Myelin Basic Protein Peptide Research Articles

Citrullination of myelin basic protein induces a Th17-cell response in healthy individuals and enhances the presentation of MBP85-99 in patients with multiple sclerosis

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Myelin Basic Protein Fragmentation by Engineered Human Proteasomes with Different Catalytic Phenotypes Revealed Direct Peptide Ligands of MS-Associated and Protective HLA Class I Molecules.

Proteasomes exist in mammalian cells in multiple combinatorial variants due to the diverse regulatory particles and exchange of catalytic subunits. Here, using biotin carboxyl carrier domain of transcarboxylase from Propionibacterium shermanii fused with different proteasome subunits of catalytic and regulatory particles, we report comprehensive characterization of highly homogenous one-step purified human constitutive and immune 20S and 26S/30S proteasomes. Hydrolysis of a multiple sclerosis (MS) autoantigen, myelin basic protein (MBP), by engineered human proteasomes with different catalytic phenotypes, revealed that peptides which may be directly loaded on the HLA class I molecules are produced mainly by immunoproteasomes. We detected at least five MBP immunodominant core regions, namely, LPRHRDTGIL, SLPQKSHGR, QDENPVVHFF, KGRGLSLSRF and GYGGRASDY. All peptides, except QDENPVVHFF, which originates from the encephalitogenic MBP part, were associated with HLA I alleles considered to increase MS risk. Prediction of the affinity of HLA class I to this peptide demonstrated that MS-protective HLA-A*44 and -B*35 molecules are high-affinity binders, whereas MS-associated HLA-A*23, -A*24, -A*26 and -B*51 molecules tend to have moderate to low affinity. The HLA-A*44 molecules may bind QDENPVVHFF and its deamidated form in several registers with unprecedently high affinity, probably linking its distinct protective phenotype with thymic depletion of the repertoire of autoreactive cytotoxic T cells or induction of CD8+ regulatory T cells, specific to the encephalitogenic MBP peptide.

Liposome-based nanoparticles impact on regulatory and effector phenotypes of macrophages and T cells in multiple Sclerosis patients

Current available treatments of Multiple Sclerosis (MS) reduce neuroinflammation acting on different targets on the immune system, but potentially lead to severe side effects and have a limited efficacy in slowing the progression of the disease. Here, we evaluated in vitro the immunomodulatory potential of a new class of nanoparticles – liposomes, constituted by a double-layer of phosphatidylserine (PSCho/PS), and double-faced, with an outer layer of phosphatidylserine and an inner layer of phosphatidic acid (PSCho/PA), either alone or in the presence of the myelin basic protein (MBP) peptide (residues 85–99) (PSCho/PS-MBP and PSCho/PA-MBP). Results showed that PSCho/PS are equally and efficiently internalized by pro- and anti-inflammatory macrophages (M1 and M2 respectively), while PSCho/PA were internalized better by M2 than M1. PSCho/PS liposomes were able to inhibit the secretion of innate pro-inflammatory cytokine IL-1β. PSCho/PS liposomes expanded Tregs, reducing Th1 and Th17 cells, while PSCho/PA liposomes were unable to dampen pro-inflammatory T cells and to promote immune-regulatory phenotype (Treg). The ability of PSCho/PS liposomes to up-regulate Treg cells was more pronounced in MS patients with high basal expression of M2 markers. PSCho/PS liposomes were more effective in decreasing Th1 (but not Th17) cells in MS patients with a disease duration >3 months. On the other hand, down-modulation of Th17 cells was evident in MS patients with active, Gadolinium enhancing lesions at MRI and in MS patients with a high basal expression of M1-associated markers in the monocytes. The same findings were observed for the modulation of MBP-driven Th1/Th17/Treg responses.These observations suggest that early MS associate to a hard-wired pro-Th1 phenotype of M1 that is lost later during disease course. On the other hand, acute inflammatory events reflect a temporary decrease of M2 phenotype that however is amenable to restauration upon treatment with PSCho/PS liposomes. Thus, together these data indicate that monocytes/macrophages may play an important regulatory function during MS course and suggest a role for PSCho/PS and PSCho/PS-MBP as new therapeutic tools to dampen the pro-inflammatory immune responses and to promote its regulatory branch.

Fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis and intestinal inflammation in multiple sclerosis.

Multiple Sclerosis (MS) has been reported to be associated with intestinal inflammation and gut dysbiosis. To elucidate the underlying biology of MS-linked gut inflammation, we investigated gut infiltration of immune cells during the development of spontaneous experimental autoimmune encephalomyelitis (EAE) in humanized transgenic (Tg) mice expressing HLA-DR2a and human T cell receptor (TCR) specific for myelin basic protein peptide (MBP87-99)/HLA-DR2a complexes. Strikingly, we noted the simultaneous development of EAE and colitis, suggesting a link between autoimmune diseases of the central nervous system (CNS) and intestinal inflammation. Examination of the colon in these mice revealed the infiltration of MBP-specific Th17 cells as well as recruitment of neutrophils. Furthermore, we observed that fecal Lipocalin-2 (Lcn-2), a biomarker of intestinal inflammation, was significantly elevated and predominantly produced by the gut-infiltrating neutrophils. We then extended our findings to MS patients and demonstrate that their fecal Lcn-2 levels are significantly elevated compared to healthy donors (HDs). The elevation of fecal Lcn-2 levels correlated with reduced bacterial diversity and increased levels of other intestinal inflammation markers including neutrophil elastase and calprotectin. Of interest, bacteria thought to be beneficial for inflammatory bowel disease (IBD) such as Anaerobutyricum, Blautia, and Roseburia, were reduced in fecal Lcn-2-high MS patients. We also observed a decreasing trend in serum acetate (a short-chain fatty acid) levels in MS Lcn-2-high patients compared to HDs. Furthermore, a decrease in the relative abundance of Blautia massiliensis was significantly associated with a reduction of acetate in the serum of MS patients. This study suggests that gut infiltration of Th17 cells and recruitment of neutrophils are associated with the development of gut dysbiosis and intestinal inflammation, and that fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis in multiple sclerosis.

Role of Helical Structure in MBP Immunodominant Peptides for Efficient IgM Antibody Recognition in Multiple Sclerosis.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81–106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76–116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81–106) (1) is recognized more efficiently by IgM antibodies than MBP (76–116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

Reactivity of Rheumatoid Arthritis-Associated Citrulline-Dependent Antibodies to Epstein-Barr Virus Nuclear Antigen1-3.

Rheumatoid arthritis (RA) is a chronic disease which causes joint inflammation and, ultimately, erosion of the underlying bone. Diagnosis of RA is based on the presence of biomarkers, such as anti-citrullinated protein antibodies (ACPA) and rheumatoid factors, along with clinical symptoms. Much evidence points to a link between the Epstein-Barr virus and RA. In this study, we analyzed ACPA reactivity to citrullinated peptides originating from Epstein-Barr nuclear antigens (EBNA1, EBNA2, and EBNA3) in order to elaborate the diagnostic potential of citrullinated EBNA peptides. Moreover, ACPA cross-reactivity to citrullinated peptides from myelin basic protein (MBP) was analyzed, as citrullinated MBP recently was described to be associated with multiple sclerosis, and some degree of sequence homology between MBP and citrullinated EBNA exists. A peptide from EBNA2, (EBNA2-A, GQGRGRWRG-Cit-GSKGRGRMH) reacted with approximately 70% of all RA sera, whereas only limited reactivity was detected to EBNA1 and EBNA3 peptides. Moreover, screening of ACPA reactivity to hybrid peptides of EBNA3-A (EPDSRDQQS-Cit-GQRRGDENRG) and EBNA2-A and peptides containing citrulline close to the N-terminal confirmed that ACPA sera contain different populations of ACPAs. No notable ACPA reactivity to MBP peptides was found, confirming that ACPAs are specific for RA, and that other factors than the presence of a central Cit-Gly motif are crucial for antibody binding. Collectively, these findings illustrate that citrullinated EBNA2 is an optimal candidate for ACPA detection, supporting current evidence that EBV is linked to RA onset.

Experimental Autoimmune Encephalomyelitis in the Mouse.

This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.

Active and Passive Immunization with Myelin Basic Protein as a Method for Early Treatment of Traumatic Spinal Cord Injury; a Meta-Analysis.

Introduction:Traumatic spinal cord injury (SCI), as a dangerous central nervous system damage, continues to threaten communities by imposing various disabilities and costs. Early adjustment of the immune system response using Myelin Basic Protein (MBP) immunization may prevent the SCI-related secondary damages. As a result, the current study is designed to review and analyse the evidence on active and passive immunization with MBP for treatment of traumatic SCI.Methods:Medline, Embase, Scopus, and Web of Science databases were systematically searched until the end of 2020. Criteria for inclusion in the current study included pre-clinical studies, which performed passive (injection of MBP-activated T cells) or active (administration of MBP or MBP-modified peptides) immunization with MBP after traumatic SCI. Exclusion criteria was defined as lack of a non-treated SCI group, lack of evaluation of locomotion, review studies, and combination therapy. Finally, analyses were conducted using STATA software, and a standardized mean difference (SMD) with a 95% confidence interval (CI) were reported.Results:Data from 17 papers were included in the present study. Finally, analysis of these data showed that passive immunization (SMD=0.87; 95%CI: 0.19-1.55; p=0.012) and active immunization (SMD=2.08, 95%CI: 1.42-2.73; p<0.001) for/with MBP both have good efficacy in improving locomotion following traumatic SCI. However, significant heterogeneity was observed in both of them. The most important sources of heterogeneity in active immunization were differences in SCI models, route of administration, time interval between SCI and transplantation, and type of vaccine used. In passive immunization, however, these sources were the model of SCI and the time interval between SCI and transplantation. Although, there was substantial heterogeneity among studies, subgroup analysis showed that active immunization improved locomotion after traumatic SCI in all tested conditions (with differences in injury model, severity of injury, method of administration, different time interval between SCI to vaccination, etc.). Conclusion:The results of the present study demonstrated that immunization with MBP, especially in its active form, could significantly improve motor function following SCI in rats and mice. Therefore, it could be considered as a potential treatment in acute settings such as emergency departments. However, the safety of this method is still under debate. Therefore, it is recommended for future research to focus on the investigation of safety of MBP immunization in animal studies, before conducting human clinical trials.

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

Peculiarities of the Presentation of the Encephalitogenic MBP Peptide by HLA-DR Complexes Providing Protection and Predisposition to Multiple Sclerosis.

Predisposition to multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system, is due to various factors. The genetic component is considered one of the most important factors. HLA class II genes contribute the most to the development of MS. The HLA-DRB1*15 allele group is considered one of the main genetic risk factors predisposing to MS. The group of HLA-DRB1*01 alleles was shown to have a protective effect against this disease in the Russian population. In this work, we compared the binding of the encephalitogenic fragment of the myelin basic protein (MBP) to two HLA-DR complexes that provide protection against and predisposition to MS: HLA-DR1 (HLA-DRB1*0101) and HLA-DR15 (HLA-DRB1*1501), respectively. We found that the myelin peptide MBP88-100 binds to HLA-DR1 at a rate almost an order of magnitude lower than the viral peptide of hemagglutinin (HA). The same was true for the binding of MBP85-97 to HLA-DR15 in comparison with viral pp65. The structure of the C-terminal part of the peptide plays a key role in the binding to HLA-DR1 for equally high-affinity N-terminal regions of the peptides. The IC50 of the myelin peptide MBP88-100 competing with viral HA for binding to HLA-DR1 is almost an order of magnitude higher than that of HA. As for HA, the same was also true for the binding of MBP85-97 to HLA-DR15 in comparison with viral pp65. Thus, autoantigenic MBP cannot compete with the viral peptide for binding to protective HLA-DR1. However, it is more competitive than viral peptide for HLA-DR15.

HLA Class II Genotype Does Not Affect the Myelin Responsiveness of Multiple Sclerosis Patients.

Background: When aiming to restore myelin tolerance using antigen-specific treatment approaches in MS, the wide variety of myelin-derived antigens towards which immune responses are targeted in multiple sclerosis (MS) patients needs to be taken into account. Uncertainty remains as to whether the myelin reactivity pattern of a specific MS patient can be predicted based upon the human leukocyte antigen (HLA) class II haplotype of the patient. Methods: In this study, we analyzed the reactivity towards myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP) peptides using direct interferon (IFN)-γ enzyme-linked immune absorbent spot (ELISPOT). Next, the HLA class II haplotype profile was determined by next-generation sequencing. In doing so, we aimed to evaluate the possible association between the precursor frequency of myelin-reactive T cells and the HLA haplotype. Results: Reactivity towards any of the analyzed peptides could be demonstrated in 65.0% (13/20) of MS patients and in 60.0% (6/10) of healthy controls. At least one of the MS risk alleles HLA-DRB1*15:01, HLA-DQA1*01:02 and HLA-DQB1*06:02 was found in 70.0% (14/20) of patients and in 20.0% (2/10) of healthy controls. No difference in the presence of a myelin-specific response, nor in the frequency of myelin peptide-reactive precursor cells could be detected among carriers and non-carriers of these risk alleles. Conclusion: No association between HLA haplotype and myelin reactivity profile was present in our study population. This complicates the development of antigen-specific treatment approaches and implies the need for multi-epitope targeting in an HLA-unrestricted manner to fully address the wide variation in myelin responses and HLA profiles in a heterogeneous group of MS patients.

Engineered regulatory T cells expressing myelin-specific chimeric antigen receptors suppress EAE progression

The expansion of polyclonal T regulatory cells (Tregs) offers great promise for the treatment of immune-mediated diseases, such as multiple sclerosis (MS). However, polyclonal Tregs can be non-specifically immunosuppressive. Based on the advancements with chimeric antigen receptor (CAR) therapy in leukemia, we previously engineered Tregs to express a T-cell receptor (TCR) specific for a myelin basic protein (MBP) peptide. These TCR-engineered specific Tregs suppressed the proliferation of MBP-reactive T effector cells and ameliorated myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Herein, we extend this approach by creating human regulatory T cells expressing functional single-chain chimeric antigen receptors (scFv CAR), targeting either MBP or MOG. These scFv CAR-transduced Tregs retained FoxP3 and Helios, characteristic of Treg cells, after long-term expansion in vitro. Importantly, these engineered CNS targeting CAR-Tregs were able to suppress autoimmune pathology in EAE, demonstrating that these Tregs have the potential to be used as a cellular therapy for MS patients.

Peptide-Conjugated Silver Nanoparticle for Autoantibody Recognition.

In this work, we considered the autoantibodies proposed as putative biomarkers of demyelination taking into account their reactivity towards myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP). These myelin proteins are among the most commonly researched targets in the immunopathology of demyelinating diseases. In this context, the development of assays for autoantibody detection can contribute as a predictive value for the early diagnosis of demyelinating diseases. Hence, we aimed to address the application of silver nanoparticles (AgNPs) as a sensing device of autoantibodies. AgNPs were synthesized via a chemical reduction method and characterized using atomic force microscopy (AFM), X-ray diffractometry, dynamic light scattering, and UV-visible spectrophotometry. The process of peptide conjugation on the nanoparticles was also analyzed. The autoantibody recognition by the peptide-conjugated AgNPs was evaluated with UV-visible spectrophotometry, atomic force spectroscopy (AFS), and color changing. AgNPs exhibited spherical morphology, low polydispersity, face-centered cubic crystal structure, and an average size of 29.3±3.0 nm. The hydrodynamic diameter variation and AFM showed that the MBP peptide induced greater agglomeration, compared to MOG peptide. The AFS measurements indicated the efficient binding of peptides to the AgNPs maintaining their activity, revealed by typical adhesion force and shapes of curves. The absorption spectrum features were more affected by the interaction with the specific autoantibodies, which also caused a visible color change in suspension providing a qualitative response. We described a preliminary study of MOG- and MBP-conjugated AgNPs which showed to be applicable in the autoantibody recognition. These have promising implication in the searching for biological markers for diagnostic purposes in the demyelination context, in which the nanoscale sensing exploitation is recent.

Systemic immunization with altered myelin basic protein peptide produces sustained antidepressant-like effects

Immune dysregulation, specifically of inflammatory processes, has been linked to behavioral symptoms of depression in both human and rodent studies. Here, we evaluated the antidepressant effects of immunization with altered peptide ligands of myelin basic protein (MBP)-MBP87-99[A91, A96], MBP87-99[A91], and MBP87-99[R91, A96]-in different models of depression and examined the mechanism by which these peptides protect against stress-induced depression. We found that a single dose of subcutaneously administered MBP87-99[A91, A96] produced antidepressant-like effects by decreasing immobility in the forced swim test and by reducing the escape latency and escape failures in the learned helplessness paradigm. Moreover, immunization with MBP87-99[A91, A96] prevented and reversed depressive-like and anxiety-like behaviors that were induced by chronic unpredictable stress (CUS). However, MBP87-99[R91, A96] tended to aggravate CUS-induced anxiety-like behavior. Chronic stress increased the production of peripheral and central proinflammatory cytokines and induced the activation of microglia in the prelimbic cortex (PrL), which was blocked by MBP87-99[A91, A96]. Immunization with MBP-derived altered peptide ligands also rescued chronic stress-induced deficits in p11, phosphorylated cyclic adenosine monophosphate response element binding protein, and brain-derived neurotrophic factor expression. Moreover, microinjections of recombinant proinflammatory cytokines and the knockdown of p11 in the PrL blunted the antidepressant-like behavioral response to MBP87-99[A91, A96]. Altogether, these findings indicate that immunization with altered MBP peptide produces prolonged antidepressant-like effects in rats, and the behavioral response is mediated by inflammatory factors (particularly interleukin-6), and p11 signaling in the PrL. Immune-neural interactions may impact central nervous system function and alter an individual's response to stress.

The 3A6-TCR/superagonist/HLA-DR2a complex shows similar interface and reduced flexibility compared to the complex with self-peptide.

T-cell receptor (TCR) recognition of the myelin basic protein (MBP) peptide presented by major histocompatibility complex (MHC) protein HLA-DR2a, one of the MHC class II alleles associated with multiple sclerosis, is highly variable. Interactions in the trimolecular complex between the TCR of the MBP83-99-specific T cell clone 3A6 with the MBP-peptide/HLA-DR2a (abbreviated TCR/pMHC) lead to substantially different proliferative responses when comparing the wild-type decapeptide MBP90-99 and a superagonist peptide, which differs mainly in the residues that point toward the TCR. Here, we investigate the influence of the peptide sequence on the interface and intrinsic plasticity of the TCR/pMHC trimolecular and pMHC bimolecular complexes by molecular dynamics simulations. The intermolecular contacts at the TCR/pMHC interface are similar for the complexes with the superagonist and the MBP self-peptide. The orientation angle between TCR and pMHC fluctuates less in the complex with the superagonist peptide. Thus, the higher structural stability of the TCR/pMHC tripartite complex with the superagonist peptide, rather than a major difference in binding mode with respect to the self-peptide, seems to be responsible for the stronger proliferative response.

Loading rate of exogenous and autoantigenic determinants on major histocompatibility complex class ii mediates resistance to multiple sclerosis

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.

Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis.

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.

Loading Rate of Exogenous and Autoantigenic Determinants on Major Histocompatibility Complex Class II Mediates Resistance to Multiple Sclerosis.

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.

PD-1 expression is upregulated on adapted Tcells in experimental autoimmune encephalomyelitis but is not required to maintain a hyporesponsive state.

T cell adaptation is an important peripheral tolerogenic process which ensures that the T cell population can respond effectively to pathogens but remains tolerant to self‐antigens. We probed the mechanisms of T cell adaptation using an experimental autoimmune encephalomyelitis (EAE) model in which the fate of autopathogenic T cells could be followed. We demonstrated that immunisation with a high dose of myelin basic protein (MBP) peptide and complete Freund's adjuvant failed to effectively initiate EAE, in contrast to low dose MBP peptide immunisation which readily induced disease. The proportion of autopathogenic CD4+ T cells in the central nervous system (CNS) of mice immunised with a high dose of MBP peptide was not significantly different to mice immunised with a low dose. However, autopathogenic T cells in mice immunised with high dose MBP peptide had an unresponsive phenotype in ex vivo recall assays. Importantly, whilst expression of PD‐1 was increased on adapted CD4+ T cells within the CNS, loss of PD‐1 function did not prevent the development of the unresponsive state. The lack of a role for PD‐1 in the acquisition of the adapted state stands in striking contrast to the reported functional importance of PD‐1 in T cell unresponsiveness in other disease models.

GDP-l-fucose synthase is a CD4+ T cell-specific autoantigen in DRB3*02:02 patients with multiple sclerosis.

Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4+ T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)-l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid-infiltrating CD4+ T cells from HLA-DRB3*-positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.