Surges of increased T cell reactivity to an encephalitogenic region of myelin proteolipid protein occur more often in patients with multiple sclerosis than in healthy subjects

We have previously shown that patients with multiple sclerosis (MS) have increased T cell responses to the immunodominant region (residues 184-209) of myelin proteolipid protein (PLP). The present study investigated whether this reactivity fluctuates over time and correlates with disease activity. We performed monthly limiting dilution assays for 12-16 mo in four healthy subjects and five patients with relapsing-remitting MS to quantify the frequencies of circulating T cells proliferating in response to PLP(41-58), PLP(184-199), PLP(190-209), myelin basic protein (MBP), MBP(82-100), and tetanus toxoid. Disease activity was monitored by clinical assessment and gadolinium-enhanced magnetic resonance imaging of the brain. There were fluctuations in the frequencies of autoreactive T cells in all subjects. Compared with healthy controls, MS patients had significantly more frequent surges of T cells reactive to the 184-209 region of PLP, but infrequent surges of T cell reactivity to MBP(82-100). There was temporal clustering of the surges of T cell reactivity to MBP(82-100) and MBP, suggesting T cell activation by environmental stimuli. Some clinical relapses were preceded by surges of T cell reactivity to PLP(184-209), and in one patient there was significant correlation between the frequency of T cells reactive to PLP(184-199) and the total number of gadolinium-enhancing magnetic resonance imaging lesions. However, other relapses were not associated with surges of T cell reactivity to the Ags tested. T cells reactive to PLP(184-209) may contribute to the development of some of the CNS lesions in MS.     

A structurally available encephalitogenic epitope of myelin oligodendrocyte glycoprotein specifically induces a diversified pathogenic autoimmune response

Multiple sclerosis is an inflammatory disease of the CNS that involves immune reactivity against myelin oligodendrocyte glycoprotein (MOG), a type I transmembrane protein located at the outer surface of CNS myelin. The epitope MOG92-106 is a DR4-restricted Th cell epitope and a target for demyelinating autoantibodies. In this study, we show that the immune response elicited by immunization with this epitope is qualitatively different from immune responses induced by the well-defined epitopes myelin basic protein (MBP) 84-96 and proteolipid protein (PLP) 139-151. Mice with MOG92-106-, but not with MBP84-96- or PLP139-151-induced experimental autoimmune encephalomyelitis developed extensive B cell reactivity against secondary myelin Ags. These secondary Abs were directed against a set of encephalitogenic peptide Ags derived from MBP and PLP as well as a broad range of epitopes spanning the complete MBP sequence. The observed diversification of the B cell reactivity represents a simultaneous spread toward a broad range of antigenic epitopes and differs markedly from T cell epitope spreading that follows a sequential cascade. The Abs were of the isotypes IgG1 and IgG2b, indicating that endogenously recruited B cells receive help from activated T cells. In sharp contrast, B cell reactivity in MBP84-96- and PLP139-151-induced experimental autoimmune encephalomyelitis was directed against the disease-inducing Ag only. These data provide direct evidence that the nature of the endogenously acquired immune reactivity during organ-specific autoimmunity critically depends on the disease-inducing Ag. They further demonstrate that the epitope MOG92-106 has the specific capacity to induce a widespread autoimmune response.     

Peptide determinants of myelin proteolipid protein (PLP) in autoimmune demyelinating disease: A review

This article reviews recent advances in understanding the role of myelin proteolipid protein (PLP) in autoimmune demyelination. It is drawn largely from work published within the last years and discusses the immunology of PLP in the historical context of what has been learned from extensive studies on the immune response to myelin basic protein (MBP). Despite the, fact that PLP is the major protein constituent of mammalian myelin, its role in autoimmune demyelination has not been widely recognized. The lack of understanding about the immunology of PLP is a direct result of the biochemical characteristics of the protein. PLP is a highly hydrophobic membrane protein with limited aqueous solubility. The hydrophobicity of PLP has thwarted, immunologic studies of the intact protein. Recent work has circumvented the technical obstacles of studying the intact protein by using soluble synthetic PLP peptides. This approach has rapidly resulted in a more definitive understanding of the immune response to PLP. Presently, the data indicate that:i) PLP is a major central nervous system (CNS) specific encephalitogen;ii) CD4+T cell reactivity to discrete PLP peptide determinants can mediate the development of acute chronic relapsing, and chronic progressive experimental autoimmune encephalomyelitis (EAE); andiii) T cell reactivity to multiple PLP determinants occurs in patients with multiple sclerosis (MS), the major human CNS demyleinating disease.Special Issue dedicated to Dr. Majorie B. Lees.     

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.     

Triosephosphate isomerase- and glyceraldehyde-3-phosphate dehydrogenase-reactive autoantibodies in the cerebrospinal fluid of patients with multiple sclerosis

Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.     

Experimental allergic encephalomyelitis mediated by murine encephalitogenic T cell lines specific for myelin proteolipid apoprotein

T cell lines specific for bovine myelin proteolipid apoprotein (PLP) were established from SJL/J mice. The line cells bore surface phenotypes of T helper/inducer cells (Lyt-1+, Lyt-2-, L3T4+) and responded well to bovine, rat, and guinea pig PLP but not to myelin basic protein. One line responded to major PLP, and another responded to both major PLP and DM-20, which are the two major intrinsic membrane proteins of the central nervous system (CNS) myelin. Intraperitoneal inoculation of 4 to 30 X 10(6) PLP-activated line cells followed by injection of pertussis vaccine induced acute inflammatory disease of the CNS, with typical clinical signs of EAE mostly in a week in recipient mice that had been treated with low-dose irradiation. Almost all animals recovered completely, and two of the 12 animals relapsed 42 or 75 days after inoculation. The lesions were restricted to the CNS and were characterized by perivascular and parenchymal infiltration of inflammatory cells, fibrin deposit, and demyelination. In the severe lesions, axons were also damaged. These observations suggest that PLP is a definite encephalitogen, and PLP-sensitized effector T cells induce inflammatory demyelination in the CNS.     

Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.     

Expression of recombinant forms of human 21.5 kDa myelin basic protein and proteolipid protein in CHO cells

MBP and PLP are major structural protein components of myelin. Both proteins play a functional role in formation of myelin sheath and in maintenance of its compaction. Immune responses to MBP and PLP have been implicated in the pathogenesis of multiple sclerosis (MS), an auto-immune disease of the central nervous system. Recombinant forms of both proteins isolated and purified from bacterial or insect cell systems are commonly used to study the specificity of auto-response in MS. We have prepared recombinant forms of MBP and PLP stably expressed in CHO cells. Several clones with proper cytoplasmic MBP or surface PLP localization were obtained and characterized by flow cytometry and indirect immunostaining. CHO cells expressing the recombinant forms of MBP and PLP can be very useful in studies on the autoimmune mechanism of MS.     

HLA-DQ6 (DQB1*0601)-restricted T cells protect against experimental autoimmune encephalomyelitis in HLA-DR3.DQ6 double-transgenic mice by generating anti-inflammatory IFN-gamma

The human MHC class II genes are associated with genetic susceptibility to multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS of presumed autoimmune origin. These genes encode for proteins responsible for shaping immune response. The exact role of HLA-DQ and -DR genes in disease pathogenesis is not well-understood due to the high polymorphism, linkage disequilibrium, and heterogeneity of human populations. The advent of HLA class II-transgenic (Tg) mice has helped in answering some of these questions. Previously, using single-Tg mice (expressing the HLA-DR or -DQ gene), we showed that proteolipid protein (PLP)(91-110) peptide induced classical experimental autoimmune encephalomyelitis only in DR3.Abeta degrees mice, suggesting that DR3 (DRB1*0301) is a disease susceptible gene in the context of PLP. Human population studies have suggested that HLA-DQ6 (DQB1*0601) may be a protective gene in MS. To test this disease protection in an experimental model, we generated double-Tg mice expressing both HLA-DR3 and -DQ6. Introduction of DQ6 onto DR3-Tg mice led to a decrease in disease incidence on immunization with PLP(91-110) peptide indicating a dominant protective role of DQ6. This protective effect is due to high levels of IFN-gamma produced by DQ6-restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis. Our study indicates that DQ6 modifies the PLP(91-110)-specific T cell response in DR3 through anti-inflammatory effects of IFN-gamma, which is protective for experimental autoimmune encephalomyelitis. Thus, our double-Tg mouse provides a novel model in which to study epistatic interactions between HLA class II molecules in MS.     

Immunity to the extracellular domain of Nogo-A modulates experimental autoimmune encephalomyelitis

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE). Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.     

Production of autoantibodies against citrullinated antigens/peptides by human B cells

Autoantibodies against citrullinated protein Ags (ACPA) are associated with the development of rheumatoid arthritis (RA). This immune response against citrullinated protein Ags, which is thought to be facilitated by certain MHC HLA-DR alleles, is highly specific for this disease and has been speculated to be involved in the pathogenesis. We have previously studied cultures of B cells for the production of Abs against HLA Ags. The aim of the current study was to examine the role of B cells in the production of ACPA in patients with RA. Peripheral blood B cells from RA patients and healthy people were cultured with EL4-B5, a murine cell line expressing human CD40L, and with T cell factors to stimulate the in vitro production of Abs by B cells isolated from peripheral blood. ACPA were produced by cultured B cells from RA patients, as determined by reactivity to cyclic citrullinated peptide (CCP). The results showed that 22% of the healthy persons tested also had B cells that could produce ACPA. Patients with HLA-DR alleles carrying the RA-associated shared epitope appeared to have more B cells with autoimmune potential for CCP than those without such HLA alleles (odds ratio 8.1, p = 0.001). In healthy individuals, anti-CCP-producing B cells were also observed more frequently if the RA-associated MHC genes were present (odds ratio 8.0, p = 0.01). Analysis of B cells in cultures may shed light on the interaction of genetic and environmental factors in the development of RA.     

A Chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.     

Cytokine Control of Inflammation and Repair in the Pathology of Multiple Sclerosis

Cytokines are secreted signaling proteins that play an essential role in propagating and regulating immune responses during experimental autoimmune encephalomyelitis (EAE), the mouse model of the neurodegenerative, autoimmune disease multiple sclerosis (MS). EAE pathology is driven by a myelin-specific T cell response that is activated in the periphery and mediates the destruction of myelin upon T cell infiltration into the central nervous system (CNS). Cytokines provide cell signals both in the immune and CNS compartment, but interestingly, some have detrimental effects in the immune compartment while having beneficial effects in the CNS compartment. The complex nature of these signals will be reviewed.     

Amelioration of established experimental autoimmune encephalomyelitis by an MHC anchor-substituted variant of proteolipid protein 139-151

Murine experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated autoimmune disorder directed against myelin proteins within the CNS. We propose that variant peptides containing amino acid substitutions at MHC anchor residues will provide a unique means to controlling the polyclonal autoimmune T cell response. In this study, we have identified an MHC variant of proteolipid protein (PLP) 139-151 (145D) that renders PLP(139-151)-specific T cell lines anergic in vitro, as defined by a significant reduction in proliferation and IL-2 production following challenge with wild-type peptide. In vivo administration of 145D before challenge with PLP(139-151) results in a significant reduction in disease severity and incidence. Importantly, we demonstrate the ability of an MHC variant peptide to ameliorate established EAE. An advantage to this treatment is that the MHC variant peptide does not induce an acute hypersensitivity reaction. This is in contrast to previous work in the PLP(139-151) model demonstrating that anaphylactic shock resulting in death occurs upon rechallenge with the encephalitogenic peptide. Taken together, these data demonstrate the effectiveness of MHC anchor-substituted peptides in the treatment of EAE and suggest their utility in the treatment of other autoimmune disorders.     

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.     

Functional activation of myelin-specific T cells by virus-induced molecular mimicry

Molecular mimicry is the process by which T cells activated in response to determinants on an infecting microorganism cross-react with self epitopes, leading to an autoimmune disease. Normally, infection of SJL/J mice with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent CNS infection, leading to a chronic progressive, CD4(+) T cell-mediated demyelinating disease. Myelin damage is initiated by T cell responses to virus persisting in CNS APCs, and progressive demyelinating disease (50 days postinfection) is perpetuated by myelin epitope-specific CD4(+) T cells activated by epitope spreading. We developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant myelin epitope, proteolipid protein (PLP) 139-151, into the coding region of a nonpathogenic TMEV variant. PLP139-TMEV-infected mice developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLP139-151-specific CD4(+) Th1 responses within 10-14 days postinfection. The current studies demonstrate that the early onset demyelinating disease induced by PLP139-TMEV is the direct result of autoreactive PLP139-151-specific CD4(+) T cell responses. PLP139-151-specific CD4(+) T cells from PLP139-TMEV-infected mice transferred demyelinating disease to naive recipients and PLP139-151-specific tolerance before infection prevented clinical disease. Finally, infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139-151-specific CD4(+) T cells could be activated in the periphery and traffic to the CNS. Collectively, infection with PLP139-151 mimic encoding TMEV serves as an excellent model for molecular mimicry by inducing pathologic myelin-specific CD4(+) T cells via a natural virus infection.     

Myelin basic protein and proteolipid protein reactivity of brain- and cerebrospinal fluid-derived T cell clones in multiple sclerosis and postinfectious encephalomyelitis

T cells were directly cloned from autopsied MS brain plaque tissue and reactivity was measured with the major encephalitogenic neuroantigens, myelin basic protein (MBP), and proteolipid protein (PLP). Control clones were simultaneously derived from the blood. The proportion of T4+ and T8+ T cell clones from the brain tissue differed from that of peripheral blood T cell clones derived at the same time, suggesting that the clones were not derived from the peripheral blood. None of 57 brain-derived T cell clones proliferated to either MBP or PLP, although they responded well to PHA and IL 2. An additional 235 clones derived from the cerebrospinal fluid and 126 clones from the peripheral blood of other subjects with multiple sclerosis also did not proliferate to MBP or PLP. In contrast, five of nine T4+ clones from the CSF of a subject with postinfectious encephalomyelitis exhibited low but clear reactivity to human MBP, supporting the possible role of MBP as the target antigen in this disease. These studies, the first to clone T cells directly from MS plaque tissue, suggest that the lack of consistent T cell reactivity to MBP or PLP in the peripheral blood of MS patients does not appear to be secondary to the sequestration of a large number of these cells in the brain.     

Strategies for the identification of loci responsible for the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is a chronic, debilitating disease, which manifests itself by de-myelination of the central nervous system (CNS). MS is predominantly found in Caucasians of European decent and is more prominent in females than males. MS is one of the most prevalent causes of disability of young adults in the world. The exact cause of MS is not known, however genetic susceptibility to MS is linked to the major histocompability complex (MHC). Self reactive CD4+ T cells, specific for CNS antigens, such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP), are detectable in MS patients along with pathogenic autoantibodies specific to these CNS antigens produced by B cells. These observations suggest that MS is an autoimmune disease. Epidemiology of MS along with the analysis of sibling pairs and twins suggest that the multiple genetic factors and their interaction with environment contribute to disease susceptibility. Recent developments and advancements in genetic analysis may aid in accurate determination of genetic risk factors for the development of MS. We review these developments, advances in technology and discuss recent results in this article. These authors contributed equally to this paper     

Neutrophils that infiltrate the central nervous system regulate T cell responses

Regulation of inflammatory responses is critical to progression of organ-specific autoimmune disease. Although many candidate cell types have been identified, immunoregulatory activity has rarely been directly assayed and never from the CNS. We have analyzed the regulatory capability of Gr-1high neutrophils isolated from the CNS of mice with experimental autoimmune encephalomyelitis. Proportions of neutrophils were markedly increased in the CNS of IFN-gamma-deficient mice. Strikingly, CNS-derived neutrophils, whether or not they derived from IFN-gamma-deficient mice, were potent suppressors of T cell responses to myelin or adjuvant Ags. Neutrophil suppressor activity was absolutely dependent on IFN-gamma production by target T cells, and suppression was abrogated by blocking NO synthase. These data identify an immunoregulatory capacity for neutrophils, and indicate that interplay between IFN-gamma, NO, and activated Gr-1high neutrophils within the target organ determines the outcome of inflammatory and potentially autoimmune T cell responses.