Cytokine secretion profile of myelin basic protein-specific T cells in multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a presumed autoimmune pathogenesis involving autoantigen-specific CD4+ T cells and cytokines. A similar frequency of T cells responding to myelin basic protein (MBP), a putative target in MS, has been observed in MS patients and controls. To dissect the differences between MBP-specific T cells in patients and controls, we have analyzed the cytokine secretion profile of such autoreactive T cells. MBP-specific T cell clones (TCC) were isolated from the peripheral blood of MS patients and controls by limiting dilution. Expression of mRNA for interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) was assessed by polymerase chain reaction whereas secretion of cytokine protein was measured by ELISA. MBP-specific TCC exhibited a heterogeneous cytokine secretion profile with clones displaying Th1, Th2 and Th0 phenotypes. A significant difference in the distribution of the cytokine profile was noted between MS patients and controls. Although the frequency of Th1 secreting MBP-reactive TCC was similar between MS patients and controls, stable MS patients had a significant association with the Th0 phenotype whereas healthy individuals were associated with the Th2 phenotype. In comparison to control TCC, MBP-specific TCC from MS patients secreted increased amounts of IFN-gamma, IL-4 and IL-10 and decreased quantities of TGF-beta. Thus, these studies suggest that there is a dysregulation in the balance between pro-inflammatory Th1 and anti-inflammatory Th2 cytokines in MS. It appears that the presence of Th1 secreting autoreactive T cells in healthy individuals may be counterbalanced by the presence of cells secreting Th2 cytokines and by the augmented production of the immunosuppressive cytokine TGF-beta, whereas in MS there is a decrease in these anti-inflammatory agents.     

Isolation of myelin basic protein–specific T cells predominantly from the memory T-cell compartment in multiple sclerosis

Myelin antigen–reactive T cells have been implicated in the pathogenesis of multiple sclerosis (MS). Myelin-reactive T cells can be isolated from control subjects as well as individuals who have MS. Experimental models of MS indicate that recently stimulated, myelin-reactive T cells have greater encephalitogenic potential than resting T cells. Activation induces changes in T-cell surface antigens that may distinguish previously stimulated, memory T cells from naive T cells. Therefore, we examined 108 myelin basic protein (MBP)-reactive T-cell lines from 7 MS and 8 control subjects to determine whether MBP-reactive T cells originated in the memory T-cell subset or in the naive subset. Isotypes of CD45 were used that designate memory or naive T cells. In subjects having MS, 84% of the MBP-reactive T cells resided in the memory T-cell subset. However, in control subjects, only 13% of MBP-specific T cells originated from the memory T-cell subset. This result suggests that a substantial proportion of MBP-reactive T cells from some individuals with MS have been previously activated in vivo. This difference provides additional support for the hypothesis that myelin antigen–specific T cells are involved in the pathogenesis of MS. Ann Neurol 1999;45:33–39     

Urinary myelin basic protein—like material as a correlate of the progression of multiple sclerosis

In the multicenter, randomized, placebo-controlled trial of alternate-day injections of recombinant interferon beta-1b in relapsing-remitting multiple sclerosis (MS), urine specimens were collected periodically from all patients (n = 64) in two of the clinical test sites over the 2 years of the study. Urine specimens were also collected over two consecutive 24-hour periods from 43 patients from a third center. Urine samples were assayed for their content of myelin basic protein-like material (MBPLM), the level of which was correlated with clinical changes, cranial magnetic resonance imaging results, and the development of progressive disease. Concordant changes in creatinine values affected some of the relationships of MBPLM. The level of urinary MBPLM correlated with a chronic progressive course and with the number of lesions and the total lesion area on cranial magnetic resonance images. A rise in the level of urinary MBPLM appeared to antedate the clinical transition from a relapsing-remitting to a chronic progressive course. By chance, the randomized entry of patients led to significant differences in urinary MBPLM levels among the three treatment groups, thus precluding correlation studies of treatment effects. However, the patient group from which 24-hour specimens were collected showed that the patients with relapsing-remitting MS changing to a chronic progressive course, and more specifically, those patients with chronic progressive MS receiving placebo, had the highest values of urinary MBPLM. These findings indicate that urinary MBPLM may offer an objective test and possibly serve as a surrogate Marchker for detecting or predicting the failure of remission or the transition to a progressive phase of MS.     

Multiple sclerosis: occurrence of myelin basic protein peptide-reactive T cells in healthy family members

Genetic factors influence the susceptibility to multiple sclerosis (MS). This disease is accompanied by augmented T cell responses to CNS myelin components such as myelin basic protein. To evaluate the familial occurrence of such T cell autoreactivity, we have studied 12 MS families including 37 healthy first-degree relatives for occurrence of numbers of interferon-gamma (IFN-γ) secreting cells among blood mononuclear after culture in presence of myelin basic protein (MBP), eight synthetic MBP peptides and the control antigen acetylcholine receptor (AChR). There were no differences between MS patients and healthy family members regarding frequencies of autoreactive T cells recognizing MBP, the eight different MBP peptides or AChR. None of the MBP peptides predominated as T cell antigen among the MS patients or their unaffected family members. In some families the highest number of MBP peptide reactive T cells were found among unaffected family members. No correlation was observed between numbers of MBP or MBP peptide reactive T cells in various subjects and their HLA-DR-DQ phenotypes. In conclusion, this study has revealed the presence of MBP and MBP peptide reactive T cells of similar frequencies in MS patients and their healthy family members.     

CSF myelin basic protein in multiple sclerosis

Cerebrospinal fluid (CSF) from 221 patients with multiple sclerosis (MS) and 85 patients with other neurological disorders (OND) was examined using a competitive radioimmunoassay for myelin basic protein (MBP) immunoreactivity. MBP was found in 46 of 55 MS patients (84%) examined within six weeks of relapse but in only 11 of 85 patients (13%) with OND. There was a significant correlation between the concentration of MBP in the CSF and relapse severity in patients seen within four weeks of the onset of symptoms (p less than 0.01). Of 44 patients in remission, MBP was detected in 12, and these patients had a significantly higher tendency to subsequent relapse (p less than 0.05). In 72 patients with progressive disease the presence of MBP in the CSF reflected the confidence of clinical diagnosis. The results of this study suggest that measurement of MBP in the CSF gives an objective method of monitoring disease activity in patient with MS.     

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.     

Human T-cell response to myelin basic protein in multiple sclerosis patients and healthy subjects

In order to explore the T-cell repertoire to myelin basic protein (BP) of both multiple sclerosis (MS) patients and healthy subjects (HS), we raised BP reactive T-cell lines from blood mononuclear cells of eight MS patients and five HS. These lines were triggered in vitro by human BP. When analyzing their patterns of recognition of human BP versus heterologous BP, we could observe differences between healthy subjects and MS patients. Whereas T-cell lines from healthy subjects developed a response to heterologous BP, which was in most cases equal or higher than that elicited by human BP, T-cell lines from most MS patients displayed a low response, or no response at all, to one or several of the heterologous BP tested. A low response to bovine BP was only observed in active cases, whereas decreased responses to rat and/or monkey BP were observed both during remission and during active disease. This may indicate that T-cell repertoire to BP in MS patients differs from that of healthy subjects. BP-reactive T-cell clones were obtained by limiting dilution from two healthy subject lines. Their pattern of response to heterologous BP as compared to human BP suggest that T-cells from the same individual can recognize different BP epitopes.     

Target epitopes of myelin basic protein specific T cell lines in multiple sclerosis

A highly efficient new method for the generation of antigen specific T cell lines (TCL) is now available. By this method we established 134 myelin basic protein (MBP) TCL from the peripheral blood of 9 patients with definite multiple sclerosis (n=69) and 8 healthy donors (n=65). The yield of MBP reactive TCL in the two groups was comparable. So far 22 MBP specific TCL from 7 patients and 24 from 7 healthy individuals have been tested for their proliferative response to a panel of four synthetic peptides representing MBP residues 7–26, 80–99, 139–153 and 148–162. Although the peptide sequences did not encompass the whole MBP, the pattern of reactivity to these peptides in patients and controls seems to be similar. Further, when multiple TCL from the same donor were analysed, no dominant recognition emerged. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Enhanced T cell responsiveness to citrulline-containing myelin basic protein in multiple sclerosis patients

Myelin basic protein (MBP), a candidate autoantigen in multiple sclerosis (MS), exists in different isoforms and charge isomers generated by differential splicing of exons and by a combination of posttranslational modifications, respectively. These various isoforms and charge isomers of MBP vary in abundance and most likely serve different functions during myelinogenesis and remyelination. The least cationic among the charge isomers of MBP is citrullinated and is referred to as MBP-C8. MBP-C8 is relatively increased in the population of MBP isomers in more developmentally immature myelin and in MS brain tissue. In a previous study, we found that MBP-C8-reactive T cells could be detected in CD4+ T cell lines (TCL) generated with MBP from both MS patients and normal controls. Here, we examined the frequency and peptide specificity of MBP-C8-specific TCL generated with MBP-C8 in MS patients and controls. Ten subjects grouped in five sets, each an MS patient and a control, were studied. In all cases, the MS patient had either a higher overall number of MBP-C8-responding lines, responded with greater sensitivity to the MBP-C8 antigen or both. Few lines responded to the MBP-C8 peptides but, if they did, they appeared to be specific to the carboxyl-half of the MBP-C8 molecule. Given the large amounts of citrullinated MBP in MS brain tissue, a preferential T cell response to MBP-C8 may be involved in the induction and perpetuation of this disease. Multiple Sclerosis (2000) 6 220 - 225     

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.     

Myelin reactive T cells after T cell vaccination in multiple sclerosis: cytokine profile and depletion by additional immunizations

Pathogenic autoreactive T cells can be targeted by T cell vaccination (TCV), a procedure in which patients are immunized with autologous attenuated pathogenic T cells. We reported previously that TCV with myelin basic protein (MBP) reactive T cell clones in multiple sclerosis (MS) patients induced T cell immune responses, resulting in a clonal depletion of MBP reactive T cells in all patients. Five years after TCV, MBP reactive T cells were observed in five out of nine MS patients. These clones had a different clonal origin from those isolated before vaccination. We have studied the cytokine profile, cytotoxicity and epitope specificity of these reappearing clones. Our data indicate that the clones express similar effector functions as those isolated before TCV, suggesting that they also could play a pathogenic role in the disease. We demonstrated that these clones can be depleted by an additional sequence of immunizations. These findings may be relevant to other T cell targeted immunotherapies for MS and other autoimmune diseases.     

髓鞘碱性蛋白基因与多发性硬化相关性的研究

目的探讨中国人群中多发性硬化（ＭＳ）的遗传易患性与髓鞘碱性蛋白基因５′端四核苷酸重复序列多态性的相关性。方法以２８例确诊ＭＳ患者及４７名正常对照为研究对象,从外周血白细胞中提取ＤＮＡ,利用聚合酶链反应技术特异性扩增ＭＢＰ基因５′端四核苷酸重复序列,并通过琼脂糖凝胶电泳分析扩增产物的多态性。结果发现四种等位基因,片段大小为１．０６ｋｂ,０．９８ｋｂ,０．６１ｋｂ和０．５４ｋｂ,以前两种多见。１．０６ｋｂ基因片段在女性中出现频率很高,达７３％,ＭＳ组与对照组无差别。男性中１．０６ｋｂ基因片段出现频率ＭＳ组（６９％）明显高于对照组（３９％）。ＭＳ组中等位基因的分布无性别差异。结论中国人群中ＭＢＰ基因第一个外显子５′端四核苷酸重复序列多态位点等位基因的分布有性别差异,１．０６ｋｂ的等位基因片段可能与ＭＳ易患性相关。     

Cellular hypersensitivity to gangliosides and myelin basic protein in multiple sclerosis

Peripheral blood lymphocytes from patients with multiple sclerosis (MS), other neurological diseases and healthy controls were investigated for the presence of cell-mediated hypersensitivity to brain gangliosides and myelin basic protein using an active E-rosette assay. Sensitivity to myelin basic protein and gangliosides was found in MS patients in acute relapse and with progressive disease, whereas no sensitivity was found in MS patients in remission. Patients with other neurological diseases showed no response to gangliosides, but sensitization to myelin basic protein was found in a patient with leucoencephalopathy and in 4 of 6 stroke patients. Healthy controls did not respond to either antigen. In MS patients a positive correlation was seen between lymphocyte responses to myelin basic protein and to gangliosides. The data suggest that in comparison to gangliosides, myelin basic protein is a weaker stimulator of active rosette-forming cells. Moreover, cellular hypersensitivity to myelin basic protein is not MS-specific and may be present as a consequence of brain damage. However, cellular hypersensitivity to gangliosides appears to be more specific to MS and may be important in the pathogenesis of the disease.     

Functional properties of myelin oligodendrocyte glycoprotein-reactive T cells in multiple sclerosis patients and controls

Autoimmune T-cell reactivity to myelin components may be implicated in the initiation or maintenance of the inflammation leading to myelin destruction in multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG), a quantitatively minor myelin protein, is an important candidate autoantigen in MS. We studied T-cell responses to recombinant MOG (extracellular domain, rMOG) and a panel of four peptides within this domain (amino acids 1-22, 34-56, 64-86 and 74-96) in MS patients and healthy controls (NS). Frequency analysis of T cells reactive to rMOG as measured by IFN-gamma ELISPOT did not reveal significant differences between MS patients and controls. MOG-reactive T-cell lines and clones (TCL/TCC) were generated by stimulating PBMC of four MS patients and three healthy subjects with a cocktail of the four MOG peptides. The functional properties of 50 MOG peptide-reactive TCL/TCC obtained were studied. All TCL were TCR alpha beta+CD4+ and 20 TCL showed reactivity to MOG peptides 1-22, 13 to 34-56, 1 to 64-86 and 16 to 74-96. No significant differences in peptide recognition were observed between MS patients and controls. The T-cell receptor (TCR) hypervariable regions of MOG-reactive TCL/TCC showed a heterogeneous usage of various TCR V(-D)-J elements. The data provide no evidence for clonal expansions within the MOG-reactive T-cell repertoire of the two study groups. Intracellular cytokine analysis demonstrated predominantly Th1-TCC (IFN-gamma+/IL-4-) in MS patients, while most MOG-reactive TCC of control subjects had a mixed Th0/Th1 phenotype. Furthermore, the MS-derived MOG-reactive TCC produced increased levels of TNF-alpha upon antigen stimulation as compared to controls. Most of the MS-derived MOG-TCC induced specific cytolysis of autologous MOG-pulsed PBMC (9/11) while none of the MOG-TCC isolated from control subjects showed this cytotoxicity (0/8). In conclusion, although the frequency of anti-MOG T cells was similar in MS patients and controls, our data indicate potential differences in the functional properties of MOG TCL in MS patients versus healthy controls which may relate to their role in the disease process.     

T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls

Myelin proteins, including myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) are candidate autoantigens in MS. It is not clear whether MS patients show a predominant reactivity to one or several myelin antigens. We evaluated the IFN-gamma production induced by MBP and MOG and selected MBP-, MOG- and PLP-peptides in MS patients and healthy controls using the IFN-gamma ELISPOT assay. Most MS patients and healthy controls showed a heterogeneous anti-myelin T-cell reactivity. Interestingly in MS patients a positive correlation was found between the anti-MOG and anti-MBP T-cell responses. No myelin peptide was preferentially recognized among the peptides tested (MBP 84-102, 143-168, MOG 1-22, 34-56, 64-86, 74-96, PLP 41-58, 184-199, 190-209). In addition the frequency of IL2R+ MBP reactive T-cells was significantly increased in blood of MS patients as compared with healthy subjects, indicating that MBP reactive T-cells exist in an in vivo activated state in MS patients. Most of the anti-MBP T-cells were of the Th1-type because reactivity was observed in IFN-gamma but not in IL-4 ELISPOT-assays. Using Th1 (IL-12) and Th2 (IL-4) promoting conditions we observed that the cytokine secretion pattern of anti-MBP T-cells still is susceptible to alteration. Our data further indicate that precursor frequency analysis of myelin reactive T-cells by proliferation-based assays may underestimate the true frequency of myelin specific T-cells significantly.     

Similar sensitivity of regulatory T cells towards CD95L-mediated apoptosis in patients with multiple sclerosis and healthy individuals

Impaired suppressive function of CD4(+)CD25(high) regulatory T cells (T(reg)) has been reported as a novel pathogenetic mechanism in Multiple sclerosis (MS). We addressed if high apoptosis sensitivity of MS-T(reg) could explain this functional T(reg) defect. T(reg) from treatment-na?ve MS patients showed high sensitivity towards CD95Ligand-mediated apoptosis and exhibited enhanced cell death to IL-2 and TCR-signal deprivation. Since susceptibility of T(reg) to cell death was similar in MS patients and healthy controls, this cannot explain the inhibitory dysfunction of T(reg) associated with MS. Furthermore, as cell death is not enhanced, therapeutic expansion of MS-T(reg)in vitro should be a reasonable and novel therapeutic option.     

Adherence of cells to myelin basic protein I. Adherence of red and white blood cells from patients with multiple sclerosis to myelin basic protein

Red blood cells (RBC) and white blood cells (WBC) of patients with multiple sclerosis (MS) show decreased adherence to myelin basic protein (MBP) immobilized on plastic surfaces compared to the binding of cells from patients with other neurological diseases (OND), or such other autoimmune diseases as psoriasis (PS), and to that of healthy controls (HC). No similar phenomenon occurred to basic and non-basic type proteins other than MBP, for example, to histone (HIS), lysozyme (LYS) and ovalbumin (OVA). Thus, decreased adherence of RBC and WBC in MS patients to MBP appears to be a unique feature of the disease if compared with OND or PS.     

Intrathecal synthesis of autoantibodies to myelin basic protein in multiple sclerosis

A solid phase radioimmunoassay was used to detect anti-myelin basic protein (MBP) antibodies in the CSF and serum of multiple sclerosis (MS) patients and controls. CSF and serum samples were assayed prior to acid hydrolysis in order to detect free anti-MBP as well as after acid hydrolysis to measure the total (free and bound) amount of antibody. An anti-MBP index controlling for serum levels as well as the degree of breakdown of the blood brain barrier was used to estimate intrathecal synthesis of anti-MBP. MS patients with acute exacerbations or chronically progressive disease have significantly elevated levels of both free and total CSF anti-MBP. The anti-MBP index is also significantly increased in MS patients with both forms of active disease. Anti-MBP antibodies are intrathecally produced in MS patients with active disease.