

Current MS therapies

Drug	Indication	Main Impact	Mechanism of Action
IFN-β-1a	Relapsing MS	Immunomodulation	Inhibits T cell division and CNS infiltration; Induces Tregs and suppressor B cells
Glatiramer acetate	Relapsing MS	Immunomodulation	Competes with peptide binding to MHC
Teriflunomide	Relapsing MS	Immunomodulation	Pyrimidine synthesis inhibitor
Dimethyl fumarate	Relapsing MS	Immunomodulation	NRF2 activation/antioxidant signaling
Diroximel fumarate	Relapsing MS	Immunomodulation	NRF2 activation/antioxidant signaling
Fingolimod	Relapsing MS	Inhibition of immune cell trafficking	Sphingosine-1-phosphate (S1P) receptor modulator
Siponimod	Relapsing MS SPMS	Inhibition of immune cell trafficking	Sphingosine-1-phosphate (S1P) receptor modulator
Ozanimod	Relapsing MS	Inhibition of immune cell trafficking	Sphingosine-1-phosphate (S1P) receptor agonist
Ponesimod	Relapsing MS	Inhibition of immune cell trafficking	Sphingosine-1-phosphate receptor 1 (S1PR1) modulator
Natalizumab	RRMS	Inhibition of immune cell trafficking	Anti-VLA-4 monoclonal antibody
Cladribine	Relapsing MS	Immunodepletion	Purine analog
Alemtuzumab	Relapsing MS	Immunodepletion	Anti-CD52 monoclonal antibody
Ocrelizumab	Relapsing MS PPMS	Immunodepletion	Anti-CD20 monoclonal antibody
Ofatumumab	Relapsing MS	Immunodepletion	Anti-CD20 monoclonal antibody

Immunomodulatory therapies represent the earliest DMTs approved for MS. Their clinical efficacy results from targeting multiple immune cell subsets (mainly T cells) to re-balance the ratio of pro-to anti-inflammatory responses.^177,178 These therapeutic agents include IFN-β, glatiramer acetate, teriflunomide and dimethyl fumarate.

Anti-trafficking therapies interfere with immune cell migration into the CNS at two different stages. The sphingosine-1-phosphate (S1P) receptor modulators fingolimod and siponimod sequester lymphocytes within lymph nodes, although S1P signaling also affects CNS-resident cells such as astrocytes.^179,180 In contrast, natalizumab directly inhibits the recruitment of circulating leukocytes into the CNS by blocking VLA-4. Of note, this mechanism of action interferes with physiologic CNS immune patrolling and has been linked to John Cunningham virus reactivation and progressive multifocal leukoencephalopathy in natalizumab-treated patients.¹⁸¹

Immunodepleting therapies target adaptive immune cells. Approved to treat RRMS and active progressive MS, cladribine (purine analogue) and alemtuzumab (anti-CD52) target T cells and B cells, whereas ocrelizumab, ofatumumab and ublituximab (anti-CD20) specifically deplete B cells.^140,182,183 In 2017, ocrelizumab became the first DMT approved for PPMS.¹⁸⁴ B-cell directed therapies targeting CD20 show strong suppressive effects on pathogenic T cell responses. However, it should be noted that patients on anti-CD20 therapy and fingolimod show reduced responses to vaccination against COVID-19.¹⁸⁵ In addition, the recombinant protein atacicept, which blocks B-cell function and proliferation, failed to control MS relapses and triggered disease exacerbations,¹⁸⁶ probably because of its activity against IL-10⁺ anti-inflammatory IgA⁺ B cells,⁴¹ which are not targeted by anti-CD20 antibodies. These findings highlight the role of B cells in the control of T cell autoimmunity in MS, as well as the challenges linked to their therapeutic targeting.

NOVEL THERAPEUTIC APPROACHES AND TARGETS

Currently available DMTs show limited success in halting MS progression.¹⁴⁴ However, numerous promising treatments are currently in advanced clinical trials. One example is that of Bruton tyrosine kinase (BTK) inhibitors, which target B cells and peripheral myeloid cells, limiting their pathogenic cross-talk with T cells.¹⁸⁷ In addition, due to their capacity to cross the BBB, some BTK inhibitors may suppress microglial pathogenic functions and promote remyelination,^188,189 offering hope for the treatment of progressive MS.

One desired feature of novel therapies for MS and other autoimmune diseases is the re-establishment of antigen-specific tolerance, without impacting protective immunity against infectious agents and tumors. However, the development of these tolerogenic approaches is not risk free, as indicated by disease exacerbation in MS patients treated with a modified myelin peptide thought to be endowed with tolerogenic activity.¹⁹⁰ Promising approaches for the induction of antigen-specific tolerance involve cell- or nanoparticle-based methods. The former includes the administration of antigen-loaded tolerogenic DCs, autoantigen-specific Treg cells or chimeric antigen receptor T cell-based approaches.^191,192 However, logistic challenges limit the clinical implementation of cell-based therapies.

Alternatively, antigen-specific immune tolerance has been induced with nanoparticles loaded with myelin antigens and tolerogenic adjuvants such as aryl hydrocarbon receptor agonists and other factors.^130,193,194 More recently, mRNA vaccines modified to prevent immune activation were shown to induce immune tolerance in EAE,¹⁹⁵ recapitulating previous observations made with DNA vaccines.¹⁹⁶ Interestingly, these nanoparticle- and mRNA-based approaches induce bystander suppression, an important feature considering the challenge imposed by epitope spreading and the heterogeneity of the autoantigens targeted in different MS patients. It remains to be seen whether the lack of an adjuvant enforcing tolerance in mRNA vaccines induces tolerogenic responses strong enough to arrest the ongoing inflammatory response in MS.

Finally, the identification of environmental factors associated with MS development opens new therapeutic avenues ranging from EBV vaccines⁴⁹ to supplementation with specific probiotics, their metabolites, or even synthetic probiotics engineered to produce therapeutic molecules.¹⁹⁷ Additional therapeutic avenues for MS could stem from studies focused on circadian biology and pollution. For example, the reported effects of melatonin and pollutants on regulatory and pro-inflammatory responses,^52,198 may provide further insight into environmentally controlled pathways that play important but less well understood roles in MS pathogenesis.²³

Conclusions

Halting MS progression remains an unmet clinical need. Recent single-cell studies have provided unique insights into MS pathology, uncovering CNS genomic signatures associated with disease.^{35,72,111,148-150,163} The integration of these findings with in situ profiling methods will enable the identification of perturbed neuroimmune interactions relevant for MS progression amenable to therapeutic targeting. It should be kept in mind, however, that age is strongly associated with MS progression, reflecting the impact of senescence on pro-inflammatory, neurodegenerative, and tissue repair responses.¹⁹⁹ For instance, senescence in progressive MS was recently linked to impaired oligodendrocyte progenitor cell maturation and remyelination.²⁰⁰ Thus, the study of aging may provide novel insights on the biology of MS progression.

In summary, we have witnessed remarkable progress in our understanding of MS pathogenesis and the development of DMTs. In addition, groundbreaking studies have uncovered environmental and genetic factors relevant for MS pathogenesis. The identification of mechanisms of disease progression, and biomarkers to monitor and target specific cell-subsets involved in MS, may provide new therapeutic strategies for MS.