Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome.

BACKGROUND: Antibodies against IgLON5, a neuronal adhesion protein of unknown function, are markers of a novel neurological disorder termed anti-IgLON5 syndrome. The disorder shows a remarkable association with the HLA-DQB1*0501 and HLA-DRB1*1001 alleles, and postmortem studies demonstrate a novel neuronal tauopathy predominantly involving the hypothalamus and tegmentum of the brainstem. The role of IgLON5 antibodies in the pathogenesis of the disease is currently unknown. Here, we have determined the target epitopes of IgLON5 antibodies, the effects of the IgLON5 antibodies in rat hippocampal neurons, and the IgG subclass responsible for these effects. METHODS: HEK293 cells expressing several deletion constructs of IgLON5 were used to determine the epitopes recognized by the serum of 15 patients with anti-IgLON5 syndrome. The role of glycosylation in immunogenicity was tested with PNGase F treatment of transfected cells. Dissociated hippocampal neuronal cultures were used to test by immunocytochemistry the effects of total IgG, IgG1, and IgG4 subclasses of IgLON5 antibodies. RESULTS: Patients' antibodies reacted with the immunoglobulin-like domain 2 of IgLON5. Glycosylation was not required for immunoreactivity. The predominant subclass of IgLON5 antibodies was IgG4 but all patients also had IgG1. The mean percentage of specific IgLON5 IgG4 and IgG1 of the samples analyzed by flow cytometry was 64 and 33 %, respectively. In cultures of hippocampal neurons, patients' antibodies caused a decrease of cell surface IgLON5 clusters that was not reversed after IgLON5 antibodies were removed from the media. The decrease of surface IgLON5 clusters correlated with the rate of antibody internalization. These effects were observed with purified IgG1 but not with the IgG4 antibodies. CONCLUSIONS: IgLON5 antibodies recognize the immunoglobulin-like domain 2 of the antigen, and the reactivity is not dependent on glycosylation. The effects observed on hippocampal neuronal cultures indicate an irreversible antibody-mediated internalization of surface IgLON5. These effects were mediated by specific IgLON5 IgG1 antibodies and suggest a pathogenic role of these antibodies in the disease.

Xylitol, an anticaries agent, exhibits potent inhibition of inflammatory responses in human THP-1-derived macrophages infected with Porphyromonas gingivalis.

BACKGROUND: Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. METHODS: Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. RESULTS: Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1ß, in a multiplicity of infection- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ-induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted antiphagocytic activity against both Escherichia coli and P. gingivalis. CONCLUSION: These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.

Adduction of cholesterol 5,6-secosterol aldehyde to membrane-bound myelin basic protein exposes an immunodominant epitope.

Myelin degradation in the central nervous system (CNS) is a clinical hallmark of multiple sclerosis (MS). A reduction in the net positive charge of myelin basic protein (MBP) via deimination of arginine to citrulline has been shown to correlate strongly with disease severity and has been linked to myelin instability and a defect that precedes neurodegeneration and leads to autoimmune attack. Recently, we have shown that lipid-derived aldehydes, such as cholesterol 5,6-secosterols atheronal A (1a) and atheronal B (1b), modulate the misfolding of certain proteins such as apolipoprotein B(100), ß-amyloid, α-synuclein, and κ- and λ-antibody light chains in a process involving adduction of the hydrophobic aldehyde to lysine side chains, resulting in a decrease in the net positive charge of the protein. In this study, we show that the presence of either atheronal A (1a) or atheronal B (1b) in large unilamellar vesicles (cyt-LUVs) with the lipid composition found in the cytosolic myelin sheath and bovine MBP (bMBP) leads to an atheronal concentration-dependent increase in the surface exposure of the immunodominant epitope (V86-T98) as determined by antibody binding. Other structural changes in bMBP were also observed; specifically, 1a and 1b induce a decrease in the surface exposure of L36-P50 relative to control cyt-LUVs as measured both by antibody binding and by a reduction in the level of cathepsin D proteolysis of F42 and F43. Structure-activity relationship studies with analogues of 1a and 1b point to the aldehyde moiety of both compounds being critical to their effects on bMBP structure. The atheronals also cause a reduction in the size of the bMBP-cyt-LUV aggregates, as determined by fluorescence microscopy and dynamic light scattering. These results suggest that formation of an imine between inflammatory-derived aldehydes, which effectively reduces the cationic nature of MBP, can lead to structural changes in MBP and a decrease in myelin stability akin to deimination and as such may make a hitherto unknown contribution to the onset and progression of MS.

Triptolide inhibits IL-12/IL-23 expression in APCs via CCAAT/enhancer-binding protein alpha.

Triptolide is a biologically active component purified from Chinese herbal plant Tripterygium wilfordii Hook F. It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect's disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-alpha (C/EBPalpha), which directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the transcription of C/EBPalpha, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPalpha inhibition of p40. Further, activation of C/EBPalpha by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3beta. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases.

Oxidative stress can alter the antigenicity of immunodominant peptides.

APCs operate frequently under oxidative stress induced by aging, tissue damage, pathogens, or inflammatory responses. Phagocytic cells produce peroxides and free-radical species that facilitate pathogen clearance and can in the case of APCs, also lead to oxidative modifications of antigenic proteins and peptides. Little information is available presently about the consequences of such modifications on the immune response. To model oxidative modification of an immunodominant antigenic peptide, we oxidized the methionine residue of the human CMV pp65(495-503) (NLVPMVATV) peptide. Such modifications of an antigenic peptide can affect MHC binding or TCR recognition. Using binding and dissociation assays, we demonstrate that oxidative modification of the CMVpp65(495-503) peptide leads to a decreased binding of the pMHC complex to the TCR, whereas binding of the peptide to the MHC class I molecule is not impaired. Additionally, we show that CD8(+) T cells have a decreased proliferation and IFN-gamma production when stimulated with oxidized CMVpp65(495-503) peptide. Spectratyping the antigen-binding site of the TCR of responding T cells demonstrates that the CMVpp65(495-503) and the CMVoxpp65(495-503) peptides preferentially stimulate BV8 T cells. Sequencing of this dominant BV family reveals a highly conserved CDR3 amino acid motif, independent of the mode of stimulation, demonstrating the recruitment of the same T cell clonotypes. Our results suggest that oxidative modification of antigenic peptides may affect T cell responses severely by binding T cell clones with different affinity. This may lead to an altered immune response against infectious agents as well as against tumor or autoantigens under oxidative stress conditions.

Immunodominance in virus-induced CD8(+) T-cell responses is dramatically modified by DNA immunization and is regulated by gamma interferon.

The phenomenon whereby the host immune system responds to only a few of the many possible epitopes in a foreign protein is termed immunodominance. Immunodominance occurs not only during microbial infection but also following vaccination, and clarification of the underlying mechanism may permit the rational design of vaccines which can circumvent immunodominance, thereby inducing responses to all epitopes, dominant and subdominant. Here, we show that immunodominance affects DNA vaccines and that the effects can be avoided by the simple expedient of epitope separation. DNA vaccines encoding isolated dominant and subdominant epitopes induce equivalent responses, confirming a previous demonstration that coexpression of dominant and subdominant epitopes on the same antigen-presenting cell (APC) is central to immunodominance. We conclude that multiepitope DNA vaccines should comprise a cocktail of plasmids, each with its own epitope, to allow maximal epitope dispersal among APCs. In addition, we demonstrate that subdominant responses are actively suppressed by dominant CD8(+) T-cell responses and that gamma interferon (IFN-gamma) is required for this suppression. Furthermore, priming of CD8(+) T cells to a single dominant epitope results in strong suppression of responses to other normally dominant epitopes in immunocompetent mice, in effect rendering these epitopes subdominant; however, responses to these epitopes are increased 6- to 20-fold in mice lacking IFN-gamma. We suggest that, in agreement with our previous observations, IFN-gamma secretion by CD8(+) T cells is highly localized, and we propose that its immunosuppressive effect is focused on the APC with which the dominant CD8(+) T cell is in contact.

Alpha 2,3-specific desialylation of human red cells: effect on the autoantigens of the Pr, Sa and Sia-l1, -b1, -lb1 series.

BACKGROUND AND OBJECTIVES: Pr1,2,3, PrM, Sa and Sia-l1, -b1, -lb1 are sialic acid (NeuNAc)-dependent antigens recognized by human cold agglutinins. Pr and Sa antigens are the O-glycans of glycophorins containing alpha 2,3NeuNAc (to galactose) and/or alpha 2,6NeuNAc (to galactosamine). The antigens of the Sia-l1, -b1, -lb1 complex are gangliosides that may carry alpha 2,3NeuNAc (to galactose) and/or alpha 2,8NeuNAc (to NeuNAc). We studied the NeuNAc groups involved in the antigens. MATERIALS AND METHODS: From 74 sera with cold agglutinins against NeuNAc-dependent antigens, anti-T-free preparations were made and tested against human red cells, treated with an alpha 2,3-specific recombinant sialidase. RESULTS: Most (51/62) Pr antigens use alpha 2,3NeuNAc, some (8/62) use alpha 2,6NeuNAc and a few (3/62) use both sialyl groups as immunodominant components on glycophorins. The immunodominant component of Sa and Sia-l1, -b1, -lb1 determinants was alpha 2,3NeuNAc in all cases. CONCLUSION: The red cell target structures for cold agglutinins against NeuNAc-dependent antigens have been identified. We propose a Pr nomenclature to reflect this. The binding of anti-Pr to gangliosides may be the basis for anti-Pr-induced peripheral neuropathy.

Anti-melanoma cytotoxic T lymphocytes (CTL) recognize numerous antigenic peptides having 'self' sequences: autoimmune nature of the anti-melanoma CTL response.

A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.

Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12.

Immunodominance (ID) of T cell epitopes is a well-documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H-2Kd, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half-life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co-injected CTL epitopes can be determined both at the peptide level (binding affinity to H-2Kd) and at the T cell level. This hypothesis is supported by our finding that interleukin-12 strongly modulates ID when it is not correlated with MHC binding.

Myelin proteolipid protein-induced Th1 and Th2 clones express TCR with similar fine specificity for peptide and CDR3 homology despite diverse V beta usage.

Myelin-specific T-helper (Th) cells which induce encephalomyelitis belong to the inflammatory Th1 subset. Th2 cells recognizing similar epitopes potentially represent specific inhibitors of encephalitogenic Th1 cells. Since the differential stimulation of antigen-specific Th2 cells may be important in the regulation of autoimmune inflammatory disorders, we have examined the fine specificity of a Th1 and a Th2 clone, induced by immunization of SJL mice with native proteolipid protein (PLP) and specific for the PLP 139-151 sequence. Stimulation of the clones by synthetic peptides containing single alanine substitutions demonstrated that L141, W144, H147, and P148 represent critical residues. Surprisingly, this pattern was identical for both subsets. Competition studies indicated indirectly that L141 and P148 may be MHC-binding residues, whereas W144 and H147 contact the TCR. Sequencing of the TCR expressed by both Th subset clones demonstrated different V beta usage as well as variation in the D-region sequence and length. Interestingly, realignment of the sequence of the CDR3 regions showed striking homology. This study demonstrates that Th1 and Th2 subsets can express very similar peptide specificities, while utilizing very different TCR V beta chains. These results suggest that the therapeutic modalities based on either peptide antagonists or antibodies specific for CDR3 may have limited effectiveness in treating autoimmune disorders, since they may also target the beneficial arm of the immune response.

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid.

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.