Search

Objective Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. Methods We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. Results Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. Interpretation The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

It is well established that the major histocompatibility complex (MHC) on chromosome 6p21 contains at least one gene that influences susceptibility to multiple sclerosis.1–6 Although this association was first identified more than 30 years ago1 through the study of class I human leukocyte antigens (HLAs), it was quickly realized that this signal was predominantly, if not exclusively, the result of linkage disequilibrium (LD) with class II HLA genes, and that these exert the primary effect on susceptibility.7, 8 The complex nature of the MHC, especially its high gene content, extreme polymorphism, and extensive LD,9 has confounded efforts to resolve the nature of the MHC association in multiple sclerosis, although progress and useful clarifications have been made, especially in recent years. In virtually every population studied, multiple sclerosis is found to be associated with the DRB1*1501 allele.10 The only exceptions are those populations where this allele has a low frequency, and analysis is therefore underpowered; but even in these situations, DRB1*1501 is generally overrepresented in cases.11 The DRB1*1501 allele is carried on a particularly extensive haplotype,12 the most common DR15 haplotype found in white Europeans. As a result, many variants from flanking genes, even some located quite a distance from DRB1, have sufficient LD with DRB1*1501 that they invariably also show evidence for association with the disease in any population where association with DRB1*1501 can be demonstrated.11, 13–17 This extensive LD has made it difficult to establish which of the variants making up this haplotype is primarily responsible for the association. The distinction between DRB1*1501 and DQB1*0602 has been particularly taxing because the LD between these closely mapped genes is especially tight in those populations where the disease is frequent, that is, white Europeans and their migrant descendants. However, recent studies in the admixed African American population indicate the supremacy of the DRB1*1501 allele.18 In the presence of one susceptibility allele it is difficult to identify effects attributable to a second allele,19 especially if the second allele exerts a more modest effect or has a low frequency, or both. However, by analyzing populations where DR15 haplotypes are less common, and by using large cohorts, it has been possible to demonstrate that the DRB1*0301 allele also confers susceptibility to multiple sclerosis, thereby confirming allelic heterogeneity at the DRB1 locus.4, 6, 18, 20 Furthermore available evidence suggests that the susceptibility effects of the DRB1*1501 allele may be modulated by other DRB1 alleles.6, 20 The relation between the MHC and multiple sclerosis is further complicated by the accumulating evidence suggesting that MHC loci mapping outside DRB1 also influence susceptibility to the disease.11, 13–16 Work in animal models suggests that clustering of susceptibility loci is a common phenomenon in complex disease,21 and it therefore appears reasonable to expect that other genes from the MHC region may influence susceptibility to multiple sclerosis. The observation of positive logarithm of odds scores in the MHC region in linkage studies stratified for the effects of DRB1 supports the existence of secondary loci,22–24 although none of these data reaches a level providing statistical confidence. In considering these linkage data, it is important to remember that early linkage studies in multiple sclerosis25–27 were significantly underpowered,28 to the point that they could not even convincingly demonstrate evidence for linkage resulting from the effects of DRB1. Confirmation of linkage in this region has been established only in more recent studies involving many hundreds of families.23, 24, 29 Given the inherently limited resolution of linkage-based studies,28 the absence of statistically significant linkage in the MHC region after exclusion of primary effects attributable to DRB1 does not exclude the presence of secondary loci. Several authors have attempted to identify secondary loci using more powerful association-based methods. Two groups have typed dense microsatellite maps of the region and both found evidence for a secondary locus maximal in a region close to HLA-A: one group identifying the marker D6S1683 just telomeric of HLA-A,11 and the second group implicating a region including HLA-A extending from MOGCA to D6S265 marker.14 Follow-up studies in Norway also found evidence implicating the D6S265 marker.16 In another smaller study, a microsatellite marker close to HLA-C (marker C1_3_2) also showed evidence for an independent effect.15 In contrast, a systematic effort to screen the MHC and flanking regions using single nucleotide polymorphisms (SNPs) found no evidence for association beyond that attributable to DRB1*1501, although this study was limited by a high genotyping failure rate (40%) and, more importantly, a distribution of markers leaving regions close to the classical loci essentially unexplored.17 In all of these studies, statistical power has inevitably been reduced by the processes required to filter out the primary effect attributable to DRB1 and the large correction required for multiple testing. Unfortunately, none of the published studies has used sufficient samples to compensate for these statistical penalties, and thus none is able to provide unequivocal evidence supporting any particular secondary locus.

Multiple sclerosis (MS) is postulated to be a Th1-type cell-mediated autoimmune disease. Thus therapies that decrease T cell interferon (IFN)-gamma production or increase interleukin (IL)-4 production would be expected to have an ameliorating effect on MS. Some progressive MS patients receiving pulse cyclophosphamide therapy developed peripheral blood eosinophilia. We investigated whether cyclophosphamide-treated patients had immune deviation toward Th2 responses. We measured cytokine production in patients receiving either monthly intravenous methylprednisolone (MP), intravenous cyclophosphamide plus methylprednisolone (CY/MP), methotrexate, IFN-beta1b, in untreated MS patients, and in healthy controls. Minimal IL-4 was secreted in untreated patients (129 +/- 62 pg/ml), methotrexate-treated patients (99 +/- 79 pg/ml), and healthy controls (50 +/- 13 pg/ml). A marked increase in IL-4 was observed in CY/MP patients (1,503 +/- 291 pg/ml). Patients treated with MP (418 +/- 160 pg/ml) or IFN-beta1b (425 +/- 167 pg/ml) showed small increases. Eosinophilia in CY/MP-treated patients (6.0 +/- 0.7%) correlated with increased IL-4. IL-10 production was also increased in CY/MP-treated patients. Both CY/MP- and MP-treated groups had decreased production of IFN-gamma compared with untreated MS. These findings demonstrate pronounced immune deviation favoring Th2-type responses after pulse cyclophosphamide therapy.

Oral administration of proteins is a long-recognized method of inducing antigen-specific peripheral immune tolerance. We previously showed that oral administration of myelin basic protein suppresses monophasic experimental autoimmune encephalomyelitis in the Lewis rat when it is given in association with immunization and prior to disease onset. As a potential therapy for human autoimmune disease, it is crucial to determine whether oral tolerance can ameliorate an ongoing immune response. We therefore asked whether oral administration of myelin antigens, after sensitization and disease expression has occurred, could affect immunological, clinical, or pathological features of experimental autoimmune encephalomyelitis. Chronic relapsing experimental autoimmune encephalomyelitis was induced in the Lewis rat and strain 13 guinea pig by immunization with whole guinea pig cord homogenate, complete Freund's adjuvant, and Mycobacterium tuberculosis. Following recovery from the first attack, animals were orally given bovine myelin, guinea pig myelin, or guinea pig myelin basic protein three times per week for up to 3 months. Animals receiving myelin products orally had decreased severity and frequency of clinical relapses, decreased delayed-type hypersensitivity responses to myelin antigens, diminished inflammation in the central nervous system (CNS), and decreased areas of CNS demyelination. In the rat, guinea pig myelin basic protein was as effective as guinea pig myelin in ameliorating the disease and also resulted in decreased serum anti-myelin basic protein antibody levels. No exacerbation of disease or worsening of pathological findings occurred in the animals given myelin products. These results demonstrate that oral administration of myelin antigens can suppress chronic relapsing experimental autoimmune encephalomyelitis and have direct relevance to therapy of human demyelinating disorders such as multiple sclerosis.

T-cell populations were investigated in the blood and cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. Individual T cells were directly cloned from the cerebrospinal fluid and blood before in vitro expansion, and their clonotypes were compared by Southern blot analysis of the rearrangement patterns of their T-cell receptor beta chain and gamma chain genes. This allowed the determination of whether two T cell clones shared the same T-cell receptor and thus arose from identical, clonally expanded (oligoclonal) progenitor T cells. As an extension of previous studies, oligoclonal T-cell clones were identified in both cerebrospinal fluid and blood populations in 5 of 9 patients with inflammatory demyelinating disease among a total of 486 blood and cerebrospinal fluid T-cell clones. In contrast, no clonally expanded T-cell populations were found among a total of 424 clones derived from either blood of 4 normal control subjects or blood and cerebrospinal fluid of 8 patients with other neurological diseases. Analysis of T-cell receptor V beta genes among 4 oligoclonal T-cell populations derived from 3 patients with multiple sclerosis demonstrated common usage of the V beta 12 gene segment. These data suggest that oligoclonal T cells share similar specificities and that clonal expansion may have resulted from specific stimulation by an antigen. Moreover, identical clones between blood and cerebrospinal fluid were observed in 3 of 9 patients with demyelinating disease, thus providing further evidence of an equilibrium between peripheral and central nervous system immune compartments.

A consistent immunological finding in patients with progressive multiple sclerosis is a loss of functional suppression. We have recently found decreases in suppressor inducer T cells in progressive multiple sclerosis as measured by two-color immunofluorescence using differentiation markers CD4 and 2H4. In the present study, we examined the relationship between functional suppression and circulating CD4+ 2H4+ T cells using a two-stage assay. (1) T cells were stimulated for 7 days with irradiated non-T cells (autologous mixed lymphocyte reaction [AMLR]) and harvested. It has previously been shown that suppressor T cells are generated during the course of the AMLR. (2) The AMLR-generated suppressor T cells were then incubated with mononuclear cells plus pokeweed mitogen, and immunoglobulin (Ig) synthesis was measured. There was less AMLR-induced suppression of IgG synthesis in patients with progressive multiple sclerosis as compared with normal subjects and patients with other neurological diseases. More importantly, there were significant correlations between decreases in circulating CD4+ 2H4+ cells and the AMLR (p = 0.009). Thus, the decreases in functional suppression and the decreases in the AMLR in multiple sclerosis appear tightly linked to CD4+ 2H4+ cells, and their measurement provides a means to monitor suppressor function phenotypically. Decreases in suppressor inducer T cells may in part explain immunoregulatory abnormalities observed in multiple sclerosis.

Based on the assumption that multiple sclerosis is an autoimmune disease, a number of clinical trials designed to suppress the immune system or to restore immune balance in multiple sclerosis have been attempted. Depending on the disease category, the clinical goals of immunotherapy differ. Therapeutic goals include improving recovery from acute attacks, preventing or decreasing the number of relapses, and halting the disease in its progressive stage. The ultimate goal of multiple sclerosis therapy is the early treatment of patients in an attempt to halt the onset of progression. Specific strategies of immunotherapy include generation of a suppressor influence, removal of helper/inducer cells, manipulation of activated T cells, manipulation of class II major histocompatibility complex-bearing cells, alteration of lymphocyte traffic, extracorporeal removal of serum factors or cells, and manipulation of antigen-specific cells. Present treatment modalities are beginning to show some efficacy of nonspecific immunosuppression, but these treatments are limited by their toxicities. As the immunotherapy of multiple sclerosis moves to the next stage in the coming years, patients at an earlier stage of their disease will have to be treated, nontoxic forms of therapy developed, clinical trials lengthened, and a laboratory monitor of the disease developed. Given the positive effects of immunotherapy seen thus far in the disease, it is possible that appropriate immunotherapeutic intervention may provide effective treatment for the disease in the future.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). In MS patients being treated with anti-T11, a murine monoclonal antibody which recognizes the sheep red blood cell receptor, it was found that peripheral blood T cells were labeled in vivo by the antibody. Furthermore, anti-T11 did not lyse cells or enter the cerebrospinal fluid (CSF). In CSF specimens obtained by serial lumbar punctures from patients with progressive MS who received five daily infusions of anti-T11, 70 +/- 12% of the T cells had mouse antibody bound to their surface by 72 to 96 hours. No in vivo staining of CSF T cells was observed in patients infused with anti-T4, a murine monoclonal antibody that was not found to label T cells in vivo. These results demonstrate that there is rapid movement of lymphocytes from the peripheral blood to the CNS in patients with progressive MS. This rapid trafficking of T cells suggests that the ongoing pathological process within the CNS may be closely linked to the peripheral immune system and may have implications for the monitoring and treatment of MS.

A single-cell cloning technique was used to analyze both phenotype and function of individual T cells in patients with multiple sclerosis (MS). Blood and cerebrospinal fluid (CSF) lymphocytes were plated at 1 cell per well, stimulated with phytohemagglutinin followed by interleukin 2, and expanded to 3 X 10(6) cells per "clone." More than 90% of the T8 clones generated from patients with MS and controls in both blood and CSF were cytotoxic precursors. There was also a slight decrease in cytotoxic T4 clones in the blood of patients with MS. The cytotoxic precursor frequencies of T cells in the CSF generally reflected those in the blood. In separate experiments, antigen reactivity was examined in lines established from blood or CSF. No reactivity to myelin basic protein or white matter was found in patients with MS or controls. Myelin basic protein-reactive clones could, however, be generated after first stimulating lymphocytes with antigen before cloning. These results suggest that changes in the T8 population from the blood of patients with MS involve cytotoxic as well as suppressor cells. Sequestration of myelin basic protein- or white matter-reactive T cells was not seen in the CSF of patients with MS, unlike reports of viral meningoencephalitis, in which large numbers of antigen-specific cells were found in the CSF. Direct single-cell clonal analysis of the CSF should provide a more sophisticated approach to the study of T cell abnormalities in patients with MS.

T-lymphocytes in the cerebrospinal fluid of patients with multiple sclerosis are predominantly CD4+ (inducer) as opposed to CD8+ (suppressor/cytotoxic) T cells. The CD4+ lymphocytes can be subdivided into populations that express high densities of the CDw29 (4B4) determinant and have helper inducer function or express high densities of CD45R (2H4) determinant and have suppressor inducer function. In the present study, we characterized the nature of these CD4+ T cells in the cerebrospinal fluid of patients with multiple sclerosis by performing flow cytometric analysis on paired samples of blood and cerebrospinal fluid. There were significantly lower percentages of CD4+2H4+ T cells in the cerebrospinal fluid than in the peripheral blood (p = 0.001, paired t test). In contrast, there were increased percentages of helper inducer (CD4+4B4+) T cells in the cerebrospinal fluid (p = 0.001, paired t test), compared with the peripheral blood. Analysis of subjects with other inflammatory disorders of the central nervous system did not show significant decreases in CD4+2H4+ T cells in cerebrospinal fluid, though in some, decreases were also observed. These results indicate that the CD4+ T cells in the cerebrospinal fluid of patients with multiple sclerosis are predominantly helper inducer, as opposed to suppressor inducer T cells, and that the relative decrease of suppressor inducer cells in the peripheral blood of multiple sclerosis patients is not due to their migration to the cerebrospinal fluid. Furthermore, the increased numbers of helper inducer cells in the cerebrospinal fluid may contribute to local autoimmune processes in the central nervous system compartment of multiple sclerosis patients.