Antibodies to MOG are transient in childhood acute disseminated encephalomyelitis.

OBJECTIVE: To study the longitudinal dynamics of anti-myelin oligodendrocyte glycoprotein (MOG) autoantibodies in childhood demyelinating diseases. METHODS: We addressed the kinetics of anti-MOG immunoglobulins in a prospective study comprising 77 pediatric patients. This was supplemented by a cross-sectional study analyzing 126 pediatric patients with acute demyelination and 62 adult patients with multiple sclerosis (MS). MOG-transfected cells were used for detection of antibodies by flow cytometry. RESULTS: Twenty-five children who were anti-MOG immunoglobulin (Ig) positive at disease onset were followed for up to 5 years. Anti-MOG antibodies rapidly and continuously declined in all 16 monophasic patients with acute disseminated encephalomyelitis and in one patient with clinically isolated syndrome. In contrast, in 6 of 8 patients (75%) eventually diagnosed with childhood MS, the antibodies to MOG persisted with fluctuations showing a second increase during an observation period of up to 5 years. Antibodies to MOG were mainly IgG 1 and their binding was largely blocked by pathogenic anti-MOG antibodies derived from a spontaneous animal model of autoimmune encephalitis. The cross-sectional part of our study elaborated that anti-MOG Ig was present in about 25% of children with acute demyelination, but in none of the pediatric or adult controls. Sera from 4/62 (6%) adult patients with MS had anti-MOG IgG at low levels. CONCLUSIONS: The persistence or disappearance of antibodies to MOG may have prognostic relevance for acute childhood demyelination.

Highly skewed T-cell receptor V-beta chain repertoire in the bone marrow is associated with response to immunosuppressive drug therapy in children with very severe aplastic anemia.

One of the major obstacles of immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA) comes from the often months-long unpredictability of bone-marrow (BM) recovery. In this prospective study in children with newly diagnosed very severe AA (n=10), who were enrolled in the therapy study SAA-BFM 94, we found a dramatically reduced diversity of both CD4+ and CD8+ BM cells, as scored by comprehensive V-beta chain T-cell receptor (TCR) analysis. Strongly skewed TCR V-beta pattern was highly predictive for good or at least partial treatment response (n=6, CD8+ complexity scoring median 35.5, range 24-73). In contrast, IST in patients with rather moderate reduction of TCR V-beta diversity (n=4, CD8+ complexity scoring median 109.5, range 82-124) always failed (P=0.0095). If confirmed in a larger series of patients, TCR V-beta repertoire in BM may help to assign children with SAA up-front either to IST or to allogeneic stem-cell transplantation.

Clonal expansions of pathogenic CD8+ effector cells in the CNS of myelin mutant mice.

Tissue damage in the CNS is critically influenced by the adaptive immune system. Primary oligodendrocyte damage (by overexpression of PLP) leads to low-grade inflammation of high pathological impact, which is mediated by CD8+ T cells. To yield further insight into pathogenesis and nature of immune responses in myelin mutated mice, we here apply a detailed immunological characterization of CD8+ T cells in PLP-transgenic and aged wild type mice. We provide evidence that T effector cells accumulate in the CNS of PLP-transgenic and wild-type mice and show a higher level of activation in mutant mice, indicated by surface markers and clonal expansions, as demonstrated by T cell receptor CDR3-spectratype analysis. Vbeta-Jbeta similarities suggest specificity against a common antigen, albeit we could not find specific responses against myelin-antigen-derived peptides. The association of primary oligodendrocyte damage with secondary expansions of pathogenic cells underlines the role of adaptive immune reactions in neurodegenerative and neuroinflammatory diseases.

Diversity of the anti-T-cell receptor immune response and its implications for T-cell vaccination therapy of multiple sclerosis.

More precise understanding of the immune response against T-cell receptors (TCRs) is a prerequisite for successful TCR vaccination therapy of multiple sclerosis and other neurological autoimmune diseases. We conducted a detailed analysis of a paradigmatic anti-TCR response, using synthetic TCR peptides and highly purified recombinant TCR V alpha and Vbeta variable chains for the selection of CD4+ T-cell lines from a healthy volunteer. The target TCR (designated TCR(HWBP-3)) was obtained from HWBP-3, an autologous CD4+ T-cell line specific for myelin basic protein. The V alpha and Vbeta chains of TCR(HWBP-3) were expressed in Escherichia coli and purified by Ni-chelate chromatography and SDS (sodium dodecyl sulphate) gel electrophoresis. Further, we synthesized a set of 13- to 22-mer peptides spanning the complementarity-determining regions (CDR) 1, 2 and 3 and the framework regions (FR) of the alpha and beta chains of TCR(HWBP-3). The TCR peptides and proteins were then used to select a panel of TCR-specific CD4+ T-cell lines from donor HW. Several T-cell lines cross-reacted with a recombinant V chain and synthetic peptide. Cross-reactive immunogenic TCR epitopes were identified in the FR1 and CDR3 regions of the TCR(HWBP-3) alpha chain and in the FR1, CDR1 and CDR2 regions of the TCR(HWBP-3) beta chain. The TCR proteins and peptides were recognized in the context of at least three different HLA-DR molecules [DR2a (DRB5*0101), DR2b (DRB1*1501) and DRB1*1401/DRB3*0202]. Notably, the majority of the TCR peptide-selected T-cell lines did not react with the full-length recombinant V chains, suggesting they recognize 'cryptic' determinants. Based on the diversity of the anti-TCR immune response, we suggest that candidate TCR peptides should be screened in vitro in functional experiments before they are clinically applied for TCR vaccination therapy.

Highly purified oligo-His tagged human recombinant alpha(1)-AChR is immunogenic in vivo and suitable for T cell stimulation in vitro in experimental and human myasthenia gravis.

Using recombinantly expressed proteins for selection of antigen-specific T cell lines carries a high risk of selecting T cells specific for contaminating proteins. This risk is especially high for very hydrophobic proteins which are notoriously difficult to purify, such as the integral membrane protein acetylcholine receptor (AChR). We prepared a highly purified recombinant AChR by adding an oligo-histidine affinity-tag to the human alpha(1)-AChR and expressing it in E. coli. This allowed purification by Ni-NTA chromatography and subsequent electroelution from preparative SDS gel as purification steps, resulting in complete purity as assessed by silver stain on SDS-PAGE. This protein preparation induced fatal experimental allergic myasthenia gravis in Lewis rats. Furthermore, the protein could be used to select T cell lines from immunized Lewis rats and patients with myasthenia gravis. However, even with this highly purified protein, one of 8 Lewis rat T cell lines and 3 of 7 human T cell lines cross-reacted to E. coli control proteins. The results show that oligo-histidine tagged, highly purified human alpha(1)-AChR is highly immunogenic in vivo and in vitro.

Pathway of detergent-mediated and peptide ligand-mediated refolding of heterodimeric class II major histocompatibility complex (MHC) molecules.

We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101. Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.

Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR.

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA-DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units +/- 1.04 SD (range 0.17-4.31). Approximately 10% of the samples had increased amounts of sCD74 (>3.0 relative units). Complexes of sCD74 and sHLA-DR were detected in all samples and their quantities were positively correlated (r=0.83, p<0.001) with the sCD74 concentrations. SDS-PAGE analysis of plasma samples with high sCD74 concentrations (>3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA-DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA-DR molecules.

Screening of inhibitors of HIV-1 protease using an Escherichia coli cell assay.

To evaluate the available peptidic and pseudopeptidic inhibitors of HIV protease for their possible in vivo activity, a screening test using Escherichia coli was established. E. coli cells carrying the plasmid pET9c-PR containing the gene for HIV-1 protease under the control of a T7-promotor are grown in the absence and in the presence of inhibitors. The action of the toxic protease produced by the cells is counteracted by the inhibitors. Provided sufficient membrane permeability of the inhibitors exists, this results in accelerated cell growth. From the peptides known to be active in an in-vitro enzyme test, most compounds inhibit HIV protease only to a limited degree in this test. However, two short peptides (Ac-Ser-Tyr-Glu-Leu and Lys-Ile-Ser-Tyr-Asp-Tyr) protect cell growth to an considerabe extent, thus indicating that they reach the E. coli cytosol and there block HIV protease. Two pseudopeptides known to be very potent in the enzyme test (SDZ PRI 053 and CIBA 61755) also inhibit HIV-1 protease strongly in this cell growth test.

Encephalitogenic potential of myelin basic protein-specific T cells isolated from normal rhesus macaques.

Myelin basic protein (MBP)-specific T cells are implicated in the pathogenesis of multiple sclerosis and are targets of selective immunotherapies. However, autoantigen-specific T cells can also be isolated from healthy individuals. Their functional potential is unknown and obviously cannot be tested in humans. We approached this question in a closely related primate species, the rhesus monkey. CD4+ T cell lines specific for MBP were isolated from normal rhesus monkeys using the same primary limiting dilution technique that is now widely used to generate human autoreactive T cell clones in vitro. Three different epitopes were recognized by three rhesus T cell lines isolated from three different monkeys. Upon activation, all lines produced interferon-gamma, interleukin-2, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor but neither interleukin-4 nor transforming growth factor-beta. The MBP-specific T cells were injected intravenously without adjuvant into the nonirradiated autologous monkey. One of the three rhesus monkeys developed an encephalomyelitis with a pleocytosis in the spinal fluid and perivascular infiltrates in the leptomeninges, spinal nerve roots and cerebral cortex. The data demonstrate that the normal immune repertoire of a primate species contains MBP-specific CD4+ T cells that are able to induce an autoimmune encephalomyelitis upon transfer into the nonirradiated autologous recipient.

The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) induces acute demyelinating experimental autoimmune encephalomyelitis in the Lewis rat.

Using a highly purified recombinant protein, mMOG, we demonstrated that autoimmune responses to the N-terminal domain (a.a 1-125) of the myelin oligodendrocyte glycoprotein (MOG) induce an acute demyelinating variant of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Immunisation with 100 micrograms of mMOG in adjuvant at the base of the tail induced mild clinical disease in 9 of 11 animals (mean clinical score 1.1). The disease was characterised histopathologically by the presence of inflammation and focal demyelinating lesions in the central nervous system (CNS). Adoptive transfer experiments suggest that this inflammatory demyelinating pathology is mediated by synergy between a weakly encephalitogenic, MOG-specific T cell response and a demyelinating, MOG-specific autoantibody response. Using in vitro selected mMOG-reactive T cell lines, the encephalitogenic T cell response to this domain of MOG was found to recognise two distinct epitopes, MOG1-20 and MOG35-55; whereas ELISA demonstrated that the immunodominant B cell epitope was located within the amino acid sequence MOG1-25. However although active immunisation with synthetic peptides corresponding to the T cell epitopes, MOG1-20 or MOG35-55, induced an inflammatory response in the CNS, this was not associated with demyelination indicating that the demyelinating antibody response recognises other, possibly conformation dependent epitopes. This study unequivocally demonstrates that MOG-specific autoimmune responses are alone sufficient to induce a demyelinating disease of the CNS and supports the proposal that MOG may play an important role in the immunopathogenesis of multiple sclerosis.

T cell receptor repertoire in polymyositis: clonal expansion of autoaggressive CD8+ T cells.

In polymyositis (PM), CD8+ T cell receptor (TCR) alpha/beta + cells invade and destroy major histocompatibility complex class I-positive muscle fibers. We combined polymerase chain reaction (PCR) and double-fluorescence immunocytochemistry to analyze the T cell receptor (TCR) repertoire expressed in muscle of PM patients. In patient 1, inverse PCR revealed a preferential usage of TCR V alpha 33.1, V beta 13.1, and V beta 5.1. Six of six TCR V alpha 33.1+ clones and five of seven V beta 13.1+ clones had identical nucleotide sequences. In contrast, the V beta 5.1+ TCRs were more heterogeneous. Similar results were obtained with an independent PCR method using primers specific for TCR V alpha 33, V beta 13, or V beta 5. No TCR sequences could be amplified from noninflammatory control muscle. Furthermore, none of the TCR sequences found in PM muscle could be detected in blood from the same patient or from a normal control subject. Immunohistochemistry confirmed that V beta 5.1 and V beta 13.1 were overrepresented in the muscle lesions of this patient. 32% of all CD8+ T cells were V beta 13.1+, and 16% were V beta 5.1+. However, approximately 60% of the CD8+ T cells that invaded muscle fibers were V beta 13.1+, whereas 10% were V beta 5.1+. In patient 2, 50% of the T cells were V beta 5.1+, and as in patient 1, these T cells were mainly located in interstitial areas. In patient 3, > 75% of the autoinvasive T cells stained with an anti-V beta 3 mAb. Sequence analysis of 15 PCR clones amplified with a V beta 3-specific primer showed that 9 (60%) sequences were identical. The results suggest that (a) a strikingly limited TCR repertoire is expressed in PM muscle; (b) there is a dissociation between the TCR usage of autoinvasive and interstitial T cells; and (c) the autoinvasive T cells are clonally expanded.

Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice.

A recombinant protein corresponding to the Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) and synthetic 15-mer peptides of the whole MOG molecule with eight amino acid overlaps were screened for their ability to induce experimental allergic encephalomyelitis (EAE) in Biozzi AB/H (H-2dq1) and SJL (H-2S) mice. Clinical and histologic evidence of EAE developed after sensitization with the recombinant MOG protein in both AB/H and SJL mice. In AB/H mice at least three MOG epitopes within residues 1-22, 43-57, and 134-148 induced clinical and histologic EAE, whereas only the sequence 92-106 was encephalitogenic in SJL mice. Histologically, the inflammatory response in the central nervous system consisted of perivascular accumulations of CD5+ T cells and F4/80+ macrophage/microglia cells equally distributed in the brain and spinal cord. The subpial/meningeal infiltration, characteristic of mouse EAE induced with spinal cord homogenate, was only observed in cases of severe clinical disease in SJL mice in which the cellular infiltrates predominated in the spinal cord. In spite of the presence of histologic lesions in AB/H mice immunized with MOG, clinical disease either rapidly resolved or was clinically silent. In contrast to immunization of SJL mice with recombinant MOG, sensitization to MOG 92-106 induced severe clinical paralysis. After recovery these animals relapsed and exhibited demyelinated lesions. This study is the first to describe encephalitogenic epitopes of MOG that induce both clinical and histologic signs of EAE in mice. These and previous findings implicating MOG as a target Ag for Ab-mediated attack in EAE suggest that such autoreactivity to MOG may be significant in the development of human demyelinating diseases such as multiple sclerosis.

Refolding of human class II major histocompatibility complex molecules isolated from Escherichia coli. Assembly of peptide-free heterodimers and increased refolding-yield in the presence of antigenic peptide.

The alpha- and beta-chains of the heterodimeric major histocompatibility complex molecules HLA-DRB5*0101 and DRB1*0101 were expressed separately in Escherichia coli. The cytoplasmic and membrane-spanning domains of both chains were replaced by oligohistidine tags to allow purification by metal chelate chromatography. The recombinant proteins were refolded to peptide-free, water-soluble heterodimers by removal of major amounts of detergents and concomitant reoxidiation of disulfide bonds. Correct conformation was documented by three criteria: (a) affinity binding experiments using the antibody L243, which is known to recognize a conformational epitope formed only by correctly associated heterodimers; (b) specific binding of peptides to the refolded molecules; (c) recognition of peptides bound to refolded HLA-DR molecules by T-cells as reflected by Ca2+ influx into T-cells and production of interferon-gamma. The refolding reaction did not absolutely depend on the presence of peptides. The yield of peptide-free heterodimers was 3.0%. However, the yield of refolded heterodimer was increased to 10% if refolding was performed in the presence of antigenic peptides.

Binding of truncated peptides to the MHC molecule IA (d).

A peptide comprising amino acids 323-339 of chicken ovalbumin is known to bind to two heterodimeric conformations of the MHC molecule IA(d), and to each of its separate alpha- and beta-chains. We report that minor C- and N-terminal truncations of the parent peptide do not alter the binding pattern. A decrease in binding activity was observed upon deletion of the histidine residues of the already truncated peptides. Peptides as short as 4 amino acids associate weakly with all four proteins.

In vitro peptide binding to the heavy chain of the class I molecule of the major histocompatibility complex molecule HLA-A2.

The heavy chain of class I molecules of the major histocompatibility complex forms the binding site for antigenic peptides. We describe the binding of a synthetic peptide to the purified heavy chain of the human major histocompatibility complex molecule HLA-A2. The peptide binding capacity is found to be markedly increased if the protein is first partly denatured by reduction of its disulfide bonds in detergent and subsequently renatured by reoxidation. In the presence of certain detergents, the heavy chain binds peptides even when the protein is partly unfolded.

Refolding of an integral membrane protein. OmpA of Escherichia coli.

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.

The invariant chain forms complexes with class II major histocompatibility complex molecules and antigenic peptides "in vivo".

The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad alpha/beta heterodimers and separated alpha and beta chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the alpha/beta heterodimers. A band at 180 kDa contained the alpha/beta heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.

Refolding and reassembly of separate alpha and beta chains of class II molecules of the major histocompatibility complex leads to increased peptide-binding capacity.

Class II molecules of the major histocompatibility complex present antigenic peptides to helper T cells. These are heterodimeric glycoproteins consisting of one alpha and one beta chain. Two different alpha/beta heterodimeric conformations as well as the separate alpha and beta chains bind specific peptides. The alpha chain is thought to have one and the beta chain two intramolecular disulfide bonds. In the present study we have reduced these disulfide bonds in the murine major histocompatibility complex molecule I-Ad, which led to the release of bound peptides from all conformations and to unfolding of the separate chains. The separate alpha and beta chains could be refolded to their native structure by reoxidation of the cysteines. Refolding was accompanied by reassembly of the separated chains to the alpha/beta heterodimer. Both the separated alpha and beta chains and the alpha/beta heterodimer bound significantly higher amounts of antigenic peptide after reduction and reoxidation, as compared to the untreated protein.

Specific binding of antigenic peptides to separate alpha and beta chains of class II molecules of the major histocompatibility complex.

Class II molecules of the major histocompatibility complex bind antigenic peptides and present them to T-helper cells. Class II molecules are heterodimers consisting of one alpha and one beta chain. Here we report that each isolated alpha and beta chain binds antigenic peptides and that this binding is specific. The specificity of peptide binding was investigated by employing the murine major histocompatibility complex haplotypes I-Ad and I-Ek and fluorescence-labeled peptides of chicken ovalbumin and pigeon cytochrome c, respectively, which are known to be specific for these haplotypes. The major histocompatibility complex molecules were incubated with these peptides and subjected to SDS/PAGE under nondenaturing conditions. The gels were then scanned for the fluorescent peptides and, after silver staining, for proteins. We found that the fluorescence-labeled peptide fragment of ovalbumin bound preferentially to the isolated alpha and beta chains of I-Ad, whereas the fluorescence-labeled peptide fragment of pigeon cytochrome c bound preferentially to the isolated alpha and beta chains of I-Ek. The alpha and beta chains of each haplotype bound their specific peptides about equally well, suggesting comparable affinities. Our results indicate that in vivo the kinetic pathway for the formation of antigenic peptide complexes with the alpha/beta heterodimers may involve the initial formation of complexes of the alpha and/or beta chains with the specific antigenic peptides.