Autoimmune demyelinating disorders of the central nervous system (CNS) are a major cause of chronic neurologic disability in young adults. They include multiple sclerosis (MS), for which the autoimmune targets of CNS-infiltrating T and B lymphocytes are not yet fully understood, myelin oligodendrocyte glycoprotein (MOG) antibody associated disorders (MOGAD) which were previously considered to be part of the MS spectrum and in which the oligodendrocyte protein MOG is a major candidate autoantigen, and neuromyelitis optica spectrum disorders (NMOSD) with an autoimmune response that targets aquaporin-4 on astrocytes. MS and MOGAD share many features including inflammatory demyelination in brain, oligodendrocyte death and infiltration by T and B lymphocytes and macrophages, and are distinguishable mainly by clinical, pathological and radiological criteria, and the presence of serum MOG IgG antibodies and predominance of brain-infiltrating CD4+ over CD8+ T cells in MOGAD. Experimental autoimmune encephalomyelitis (EAE) models show many of the immune and pathological features of MOGAD and possibly also of MS. CD4+IFN-γ- (Th1) and CD4+IL-17-producing (Th17) specific for MOG35-55 peptide are sufficient to induce inflammatory demyelination in EAE, while MOG-specific antibodies further enhance demyelination in the context of myelin-specific CD4+ T cell inflammation. Therapeutic strategies that aim to re-establish peripheral tolerance selectively in myelin specific CD4+ T cells are therefore a promising way forward for therapy in human autoimmune demyelination.
Early studies to induce peripheral T cell tolerance in MS focused on controlling myelin-specific T cells by directly targeting the T cell receptor (TCR). Delivery of myelin protein or peptide sequences, mainly based on myelin basic protein (MBP), by different administration routes, as DNA vaccines, or in altered forms, all showed encouraging results in animal models, but did not perform well in clinical studies in MS patients. More recent approaches aim to harness immune modulatory properties of the innate immune system, particularly through generation of alternatively-activated tolerogenic dendritic cells (DC) and other myeloid antigen-presenting cells (APC) that are essential for peripheral immune tolerance and preventing autoimmunity by limiting effector T cells and inducing regulatory T cells (Treg). Encephalitogenic peptides chemically-coupled to syngeneic mouse splenocytes or microparticles inhibit EAE in a peptide-specific manner via induction of macrophage-mediated immunomodulatory mechanisms, and coupled to autologous human PBMC reduce antigen-specific T cell responses in MS patients. Also, mouse and human MHC-peptide constructs treat EAE, and enhance type 2 (“M2”) macrophages and repair in the CNS. Direct targeting of T cell antigens to immature DC and macrophages using ligands for C-type lectin receptors such as DEC-205, DCIR2, or mannose receptor (CD206, MR), is another promising approach. Recently, a clinical study in patients with MS and NMOSD showed that intravenous administration of tolerogenic DC loaded with CNS antigens is safe and feasible. The therapeutic efficacy of APC targeting approaches in CNS demyelinating diseases remains to be shown.
We previously showed that MOG35-55 conjugated to oxidized mannan polysaccharide (OM-MOG) protects animals against the clinical and pathological features of MOG-EAE in a peptide-specific manner across different MHC class II (MHCII) types in prophylactic and therapeutic applications. Protection is associated with the maturation of functionally deficient Th1 and Th17 cells, but the mechanism of tolerance has remained elusive. Here we show that OM-MOG both protects against and treats MOG-EAE in humanized HLA-DR2b transgenic mice expressing the human MHCII MS candidate susceptibility genes DRA*0101 and DRB1*1501 (DR2b.Ab° mice). OM-MOG treatment rapidly and almost completely reverses clinical symptoms, reducing inflammatory infiltrates, microglia activation, demyelination, and axon damage in the spinal cord of DR2b.Ab° mice. Supporting studies in B6 mice showed that OM-MOG treatment is associated with a peripheral type 2 myeloid cell response, induction of T cell anergy, preservation of axons within lesions and increased expression of genes associated with recovery of myelin and neurons in the spinal cord. In a Hellenic cohort of MS patients, a high proportion showed peripheral T cell proliferation responses to hMOG35-55, as well as other myelin peptide antigens, across different HLA-DRB1 genotypes. The results suggest that patients with CNS demyelinating diseases in which the autoimmune targets are known might be candidates for peptide-specific therapy with OM-peptides independent of HLA-DRB1 genotype.
The protocol for sampling blood from MS patients and healthy individuals for T cell proliferation assays was reviewed and approved by the Ethics committee of the Aeginition Hospital of the National Kapodistrian University of Athens as being consistent with the Declaration of Helsinki (Protocol No: 7BΣH46Ψ8N2-B66, 13/05/2015). The donors signed a written informed consent before donating blood for this study. Considering the core association of the HLA-DRB1*1501 allele with MS risk, clinical course and therapeutic response, including in the Hellenic population, we genotyped patients for HLA-DRB1 and included individuals carrying the DRB1*15 allele in our sample. DNA extraction was performed with the “QIAamp Blood Maxi” commercial kit (QIAGEN, Germany) while DRB1 genotyping was performed using a commercial kit based on the PCR-SSO (Polymerase-Chain-Reaction, Sequence-Specific Oligonucleotide) technique. This method depends on reverse hybridization (Line Probe Assay, INNO-LiPA, Low Resolution, DRB1 Amp Plus, Innogenetics, Fujirebio, Europe) according to the manufacturer’s protocol, for all the specificities included in the HLA Nomenclature of 2012. PBMC were isolated and analyzed for antigen-specific T cell responses by proliferation assay with thymidine incorporation, based on a previously described method. Briefly, blood was collected in EDTA-coated tubes, diluted 1:1 with RPMI containing Glutamax and stored at 4°C for 3 days. PBMC were isolated by Biocoll (Biochrom) density gradient centrifugation, seeded in 96-well plates at 1.5 x 10 5 cells/well in RPMI with 5 μM of each peptide. Forty-eight wells were incubated per peptide, 24 or 48 wells with medium alone as negative control and 6 wells with plate bound anti-CD3 mAb (2 μg/ml) (clone HIT3a; BD Biosciences, San Diego, CA, USA), or phytohaemagglutinin (PHA; 5 μg/ml), as positive control. On day 7 cells were pulsed with 1 μCi/well [3 H]-thymidine (Amersham Radiochemicals) for 16 h. Thymidine incorporation was measured using a β scintillation counter and cell proliferation was calculated as described above. For each sample, any of the 48 replicas showing CPM higher than the mean + 3 SD of all the unstimulated cells were considered as positive. Peptide-specific responses were considered positive with two or more positive replicas.
Table 1 Clinical and demographic data of multiple sclerosis (MS) and healthy control blood donors.
Humanized HLA-DR2b transgenic mice expressing chimeric constructs in which the peptide binding part of mouse I-E are replaced by the corresponding domains of human DR2b molecules (DRA*0101 and DRB*1501), were crossed with mice that lack all mouse MHCII genes, to generate the DR2b.Ab° mice used in this study. DR2b.Ab° mice were maintained on a mixed C57BL/6 (B6)/DBA genetic background. MOG-specific T cell receptor (2D2) transgenic mice on a B6 genetic background have been described previously (Tcra2D2, Tcrb2D2)1Kuch/J; The Jackson Laboratory). Inbred B6 mice (Harlan) were used for supporting experiments. Mice were kept under specific pathogen-free conditions in the Department of Animal Models for Biomedical Research of the Hellenic Pasteur Institute. All animal experiments complied with ARRIVE guidelines and were carried out in accordance with the local Ethical Committee guidelines on the use of experimental animals at the Hellenic Pasteur Institute, were approved by the national authorities and conformed to EU Directive 2010/63/EU for animal experiments.
Peptides MBP13-32, MBP83-99, MOG1-20, mouse and human MOG35-55 (S42 in MOG, P42 in hMOG, respectively), PLP139-154, PLP178-191, and (KG)5-MOG35-55 were synthesized, and (KG)5-MOG35-55 was conjugated to OM as previously described. For this study we used OM, in which mannose polysaccharides are converted to polyaldehydes by sodium periodate treatment, and lack complement-activating activity. OM-MOG was administered to mice using prophylactic and therapeutic protocols. In the prophylactic protocol, groups of 6- to 8-week-old female DR2b.Ab° or B6 mice were injected intradermally (i.d.) on the flanks with 100 μl of carbonate-bicarbonate buffer pH 9.0 (vehicle) containing OM-MOG (30 μg MOG peptide equivalent/injection and 700 μg OM equivalent/injection) or vehicle control. Three consecutive injections were performed at 15-day intervals, and EAE was induced 15 days after the last injection. In the therapeutic protocol, groups of female DR2b.Ab° or B6 mice were injected as above, starting after disease onset when the clinical score of each individual mouse reached 2 (between days 12-14 post-immunization for EAE), and repeated every 2 days for a total of 5 injections per mouse.
MOG-EAE was induced in groups of at least 6- to 8-week-old (for therapeutic administration), or 12- to 14-week-old (after prophylactic administration), female DR2b.Ab° mice, or Ab° control mice, by s.c. tail-base injection of 200 μg MOG in 100 μl saline emulsified in an equal volume of complete Freund's adjuvant (CFA) (Sigma-Aldrich). CFA was supplemented with 400 μg/injection of H37Ra Mycobacterium tuberculosis (D
