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We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.

Using retrospectively collected outcome data for treatment naive subjects treated with either glatiramer acetate (GA) ( n = 332) or interferon beta (IFN β) ( n = 424), we replicated the lack of a significant difference in efficacy between these treatments. Further, for both treatments, we observed a decline in the hazard of a relapse over time, which may suggest the existence of subsets of subjects with differential responses to each treatment. The HLA DRB1*1501 allele explained some of this variation in event-free survival while on GA, and we found suggestive evidence that an IRF8 polymorphism influences event-free survival in IFN β treated subjects.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). In the recent years, accumulating evidence has supported an immunosuppressive role for regulatory T cells (Tregs). Most studies in the context of autoimmunity have focused on the defects of the CD4+CD25 high Tregs. However, we recently demonstrated an altered function of Tr1 Treg cells in MS, characterized by a lack of IL-10 secretion. Therefore, several major regulatory T cell defects are involved in human autoimmune disease. Hence, the induction of Tregs or the stimulation of Treg activity may be beneficial for the treatment of such diseases.

Acute disseminated encephalomyelitis (ADEM), a postinfectious illness of the central nervous system (CNS), is thought to be an autoimmune disease. Here, we characterized the cytokines secreted by myelin-reactive T cells generated from patients with ADEM. The frequency of MBP-reactive T cell lines was ten-fold higher in patients with ADEM compared to patients with encephalitis and normal subjects. Whereas there was no significant IFN-gamma secretion, the predominant cytokine secreted by MBP-reactive T cell lines was IL-4 in patients with ADEM. In contrast, IL-4 secretion was only rarely detected in the controls. The presence of high frequencies of MBP-reactive IL-4 secreting T cells in subjects with ADEM during their recovery phase may be similar to myelin reactive IL-4 secreting T cells observed during the spontaneous recovery of animals with EAE.

The presence of autoantibodies to the immunodominant antigen, myelin basic protein (MBP), in the serum and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) has been poorly characterized. Many studies report detectable levels of autoantibodies to myelin basic protein though other studies, using similar techniques, report their absence. We compared a solution-phase assay that has detected clinically relevant autoantibodies in diabetes and other autoimmune diseases to solid phase assays similar to those used in previous reports. The solution-phase assay consistently measured autoantibodies to MBP in serum from human subjects with Semple rabies vaccine (SRV)-induced demyelinating disease and from MBP-immunized animals. A solid phase assay detected MBP autoantibodies in the serum of a fraction of patients with MS. Autoantibodies capable of binding to MBP in the solution-phase were not detected in the CSF or serum of patients with MS. Additional solution-phase measurements revealed that anti-MBP antibodies from individuals with SRV-induced demyelinating disease demonstrated a binding affinity profile consistent with that of polyclonal antibodies with a range of affinities from low to high. In contrast, antibodies to MBP in the serum of MS patients detected by ELISA did not bind soluble MBP in the same assay. These results indicate that the humoral response in patients with MS does not include moderate- or high-affinity autoantibodies to MBP.

Myelin oligodendrocyte glycoprotein (MOG) has attracted considerable attention due to its possible role in multiple sclerosis (MS). Previous studies have shown that MOG-reactive T cells are more commonly detected in MS patients than controls. In this report, we studied human MOG-reactive T cell clones generated from healthy individuals as well as MS patients. Our results indicate that immunoaffinity-purified MOG, which was routinely used in prior studies, is contaminated by anti-MOG antibody (mouse IgG), despite the inability to detect IgG by Western blotting. Here, we used continuous SDS-PAGE, which enabled us to isolate highly purified MOG devoid of contaminating mouse IgG.

Multiple sclerosis (MS) is best understood as an inflammatory disease of the central nervous system (CNS) white matter characterized by demyelination, focal T cell and macrophage infiltrates, axonal injury and loss of neurological function. Our current understanding invokes proinflammatory cells and mediators that may be triggered by environmental factors to mediate disease in a genetically susceptible host. Five major themes which have been associated with the pathogenesis of MS lesions will be discussed: (1) The differential activation states of myelin-reactive T cells from MS patients vs. normal individuals, (2) the selective expression of chemokines, adhesion molecules and matrix metalloproteinases, (3) the proposed roles of the B7 costimulatory pathway, (4) the proinflammatory cytokines and (5) the role of molecular mimicry.

The objective of this study was to determine whether autoreactive T cells in patients with multiple sclerosis (MS) are polarized and committed in their differentiation to a stable cytokine phenotype or whether the cytokine secretion can be altered. We examined the cytokines secreted by myelin basic protein (MBP) as compared to tetanus toxoid-reactive (TT) T cells in 12 patients with relapsing remitting MS (RR-MS), 9 patients with chronic progressive MS (CP-MS), and 14 normal individuals. A total of 5094 short term T cell lines to MBP and TT were generated in the presence of growth conditions promoting Th1 (IL-12/alpha-IL-4 mAb) or Th2 (IL-4/alpha-IL-12 mAb) cytokine secretion. Antigen-specific cytokine secretion from normals and MS patients could be shifted to a Th1 or Th2 type phenotype depending upon culture conditions, indicating that the phenotype of MBP reactive T cells can be altered even in longstanding chronic progressive MS. There were no significant differences in the cytokine patterns secreted by MBP reactive T cells in patients with MS as compared to normal individuals. However, CP-MS patients tended to have fewer MBP reactive T cells secreting IL-4 when cultured with IL-12/anti-IL-4 mAb and more IFN-gamma secreting MBP reactive T cells when cultured with IL-4/anti-IL-12 mAb as compared to both normal controls and RR-MS, suggesting that cells from these patients might be more polarized or that fewer undifferentiated MBP-reactive cells are present in these individuals. The most striking observation was that in contrast to the RR-MS patients and normal controls, almost none of the MBP reactive T cells secreting cytokines in CP-MS incorporated 3[H]thymidine. This may be due to chronic in vivo stimulation in the presence of IL-12, or because these T cells may have entered a terminally differentiated state. Nonetheless, the ability to alter the cytokine secretion of autoreactive T cell lines even in longstanding autoimmune disease indicates that cytokine therapy might have therapeutic benefits by switching the function of myelin reactive T cells such that they are non-pathogenic.

Human microglia constitute the primary residential antigen presenting cells (APCs) in the central nervous system (CNS) and have the capacity of activating myelin reactive T-cells. T-cell activation requires two signals: first is the interaction of the T-cell receptor with the MHC-antigen complex and, secondly, contact of the CD28/CTLA4 T-cell surface molecules with the B7 family of costimulatory molecules on the APCs. We have previously shown high expression of B7.1 in early multiple sclerosis (MS) plaques, suggesting that acute T-cell-mediated CNS inflammation may require local B7.1 upregulation. We have now examined the expression of B7.1 and B7.2 costimulatory molecules on resting ex-vivo human microglia isolated directly from biopsy specimens. We found constitutive expression of B7.2 but not B7.1 on resting microglia, suggesting that B7.2 expression may lead to downregulation of pro-inflammatory Th1 T-cell responses in the normal brain.

Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP). The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as single, 18.5-kDa species as judged by SDS-PAGE. The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP. Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration. Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purifed from protease-rich central nervous system tissue. Purified recombinant MBP produced in e. coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed. Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for investigation of antigen-driven immune tolerance are discussed.

We examined immune function and changes in T cell populations over a 1-year period in a series of progressive multiple sclerosis (MS) patients treated with different regimens of cyclophosphamide/ACTH as part of the Northeast Multiple Sclerosis Treatment Group. Our studies were designed to determine the effect of different cyclophosphamide/ACTH regimens on T cell populations and functional immune assays and to determine whether immune measures could be identified to predict which patients responded favorably to treatment. Cyclophosphamide/ACTH infusions significantly decreased the proportion of peripheral blood CD4+ T cells at 2, 6 and 12 months following treatment while there was a tendency for increased CD8 expression. This was associated with significant decreases of CD4 CD8 ratios at 2, 6 and 12 months following treatment compared to pretreatment. No changes in CD3+ T cells were observed while there were increased percentages of CDw26 (Ta1) positive and IL-2 positive T cells following treatment. The only T cell populations predictive of improvement were percentages of either CD3+ or CD4+ cells where increased percentages of either these populations at 2 months following cyclophosphamide/ACTH infusions were associated with improvement at both 6 and 12 months. In terms of functional immune measures, we found that cyclophosphamide/ACTH treatment decreased the level of proliferation in the allogeneic mixed lymphocyte reaction (MLR) at 2 months and of spontaneous proliferation of mononuclear cells at 12 months following therapy. Changes in spontaneous proliferation were predictive of clinical improvement at 12 months in that subjects with improved scores on the disability status scale (DSS) had decreases in spontaneous proliferation at 12 months as compared to pretreatment, whereas those stable or worse did not change significantly. Thus, our studies have demonstrated specific alterations in immune function following immunosuppression with cyclophosphamide/ACTH and suggest that certain immune measures may be linked to a positive clinical response and thus associated with disease progression in MS.

We have found an alteration in T-cell subsets in patients with active multiple sclerosis, specifically an increase in the T4: T8 ratio. These findings have been reproducibly obtained over the past four years, occurring in the majority of acute patients tested early in the course of an attack and in between 25 and 40% of chronic progressive patients, depending on their stage of illness. These changes correlate with pleocytosis in spinal fluid and with other abnormalities of immune function, such as spontaneous immunoglobulin production. They have been helpful in assessing disease activity in patients being treated on a variety of protocols and as part of research studies of immunoregulatory abnormality in multiple sclerosis, but have not been helpful as a diagnostic test for multiple sclerosis. The decrease of these cells in the peripheral blood of patients with active disease may be secondary to migration of these cells to the central nervous system, where they are sequestered.

The autologous mixed lymphocyte reaction (AMLR) measures the T-cell response to antigens on the surface of autologous non-T-cells. Studies have shown a decreased ability of T-cells to proliferate during the AMLR in a number of autoimmune and viral disorders. The AMLR was studied in 56 patients with multiple sclerosis (MS), a presumed autoimmune disease of the central nervous system, and the response was correlated with T-cell subsets. The AMLR was decreased in active MS patients (3 117 +/- 573 cpm) as compared to inactive patients (5 896 +/- 1 800 cpm) (P less than 0.05) and normal controls (7 782 +/- 1 725 cpm) (P less than 0.01). There was no significant difference between inactive patients and normal controls (P greater than 0.1). A more pronounced difference was also seen when patients with active disease associated with peripheral blood T4/T8 ratios greater than 3.0 (1 045 +/- 223 cpm) were compared with normal controls (P less than 0.01). In addition, MS patients with T4/T8 ratios of less than 1.0 had an increased AMLR (9 256 +/- 1 762 cmp) as compared to patients with either a high T4/T8 ratio (P less than 0.001) or normal ratio (P less than 0.05). Thus, multiple sclerosis patients with clinically active disease have a decreased AMLR which correlates with high T4/T8 ratios in a subgroup of patients.

The frequency of virus-specific T cells in the cerebrospinal fluid of a patient with viral infection of the brain and meninges was determined by using a single-T-cell cloning technique where a representative sampling of T cells was cloned from cerebrospinal fluid of a patient with varicella zoster viral (VZV) meningoencephalitis. That the derived T-cell clone were in fact clonal was shown by demonstrating, on Southern blot analyses, unique rearrangements of the T-cell antigen-receptor β-chain genes of each clone. Five out of the 15 of the T4 + (CD4), 0/4 of the T8 + , and 0/1 of the T4 + T8 + T-cell clones proliferated to VZV, while no clones proliferated to mumps virus or myelin basic protein. There was no clonal expansion of any VZV-reactive T cell in this patient's cerebrospinal fluid. As VZV meningoencephalitis is thought to be due to the reactivation of a dormant herpes zoster viral infection, it can be regarded as a secondary immune response. The presence of different T-cell receptor β-chain gene rearrangements in each T-cell clone suggests that the T-cell response was polyclonal. These results demonstrate that a high frequency of polyclonal, T4 + antigen-specific T cells can be found in a naturaly occurring, localized, immune response.

Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9/30 for MAG, 0/17 for PLP, and 8/81 for MBP. No control subjects responded to MAG or PLP, and only 1/29 control subjects responded to MBP. Three of the patients that responded to MAG also responded to MBP. Although the mean proliferative response to MAG and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The OND group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to MAG (P less than 0.05), and OND vs. controls and active MS that responded to PLP (P less than 0.025). The greatest individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases.