Autoantigen conformation influences both B- and T-cell responses and encephalitogenicity.

It has become increasingly clear that only antibodies recognizing conformation-dependent epitopes of myelin oligodendrocyte glycoprotein (MOG) have a demyelinating potential in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Nevertheless, for the induction of EAE, most studies to date have used MOG peptides or bacterially expressed MOG, neither of which contain the tertiary structure of the native antigen. Non-refolded recombinant human MOG does not induce EAE in DA rats. Therefore, we refolded this protein in order to assess the influence of MOG conformation on its pathogenicity in DA rats. DA rats immunized with refolded human MOG developed severe acute EAE. As expected, rats immunized with the refolded protein had a higher amount of conformational MOG antibodies present in serum. But in addition, a striking effect of MOG refolding on the generation of T-cell responses was found. Indeed, T-cell responses against the encephalitogenic MOG 91-108 epitope were greatly enhanced after refolding. Therefore, we conclude that refolding of MOG increases its pathogenicity both by generating conformation-dependent MOG antibodies and by enhancing its processing or/and presentation on MHC molecules. These data are important in regard to investigations of the pathogenic potential of many (auto)antigens.

Maternal MHC regulates generation of pathogenic antibodies and fetal MHC-encoded genes determine susceptibility in congenital heart block.

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1(av1) (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1(l)). Different anti-Ro52 Ab fine specificities were generated in RT1(av1) versus RT1(l) animals. Maternal and fetal influence was determined in an F(2) cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1(l) than RT1(av1) pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1(l) pups more frequently developed heart block than RT1(av1) pups after passive transfer of RT1(av1) anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52-positive pregnancies.

Naturally presented peptides on major histocompatibility complex I and II molecules eluted from central nervous system of multiple sclerosis patients.

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.

Advanced intercross line mapping suggests that ncf1 (ean6) regulates severity in an animal model of guillain-barre syndrome.

We here present the first genetic fine mapping of experimental autoimmune neuritis (EAN), the animal model of Guillain-Barré syndrome, in a rat advanced intercross line. We identified and refined a total of five quantitative trait loci on rat chromosomes 4, 10, and 12 (RNO4, RNO10, RNO12), showing linkage to splenic IFN-gamma secretion and disease severity. All quantitative trait loci were shared with other models of complex inflammatory diseases. The quantitative trait locus showing strongest linkage to clinical disease was Ean6 and spans 4.3 Mb on RNO12, harboring the neutrophil cytosolic factor 1 (Ncf1) among other genes. Polymorphisms in Ncf1, a member of the NADPH oxidase complex, have been associated with disease regulation in experimental arthritis and encephalomyelitis. We therefore tested the Ncf1 pathway by treating rats with a NADPH oxidase complex activator and ameliorated EAN compared the oil-treated control group. By proving the therapeutic effect of stimulating the NADPH oxidase complex, our data strongly suggest the first identification of a gene regulating peripheral nervous system inflammation. Taken together with previous reports, our findings suggest a general role of Ncf1 and oxidative burst in pathogenesis of experimental autoimmune animal models.

Characterization of the encephalitogenic immune response in a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) can be actively induced with the extracellular domain of myelin oligodendrocyte glycoprotein (MOG 1-125). MOG-EAE closely mimics multiple sclerosis (MS) especially as far as demyelination, lesion formation and axonal pathology are concerned. MOG 91-108 is the encephalitogenic stretch within MOG 1-125 in two EAE-susceptible MHC congenic LEW rat strains [LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n))] and DA (RT1(av1)) rats. In LEW.1AV1 rats, disease could be induced with MOG 96-104 and to a lesser extent with MOG 98-106, whereas in LEW.1N rats, only MOG 98-106 was pathogenic. Both peptides bound well to their restricting MHC class II molecules, i.e., RT1.D(n) in the LEW.1N rat and RT1.B(a) in the LEW.1AV1 rat. TCR spectratyping of MOG 91-108 immunized LEW.1N, LEW.1AV1 and DA rats revealed that MHC class II determined the TCRBV preference of CNS infiltrating T cells. The data demonstrate that the most critical factor in inducing MS like pathology is presentation of autoantigenic peptides on MHC class II molecules resulting in demyelination and axonal pathology.

Enhanced glucocorticoid receptor signaling in T cells impacts thymocyte apoptosis and adaptive immune responses.

To study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses.

CDR3 sequence preference of TCRBV8S2+ T cells within the CNS does not reflect single amino acid dependent avidity expansion.

To investigate the influence of antigen and restricting MHC class II molecule on the T cell repertoire, we varied the peptide source by immunizing either with myelin basic protein (MBP) (rat)63-88 or MBP(GUINEA PIG (GP))63-88, which differ in the core region of the peptide binding site at position 79 by a single exchange of threonine (T) to serine (S) and by altering the MHC by immunizing MHC congenic LEW (RT1(1)) and LEW.1W (RT1u) rats. In both MHC haplotypes both peptides lead to oligoclonal dominance of TCRBV8S2 expressing T cells within the central nervous system (CNS) as assessed by complementary determining region 3 (CDR3) spectratyping. In contrast cytofluorometric analysis indicated that only MBP(GP)63-88 in context with the RT1(l) haplotype of the LEW rat lead to strong expansions of TCRBV8S2 expressing T cells within the CNS. Importantly, the small conservative change from S to T at position 79 within MBP63-88 had a strong influence both on the encephalitogenic potential of the peptide and on the number of TCRBV8S2+ T cells infiltrating the CNS. These results demonstrate that even minor changes in only one side chain of an amino acid within an (auto)antigen can dramatically alter TCR avidity for certain MHC class II/peptide complexes.

Peptide motif for the rat MHC class II molecule RT1.Da: similarities to the multiple sclerosis-associated HLA-DRB1*1501 molecule.

Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW.1AV1 rats share the RT1av1 haplotype. So far, no MHC class II peptide motif of RT1.Da molecules has been described. Sequence alignment of the beta chain of the rat MHC class II molecule RT1.Da with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.Da and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.Da, after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.Da. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.Da molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.Da molecules. Based on these studies we could define a peptide-binding motif for RT1.Da characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.Da. This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases.

Antibodies against glycosylated native MOG are elevated in patients with multiple sclerosis.

Antibodies against native glycosylated myelin-oligodendrocyte-glycoprotein (MOG) were measured by ELISA in patients with multiple sclerosis (MS) and controls. Anti-MOG IgM antibodies were elevated during the first demyelinating event. Higher MOG-specific IgG antibodies were found in patients during relapses and in secondary chronic progressive MS compared to patients in remission and healthy controls. Antibodies against native MOG may be a potential biomarker for MS.

MHC and non-MHC gene regulation of disease susceptibility and disease course in experimental inflammatory peripheral neuropathy.

With a panel of rat strains, we demonstrate a strong impact of the MHC genotype on susceptibility and disease course in experimental autoimmune neuritis induced with peripheral nerve myelin or the P2 peptide 58-81 (KNTEISFKLGQEFEETTADNRKTK). Beside the MHC genotype, non-MHC genes determined disease susceptibility and resistance. The type of disease induced with P2 58-81 was strongly correlated to the strength of the MHC class II isotype interaction with P2 58-81. These findings suggest a link between susceptibility and acute versus chronic disease courses on one hand and the strength of the MHC class II molecule/peptide affinity on the other hand.

MHC class II isotype- and allele-specific attenuation of experimental autoimmune encephalomyelitis.

Most autoimmune diseases are associated with certain MHC class II haplotypes. Autoantigen-based specific immune therapy can lead either to beneficial or, in the context of inflammatory conditions, detrimental outcomes. Therefore, we designed a platform of peptides by combinatorial chemistry selected in a nonbiased Ag-independent approach for strong binding to the rat MHC class II isotype RT1.D(n) allelic product of the RT1(n) haplotype that is presenting autoantigen in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LEW.1N rats. Peptide p17 (Ac-FWFLDNAPL-NH(2)) was capable of suppressing the induction of and also ameliorated established experimental autoimmune encephalomyelitis. MHC class II isotype and allele specificity of the therapeutic principle were demonstrated in myelin basic protein-induced experimental autoimmune encephalomyelitis in LEW rats bearing the RT1(l) haplotype. A general immunosuppressive effect of the treatment was excluded by allogeneic heart transplantation studies. In vitro studies demonstrated the blocking effect of p17 on autoantigenic T cell responses. We thus demonstrate a rational design of strong MHC class II-binding peptides with absolute isotype and allele specificity able to compete for autoantigenic sequences presented on disease-associated MHC class II molecules.

Lack of pathogenicity of immunodominant T and B cell determinants of the nicotinic acetylcholine receptor epsilon-chain.

The nicotinic acetylcholine receptor (nAChR) is the autoantigen in seropositive myasthenia gravis (MG) that is a T cell-dependent B cell-mediated autoimmune disorder. We tested the immunogenicity and myasthenogenicity of the extracellular and first transmembrane domain of the epsilon-chain(1-221) of the nAChR in inbred and MHC congenic rat strains. Immunodominant T and B cell determinants did not induce experimental autoimmune myasthenia gravis (EAMG), although immunization resulted in strong Th1 and B cell responses, which could be mapped with overlapping peptides of the nAChR epsilon-subunit in eight different rat strains. Our data underscores the concept that immunodominant autoantigen-specific T and B cell responses can lack pathogenicity in autoimmune disease and might be of relevance for the physiological integrity of the organism.

Immunogenicity of Torpedo acetylcholine receptor in the context of different rat MHC class II haplotypes and non-MHC genomes.

The nicotinic acetylcholine receptor (nAChR) is the autoantigen in seropositive myasthenia gravis (MG), a T-cell-dependent B-cell-mediated autoimmune disease. The nAChR is a pentameric transmembrane receptor comprising alpha alpha beta gamma delta chains. During early postnatal development the nAChR gamma chain is replaced by the nAChR epsilon chain. We tested the myasthenogenicity in experimental autoimmune myasthenia gravis (EAMG) of the native nAChR derived from the electric ray Torpedo californica (T-nAChR) in various inbred and MHC -congenic rat strains. Differences in the disease course emerged dependent on the MHC haplotype and non-MHC genes. Interestingly, no tested rat strain was completely resistant to EAMG, but there were strong differences in disease severity mainly depending on the MHC haplotype. In the LEW non-MHC genome, the B-cell response and the severity of EAMG were dependent on the expressed MHC haplotype. This study underscores the influence of genetic factors on disease severity, disease course and on the degree of the emerging antibody responses in EAMG.

Suppressive DNA vaccination in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis involves a T1-biased immune response.

Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG(91-108) is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n)) rats. We examined the effects of DNA vaccines encoding MOG(91-108) in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-gamma, TNF-alpha, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.

High immunogenicity of intracellular myelin oligodendrocyte glycoprotein epitopes.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS with associated axonal loss. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin oligodendrocyte glycoprotein (MOG) induces a type of experimental autoimmune encephalomyelitis in animals which is very MS-like since there are demyelinating CNS lesions and axonal loss. This underscores the potential role of MOG in MS pathogenesis. We performed a T cell reactivity pattern analysis of MS patients at the onset of relapse or progression of neurological deficits and controls that were stratified for the genetic risk factor HLA-DRB1*1501. For the first time, we show that there is an HLA-DR-restricted promiscuous dominant epitope for CD4(+) T cells within the transmembrane/intracellular part of MOG comprising aa 146-154 (FLCLQYRLR). Surprisingly, controls had broader T cell reactivity patterns toward MOG peptides compared with MS patients, and the transmembrane and intracellular parts of MOG were much more immunogenic compared with the extracellular part. Measurements of in vitro binding affinities revealed that HLA-DRB1*1501 molecules bound MOG 146-154 with intermediate and HLA-DRB1*0401 molecules with weak affinities. The binding of MOG 146-154 was comparable or better than myelin basic protein 85-99, which is the dominant myelin basic protein epitope in context with HLA-DRB1*1501 molecules in MS patients. This is the first study in which the data underscore the need to investigate the pathogenic or regulatory role of the transmembrane and intracellular part of MOG for MS in more detail.