Abstract

Experimental autoimmune encephalomyelitis (EAE) is the principal animal model for multiple sclerosis (MS). Here we demonstrate that the induction of EAE generates successive waves of clonally expanded CD4, CD8, and γδ T cells in the blood and central nervous system. In addition, we also demonstrate major expansions of CD8 T cells in the blood and cerebrospinal fluid of patients with MS. In EAE, we find that most expanded CD4 T cells are specific for myelin peptide MOG_35-55. In contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8 T cells inhibit disease by suppressing the proliferation of MOG-specific CD4 T cells. Overall, our results suggest that the induction of autoreactive CD4 T cells triggers an opposing mobilization of regulatory CD8 T cells.

Abstract

B cells play a central role in multiple sclerosis (MS) pathology. B and plasma cells, and their antibody products, are found at elevated levels in MS patient cerebrospinal fluid and demyelinating lesions, and B cell-predominant germinal center-like aggregates are observed in relapsing and secondary progressive patients MS patients. In Phase 3 clinical trials, relapsing MS patients treated with anti-CD20 monoclonal antibodies demonstrate reduced MRI lesion activity, clinical relapses, and disability progression; and early Phase 2 clinical studies of Bruton’s tyrosine kinase inhibitors show reduction in active MRI lesions. B and plasma cells may modulate MS disease activity through multiple mechanisms including antigen presentation, pro- and anti-inflammatory cytokine secretion, and auto-antibody production. Molecular and functional analyses of B cell populations have indicated that B cell subpopulations and mature antibody producing cells (plasmablasts and plasma cells) display complex interactions with other immune cell populations to modulate MS disease activity in the periphery and within the central nervous system. These effects are further modified by secreted immunoglobulins acting independently or in concert with other cellular immune responses. Sophisticated translational investigations and experimental models are primed to expand our understanding of MS B cell pathophysiology and advance the development of new B cell therapeutics.

Background

B cell depleting therapies are highly effective treatments for multiple sclerosis (MS). B cells are more numerous in active than inactive lesions, and their intrathecal clonal expansion and oligoclonal band production are hallmarks of MS. B cells also present antigens to T cells and secrete inflammatory cytokines. The antigenic specificity of individual B cells in cerebrospinal fluid (CSF) obtained from patients with early MS may help further clarify the role of B cells in MS biology.

Objectives

To determine the viral and autoantigen repertoire of CSF-derived, class-switched B cells from untreated, early MS patients.

Methods

We performed single cell immunoglobulin sequencing on CSF plasma cells, plasmablasts, and class switched memory B cells from 9 untreated patients: five with relapsing remitting MS (RRMS) and four with clinically isolated syndrome (CIS). The interval between the first attack and lumbar puncture ranged from 1 - 222 days (median 67 days). Brain and spinal cord MRIs performed concurrently with lumbar punctures revealed 5/9 patients with gadolinium enhancing lesions.

Using paired heavy and light chain immunoglobulin sequences, we generated 75 monoclonal antibodies (mAbs) and screened them on a suite of unbiased antigen discovery platforms: 1) mouse brain tissue staining, 2) whole human proteome programmable phage display, 3) pan-viral programmable phage display, 4) mouse and human brain immunoprecipitation mass spectrometry.

Results

The mAbs showed diverse antigen specificities. Candidate antigens were primarily ubiquitously expressed, intracellular proteins; however, a minority were macromolecules associated with the plasma membrane and/or enriched in brain tissue. Shared antigenic targets were occasionally identified within subjects but were rarely identified across subjects, with the latter including cytoskeletal proteins. For two mAbs, high-confidence antigens with prima facie relevance to MS were identified: 1) a white matter-restricted lipid species, and 2) an Epstein-Barr virus-interacting host protein.

Conclusions

Using our panel of 75 mAbs derived from plasma cells, plasmablasts, and class-switched memory B cells found in the CSF of early, untreated RRMS/CIS patients, we identified a diverse repertoire of antigenic targets, with a majority comprised of intracellular host proteins.

Background

Both adaptive and innate immune cells infiltrate the CNS during multiple sclerosis (MS) and in its animal model experimental autoimmune encephalomyelitis (EAE). Pathogenic T cells are known as key drivers of the disease while treatment strategies specifically targeting inflammatory processes directly within the CNS parenchyma behind the blood-brain-barrier are still missing.

Objectives

In this project we aimed to unravel CNS-internal mechanisms to counteract the disease in both the murine and the human system by modulating T cell pathogenicity in the CNS.

Methods

We used intravital 2-photon microscopy to visualize T cells and their interactions with neurons and microglia in the CNS of EAE-diseased animals and in organotypic slice cultures. Using immunohistochemistry and flow cytometry we further analyzed surface molecules on T cells and myeloid cells to gain a deeper understanding of the molecular pathways behind the CNS-response to T cell infiltration. Human T cells isolated from blood and cerebrospinal fluid of MS patients were analyzed ex vivo to allow translation to the human system.

Results

We discovered that detrimental effects of pathogenic Th17 cells can be mediated via a cell-to-cell-interaction-dependent vesicular glutamate release pathway that induces damage in neurons. In a counteractive mechanism, microglia attempted to remove fully viable invaded pathogenic T cells from the CNS tissue during the disease. This T cell engulfment was mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc) and enhancement of GlcNAc signaling in the CNS ameliorated disease outcome. In human subjects, we could show that GlcNAc was highly exposed on T cells in the CNS of MS patients. Of note, this GlcNAc exposure on CNS-infiltrated T cells was significantly increased as compared to peripheral T cells.

Conclusions

Myeloid cells are able to directly react to pathogenic T cell infiltration by engulfing living T cells in a GlcNAc-dependent manner. Increased GlcNAc-exposure on T cells in the human CNS reflects high susceptibility of infiltrated T cells to get eliminated by myeloid cells, suggesting that pharmacological enhancement of T cell engulfment could particularly affect T cells in the target organ of MS.

Background

Several lines of evidence indicate essential roles for B cells in the pathogenesis of multiple sclerosis (MS). B cells act as potent antigen-presenting cells and throughout the chronic course of MS, B cell-follicle like structures can be found in the meninges of MS patients. However, whether and how B cells interact with CNS-resident cells, such as microglia and astrocytes to possibly modulate chronic progression of MS remains unclear.

Objectives

In the present study, we aimed at analyzing the interaction of B cells with CNS-resident cells in modulation of chronic CNS inflammation.

Methods

Primary microglia and astrocytes were generated from newborn C57BL/6 mice and were incubated with activated B cells or their supernatants. IL-6 and IL-10 production was abolished by genetic ablation or neutralization of IL-6 or IL-10 using specific antibodies. Thereafter, CNS resident cells were co-cultured with MOG-specific T cells. Further, C57BL/6 mice were depleted of B cells by 3 weekly subcutaneous injections of 0.2 mg murine anti-CD20 prior to immunization with MOG peptide p35-55, a setting in which B cells remain naïve. Microglial and astrocytic activation/modulation was assessed by ELISA, flow cytometry, immunohistochemistry and qRT-PCR.

Results

Incubation of primary microglia or astrocytes with IL-10-neutralized B cell supernatant or co-culture with IL-10-deficient B cells resulted in increased pro-inflammatory cytokine production, an upregulation of co-stimulatory molecules as well as an enhanced capacity to activate T cells as antigen-presenting cells. In vivo, depletion of naïve B cells worsened clinical severity of experimental encephalomyelitis (EAE) and increased the number of CNS infiltrating immune cells. Exacerbation was associated with an enhanced expression of molecules involved in antigen-presentation on microglia cells as well as an upregulation of pro-inflammatory gene products in astrocytes.

Conclusions

These findings highlight that B cells substantially alter the functional status of microglia and astrocytes in chronic CNS inflammation. Specifically, B cell-derived IL-10 is capable of diminishing the inflammatory responses of CNS-resident microglia and astrocytes. Our observation suggests that regulatory B cell function may be important in controlling CNS intrinsic inflammation associated with clinical progression.