Roland Martin

Immunology of Multiple Sclerosis.

Multiple sclerosis (MS) is considered a prototypic autoimmune disease of the central nervous system (CNS). A complex genetic background with the HLA-DR15 haplotype as the main genetic risk factor and over 100 mostly immune-related minor risk alleles as well as several environmental factors contribute to the etiology of MS. With respect to pathomechanisms, autoimmune inflammation in early MS is primarily mediated by adaptive immune responses and involves autoreactive T cells, B cells, and antibodies, while the later, chronic stages of MS are characterized by a compartmentalized immune response in the CNS with activated microglia and macrophages. A host of immune cells and mediators can contribute to the autoimmune process, but CNS-related factors such as localization of lesions, vulnerability of oligodendrocytes, neurons/axons, and secondary metabolic changes all play a role in the heterogeneous expression of the disease, including different pathologic lesion patterns, neuroimaging findings, disease courses, and severity and response to treatment.

Antigen-Specific Tolerance by Autologous Myelin Peptide–Coupled Cells: A Phase 1 Trial in Multiple Sclerosis

Antigen-coupled cells result in antigen-specific tolerization for treatment of multiple sclerosis. Multiple Sclerosis Therapy Attached at the Hip In multiple sclerosis (MS), a patient’s own immune cells are thought to attack antigens in the brain and spinal cord. One approach to prevent this attack is through tolerization: harnessing one way the body itself attempts to prevent autoimmunity. Ideally, this would happen in an antigen-specific way so that autoimmunity is blocked, while the protective functions of the immune system remain intact. There has been considerable success inducing antigen-specific tolerance in mouse models of MS by chemically coupling the antigen of choice to carrier cells. Now, Lutterotti et al. take this approach into human patients. The authors coupled peripheral blood mononuclear cells from MS patients with seven different myelin peptides thought to be potentially antigenic in MS. Patients who had T cell responses restricted to at least one of the peptides tested were selected. Indeed, patients who received the highest doses of antigen-coupled cells demonstrated decreases in antigen-specific T cell responses after therapy. Although the patient numbers are small in this first-in-human study, the safety, feasibility, and early results suggest that this approach may provide a promising avenue for future trials. Multiple sclerosis (MS) is a devastating inflammatory disease of the brain and spinal cord that is thought to result from an autoimmune attack directed against antigens in the central nervous system. The aim of this first-in-man trial was to assess the feasibility, safety, and tolerability of a tolerization regimen in MS patients that uses a single infusion of autologous peripheral blood mononuclear cells chemically coupled with seven myelin peptides (MOG1–20, MOG35–55, MBP13–32, MBP83–99, MBP111–129, MBP146–170, and PLP139–154). An open-label, single-center, dose-escalation study was performed in seven relapsing-remitting and two secondary progressive MS patients who were off-treatment for standard therapies. All patients had to show T cell reactivity against at least one of the myelin peptides used in the trial. Neurological, magnetic resonance imaging, laboratory, and immunological examinations were performed to assess the safety, tolerability, and in vivo mechanisms of action of this regimen. Administration of antigen-coupled cells was feasible, had a favorable safety profile, and was well tolerated in MS patients. Patients receiving the higher doses (>1 × 109) of peptide-coupled cells had a decrease in antigen-specific T cell responses after peptide-coupled cell therapy. In summary, this first-in-man clinical trial of autologous peptide-coupled cells in MS patients establishes the feasibility and indicates good tolerability and safety of this therapeutic approach.

Exploring the origins of grey matter damage in multiple sclerosis

Multiple sclerosis is characterized at the gross pathological level by the presence of widespread focal demyelinating lesions of the myelin-rich white matter. However, it is becoming clear that grey matter is not spared, even during the earliest phases of the disease. Furthermore, grey matter damage may have an important role both in physical and cognitive disability. Grey matter pathology involves both inflammatory and neurodegenerative mechanisms, but the relationship between the two is unclear. Histological, immunological and neuroimaging studies have provided new insight in this rapidly expanding field, and form the basis of the most recent hypotheses on the pathogenesis of grey matter damage.

T Lymphocyte Priming by Neutrophil Extracellular Traps Links Innate and Adaptive Immune Responses

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.

Identification of a Novel Risk Locus for Multiple Sclerosis at 13q31.3 by a Pooled Genome-Wide Scan of 500,000 Single Nucleotide Polymorphisms

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with an important genetic component and strongest association driven by the HLA genes. We performed a pooling-based genome-wide association study of 500,000 SNPs in order to find new loci associated with the disease. After applying several criteria, 320 SNPs were selected from the microarrays and individually genotyped in a first and independent Spanish Caucasian replication cohort. The 8 most significant SNPs validated in this cohort were also genotyped in a second US Caucasian replication cohort for confirmation. The most significant association was obtained for SNP rs3129934, which neighbors the HLA-DRB/DQA loci and validates our pooling-based strategy. The second strongest association signal was found for SNP rs1327328, which resides in an unannotated region of chromosome 13 but is in linkage disequilibrium with nearby functional elements that may play important roles in disease susceptibility. This region of chromosome 13 has not been previously identified in MS linkage genome screens and represents a novel risk locus for the disease.

Retinal Damage in Multiple Sclerosis Disease Subtypes Measured by High-Resolution Optical Coherence Tomography

Background. Optical coherence tomography (OCT) has facilitated characterisation of retinal alterations in MS patients. Only scarce and in part conflicting data exists on different MS subtypes. Objective. To analyse patterns of retinal changes in different subtypes of MS with latest spectral-domain technology. Methods. In a three-centre cross-sectional study 414 MS patients and 94 healthy controls underwent spectral-domain OCT examination. Results. Eyes of MS patients without a previous optic neuritis showed a significant reduction of both retinal nerve fibre layer (RNFL) thickness and total macular volume (TMV) compared to healthy controls independent of the MS subtype (P < 0.001 for all subtypes). RNFL thickness was lower in secondary progressive MS (SPMS) eyes compared to relapsing-remitting MS (RRMS) eyes (P = 0.007), and TMV was reduced in SPMS and primary progressive MS (PPMS) eyes compared to RRMS eyes (SPMS: P = 0.039, PPMS: P = 0.005). Independent of the subtype a more pronounced RNFL thinning and TMV reduction were found in eyes with a previous optic neuritis compared to unaffected eyes. Conclusion. Analysis of this large-scale cross-sectional dataset of MS patients studied with spectral-domain OCT confirmed and allows to generalize previous findings. Furthermore it carves out distinct patterns in different MS subtypes.

Non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes IL-17 producing mucosal-associated invariant T cells in multiple sclerosis

Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. Non-myeloablative conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+ FoxP3+ T cells and CD56(high) natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+ CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161(high) proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161(high)CD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab.

Dendritic cells signal T cells in the absence of exogenous antigen.

Interactions with self–major histocompatibility complex molecules on dendritic cells (DCs) are important for the survival of mature CD4+ T cells. We have followed the DC-mediated signal from the T cell surface to the nucleus and identified a pattern of activation that correlates with increased in vitro survival. This response is induced exclusively by DCs and is likely associated with a modulation of the T cell activation threshold. We have also found that DC-mediated activation results in antigen-independent cytokine gene expression, which points to a new role for DCs in shaping the cytokine milieu. Such antigen-independent activation of T cells may play a role in protective immunity, but may also induce and perpetuate autoimmune states such as multiple sclerosis.

Neutrophils in multiple sclerosis are characterized by a primed phenotype

Neutrophils are armed with proteases with indiscriminate histotoxic potential, and to minimize tissue injury, their activation involves priming with inflammatory mediators before cells are fully activated in a second step. Here, we show that neutrophils in multiple sclerosis patients are more numerous and exhibit a primed state based on reduced apoptosis, higher expression of TLR-2, fMLP receptor, IL-8 receptor and CD43, enhanced degranulation and oxidative burst as well as higher levels of neutrophil extracellular traps in serum. The chronic inflammatory environment in multiple sclerosis probably underlies this inappropriate neutrophil priming, which may result in enhanced neutrophil activation during infection.

Natalizumab treatment perturbs memory- and marginal zone-like B-cell homing in secondary lymphoid organs in multiple sclerosis

Natalizumab, an antibody against the α4 subunit of α4 integrins, has been approved for multiple sclerosis (MS) therapy based on its high efficacy and safety profile. However, natalizumab has been associated with the development of progressive multifocal leukoencephalopathy (PML), a disorder caused by JC virus (JCV) infection. In order to improve our understanding of the mechanism of action of natalizumab and to identify possible risk factors for PML development, we have characterized in detail the cell blood composition in MS patients treated with natalizumab for more than 30 months. Natalizumab induced the release of lymphoid‐ but not myeloid precursor cells, which resulted in a chronic increase ofT‐, NK‐ and particularly B cells. While the percentage of recent thymic emigrants (RTEs), naϊve, effector or memory T cells remained unchanged during treatment, a higher percentage of memory‐ and marginal zone (MZ)‐like, but not of naϊve B cells, was observed, which most likely is due to a decreased retention of these cells within the splenic MZ. The ability of natalizumab to influence B‐cell migration and homeostasis through the splenic MZ, where JCV has been detected, adds to the list of natalizumab effects and may contribute to PML development by disseminating JCV.