Glatiramer acetate-specific human CD8(+) T cells: increased IL-4 production in multiple sclerosis is reduced by glatiramer acetate treatment

Glatiramer acetate (GA) is an approved drug for therapy of relapsing remitting MS that acts as a T cell antigen. Here, we report the cloning of HLA restricted, GA-specific human CD8(+) T cells. In addition, we analyzed the cytokine profile of GA-reactive CD8(+) T cell lines. Unexpectedly, IL-4 was increased in untreated MS patients as compared to healthy individuals (p<0.001). In GA-treated patients, however, IL-4 (p<0.001), IL-10 (p<0.001) and TNF-alpha (p<0.001) were decreased. Thus, while GA is known to induce a TH2 bias in CD4(+) T cells, we detected a distinct pattern in GA-reactive CD8(+) T cells.     

Effect of combined IFNbeta-1a and glatiramer acetate therapy on GA-specific T-cell responses in multiple sclerosis

The combined treatment with interferon beta (IFNbeta) and glatiramer acetate (GA) is of current interest in multiple sclerosis (MS). The therapeutic effect of GA in MS is believed to be mediated by GA-specific Th2 cells. IFNbeta has a significant anti-proliferative effect on GA-induced lymphoproliferation in vitro. Therefore, we examined the possibility that IFNbeta may interfere with the generation and phenotype of GA T-cell responses in MS patients receiving combined therapy. Sixty-six GA-specific T-cell lines (TCL) were generated ex vivo from five MS patients enrolled in an open-label dinical trial of combined IFNbeta/GA treatment. Controls included 83 pretreatment and 131 on-treatment GA-TCL from 11 MS patients treated with GA only, and five GA-TCL generated from four patients receiving IFNbeta-1a monotherapy. IFNgamma and IL-5 (markers of Th1 and Th2 responses, respectively) were assayed by ELISA in GA-TCL supematants. Th1/Th2 bias was defined by the IFNgamma/IL-5 level ratio ( >2 = Th1 bias, <0.5 = Th2 bias, and 0.5-2 = Th0 bias). The frequency with which GA-reactive TCL were generated was 37.0% for the patients in the combination trial compared to 33.3% in the patients receiving GA alone. The mean stimulation index of the GA-TCL was 8.41 (range 2-42) for the combination compared to a mean of 6.29 (range 2-37) for the GA-treated group--a nonsignificant difference. Mean GA-TCL IFNgamma production was significantly lower in all treatment groups compared to pretreatment IL-5 levels were enhanced in all treatment groups compared to pretreatment levels, but the change was not statistically significant. The Th1/Th0/Th2 distribution of GA-TCL was 7%/30%/63% for the GA+IFNbeta group, 8%/9%/83% for the GA group, compared to 48%/21%/31% pre-GA treatment. All five GA-TCL from the IFNbeta-1a monotherapy patients were Th2-biased. We conclude that IFNbeta-1a does not affect the generation of GA-reactive T cells in vivo. Although more Th0 G4-TCL occurred with combination therapy than with G4 treatment alone, both groups shared an overall Th2 bias. Therefore, we speculate that combined therapy is unlikely to reduce the efficacy of GA treatment in MS.     

Glatiramer acetate induces a Th2-biased response and crossreactivity with myelin basic protein in patients with MS

Glatiromer acetate (GA) is an approved treatment for multiple sclerosis (MS). The proposed mechanism of action is the induction of GA-specific T cells characterized by protective anti-inflammatory Th2 response. We tested this hypothesis in 11 MS patients treated with GA from 1-19 months. Interferon-gamma and IL-5 (markers of Th1 and Th2 responses respectively) were assayed by ELISA in GA-specific T-cell lines (TCL) supernatants. Th1/Th2 bias was defined based on the ratio of IFN-gamma/IL-5 secretion. Fifty-eight pre-treatment and 75 on-treatment GA-specific TCL were generated. On-treatment mean IL-5 levels in GA-TCL increased significantly, whereas those for IFN-gamma were markedly reduced. Consequently, the ratio of IFN-gamma IL-5 also shifted in favor of a Th2 response. The percentage of GA-TCL classified as Th1 was decreased, whereas those classified as Th2 increased on-treatment as compared to pre-treatment. Some GA-specific TCL, (approximately 25%) generated during treatment secreted predominantly IL-5 in response to MBP and the immunodominant MBP peptide 83-99, indicating that these crossreactive antigens can act as partial agonists for GA-reactive TCL. These results strongly suggest that the mechanism of action of GA in MS involves the induction of crossreactive GA-specific T cells with a predominant Th2 cytokine profile.     

Clinical and immune responses correlate in glatiramer acetate therapy of multiple sclerosis

Glatiramer acetate (GA) treatment for relapsing remitting multiple sclerosis (RRMS) leads to decreased GA-specific proliferative responses and a Th2 cytokine shift. To study a possible correlation between immunological and clinical responses to GA therapy, we prospectively followed RRMS patients clinically, by magnetic resonance imaging and by primary immunological assays. Fluctuation of GA-specific proliferative responses was significantly lower in treatment responders than in untreated patients, and GA-specific proliferative responses were increased during relapses. These associations suggest a possible causal relationship between immunological and clinical responses to GA therapy. Primary proliferation assays may thus be a useful marker for treatment response.     

Multiple sclerosis: expression of CD1a and production of IL-12p70 and IFN-gamma by blood mononuclear cells in patients on combination therapy with IFN-beta and glatiramer acetate compared to monotherapy with IFN-beta

Current therapy of multiple sclerosis (MS) with interferon-beta (IFN-beta) or glatiramer acetate (GA) has modest effects on the course of MS. Both compounds affect several immune variables, like expression of cell surface molecules and cytokine levels. Here we compared untreated MS, therapy with IFN-beta alone and combined with GA, and healthy controls (HC), regarding expression on HLA-DR+ blood mononuclear cells (MNC) of CD1a that is a cell surface molecule with capacity to present glycolipids to T cells, and of CD80 and CD86 which are costimulatory molecules that activate Th1 and Th2 responses. Cytokine production by MNC was also measured. Flow cytometry and ELISA were used. Cross-sectional comparisons revealed that untreated MS patients had higher CD1a+ HLA-DR+ MNC and lower IL-10 production compared to patients treated with IFN-beta or IFN-beta+GA or HC. Untreated MS patients also had higher spontaneous IFN-gamma and IL-12p70 production compared to MS patients treated with IFN-beta+GA or HC, but not when compared to MS patients on monotherapy with IFN-beta. Low CD1a+ HLA-DR+ MNC and low spontaneous production of IL-12p70 and IFN-gamma were more pronounced in patients treated with IFN-beta+GA than with IFN-beta alone. In order to clarify whether these changes reflect disease activity or treatment effects, we performed a follow up study. Nineteen MS patients with disease progression, despite monotherapy with IFN-beta for more than one year; were re-examined after one to three and four to six months of treatment with IFN-beta+GA. This combination therapy was associated with normalization of CD1a+ HLA-DR+ MNC, IL-12p70 and IFN-gamma. It remains to be shown whether these immunological changes imply a clinical benefit. Follow up studies of immune variables versus clinical effects during combined therapy of MS with IFN-beta+GA are ongoing.     

Increased numbers of mononuclear cells from blood and CSF expressing interferon-gamma mRNA in multiple sclerosis are from both the CD4+ and the CD8+ subsets

Activated, cytokine-producing lymphocytes may regulate central nervous system (CNS) inflammation in multiple sclerosis (MS). We utilize a novel combination of in situ hybridization (ISH) and immunocytochemical staining of peripheral blood lymphocytes (PBLs) to identify spontaneously interferon-gamma (IFNgamma) mRNA expressing cells as CD4+ or CD8+. A major proportion of the IFNgamma mRNA expressing lymphocytes belonged to the CD4+ lineage, which concords with the cellular composition of MS brain lesions, findings in experimental models and the HLA class II haplotype association in MS. There were also significantly more CD8+ IFNgamma mRNA expressing lymphocytes in the MS patients compared with healthy controls, further suggesting the contribution of activated cells from this lineage in the inflammatory response in MS. Both CD4+ and CD8+ IFNgamma mRNA expressing cells were enriched in the cerebrospinal fluid (CSF) as compared with the peripheral blood of the MS patients. Combined with emerging genetic data on HLA class I influences, our data argues for a joint role of activated CD8+ and CD4+ cells in the pathogenesis of MS.     

Restricted BV gene usage by factor VIII-reactive CD4+ T cells in inhibitor-positive patients with severe hemophilia A

In the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitro stimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy     

Migration of T-cell subsets in multiple sclerosis and the effect of interferon-beta1a

OBJECTIVES: Migration of inflammatory cells across the blood-brain barrier is a central event in the formation of multiple sclerosis (MS) lesions and is known to be enhanced in MS patients. This study investigates the migration of CD4+ and CD8+ T-cell subsets and the effects of interferon-beta1a (IFN-beta1a) treatment on migration and matrix metalloproteinase-9 (MMP-9) production of these T-cell subsets. MATERIALS AND METHODS: An ex vivo transwell system was established to compare the migratory behaviour of lymphocytes isolated from normal controls and untreated MS patients. In addition, MS patients were investigated longitudinally after initiation of IFN-beta1a treatment. RESULTS: Migration of CD4+ T cells (P < 0.05), but not of CD8+ T cells, was enhanced in untreated MS patients compared with controls and was normalized by treatment with IFN-beta1a. In addition, IFN-beta1a treatment reduced MMP-9 production of CD4+ but not CD8+ T cells. CONCLUSION: Our results indicate that CD4+ T cells, but not CD8+ T cells, contribute to the enhanced ex vivo migration observed in MS.     

CD4+ lymphocyte subsets in the cerebrospinal fluid of multiple sclerosis and non-inflammatory neurological diseases

Summary We studied paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples from 18 inactive multiple sclerosis (MS) patients and 10 with non-inflammatory neurological diseases. By means of a dual-colour cytofluorimetric micromethod we were able to count 1500 cells on average in each CSF sample. We found a significant reduction of CD45RA+ and CD4+CD45RA+ cells in the CSF of MS patients. Similarly, CD45RA+ and CD4+CD45RA+ CSF/PB ratios were lower compared with controls. The reduction of suppressor-inducer T-cells did not correlate with CD8+ cell levels in the CSF. The CD4+ subset ratio (CD4+CD45RA–/CD4+CD45RA+) was significantly increased in the CSF of MS patients. Our data suggest that the reduction of CD4+CD45RA+ cells in the PB is not due to a segregation of such cells in the CSF. Conversely, CSF changes reflect changes in the PB similar to these found for other T-cell subsets.     

Expression of costimulatory molecules on peripheral blood mononuclear cells in multiple sclerosis

OBJECTIVE: To determine whether there was aberrant expression of costimulatory molecules and their receptors on peripheral blood mononuclear cells of MS patients and healthy individuals and whether expression correlated with disease status. METHODS: Forty-six patients with MS and 29 healthy individuals were analyzed by direct 2-color immunofluorescence flow cytometry for expression of CD80, CD86, CD28 and CTLA-4 on T and B cells and monocytes. RESULTS: Expression of CD80 on CD4+ T cells was upregulated in progressing MS patients compared to stable MS patients and controls. Marked increase in the expression of CD80 and CD86 was seen on both CD4+ and CD8+ T cells from an MS patient with rapidly progressing disease. No difference in the expression of the costimulatory molecules or their ligands was seen between IFN-beta treated and non-treated MS patients. CONCLUSIONS: Aberrant expression of costimulatory molecules occurs on T lymphocytes in MS patients with progressing disease.     

Restricted use of VH4 Germline segments in an acute multiple sclerosis brain

Multiple sclerosis (MS) cerebrospinal fluid and brain contain increased IgG and oligoclonal bands. Whether this oligoclonal and polyclonal IgG is directed against a disease-relevant antigen remains unknown. To distinguish between random activation versus a targeted B-cell response, we analyzed the IgG heavy chain variable region (V_H) repertoire expressed in different lesions of an acute MS brain. To obtain a representative sample of the V_H repertoire, we constructed directional complementary DNA libraries from plaque-periplaque messenger RNA and amplified V_H regions from the library by nested polymerase chain reaction. When MS V_H sequences were aligned to germline segments, about 60% of different V_H sequences in the acute MS brain were V_H4 germline segments, significantly greater than the known approximately 20% V_H4 germline prevalence. Specific V_H sequences were overrepresented and expressed at multiple plaque sites. Within some overexpressed populations, there were distinct sequence differences (clonal variants) indicative of clonal expansion. Alignment of V_H sequences to their closest germline counterparts revealed extensive somatic mutation and the preferential accumulation of amino acid replacement mutations in complementarity determining regions. These observations suggest the limited B-cell response found in this acute MS brain was antigen driven.     

Combination treatment of Glatiramer Acetate and Minocycline affects phenotype expression of blood monocyte-derived dendritic cells in Multiple Sclerosis patients

The effects of Glatiramer Acetate (GA) in combination with Minocycline (MIN), a second-generation tetracycline, have been investigated on the course of EAE in mice, resulting in a significant reduction in disease severity and burden with attenuation of the inflammation, axonal loss and demyelination. Here we investigate the effects of combination therapy with GA and MIN on the induction, maturation and phenotyping of blood monocyte-derived dendritic cells (DCs) in Multiple Sclerosis (MS) patients. Hence the expressions of HLA-DR, CD11c, CD83 and CD1a were studied by flow cytometric analysis on immature (iDCs) and mature DCs (mDCs) from untreated and GA treated MS patients. Thirteen relapsing-remitting MS patients and 13 healthy controls (HCs) were included in the study. Ten of the MS patient group were re-tested after a 2 month period of GA treatment. The marker expressions on DC from untreated MS and HCs were studied in vitro in the absence or presence of GA and GA+MIN; and on DCs from GA treated MS patients without and with the in vitro addition of MIN. We found that in vitro GA alone or in combination with MIN downregulated DCs antigen presentation capability (HLA-DR), whereas the combination treatment only affected also myeloid DCs activation (CD83) in both MS and HCs. Prolonged GA treatment (in vivo for 2 months) affected antigen presentation capability by DCs, whereas when treated in vitro with MIN these cells also tended to reduce activation marker expression and myeloid phenotype acquisition (CD11c). The present data demonstrate possible combination effects of GA and MIN on peripheral blood monocyte-derived DCs in MS patients.     

Expansion of regulatory CD8+ T-lymphocytes and fall of activated CD8+ T-lymphocytes after i.v. methyl-prednisolone for multiple sclerosis relapse

INTRODUCTION: Multiple sclerosis (MS) is a multifocal chronic inflammatory demyelinating disease of the central nervous system. Axonal damage correlates with the presence of macrophages and CD8+ T-lymphocytes at brain lesions. The gold standard of therapy at MS relapse are iv glucocorticoids (GC). The aim of the study was to assess the changes on the different subsets of circulating CD8+ T-lymphocytes at relapse and after iv GC therapy. PATIENTS AND METHODS: We consecutively studied 20 patients at MS relapse before and at day 5 after initiation of i.v. methyl-prednisolone (MP) therapy (1 g/day for 3-5 days). CD4+ and CD8+ T-lymphocytes subsets were studied by multiparametric flow-cytometry. As control group, 18 healthy subjects were studied. RESULTS: Treatment with i.v. MP suppressed activated (CD8+CD38+HLA-DR+, p=0.05) and effector memory (CD8+CD27-CD45RO+) T-lymphocytes (p=0.07). By contrast, an increase of na?ve (CD8+CD27+CD45RO-) (p=0.07) and regulatory CD8+CD25+ T-lymphocytes was observed (p<0.002). At MS relapse, there was an inverse correlation between regulatory CD8+CD25+CD28- T-lymphocytes and activated CD4+ (r = -0.6; p=0.012) and CD8+ (r = -0.66; p=0.004) T-lymphocytes. After i.v. MP treatment, positive correlation between regulatory CD4+CD25+high T-lymphocytes and CD8+CD25+ T-lymphocytes was observed (r=0.74; p<0.0001). CONCLUSIONS: Our data suggest that i.v. MP may contribute to changes observed on the differentiation of CD8+ T-lymphocytes, namely blocking their complete maturation, and expansion of regulatory CD8+ T-lymphocytes. We hypothesize an additional effect of i.v. MP in inhibiting axonal damage which may add a neuroprotective effect on MS relapse.     

CD5 B cells and CD48 T cells in neuroimmunological diseases

Using 2- and 3-colour FACS analysis we found increased levels of fetal-type CD5+ B cells and CD4-8- T cells in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and aseptic meningitis (AM) compared to control probands with muscular tension headache (TH). Similar differences were found for CD5+ B cells in peripheral blood, but at lower levels. CD4-8- T cells in blood exceeded those in CSF in all patient groups, with the exception of relapsing remitting MS, revealing the highest values in AM. There was a positive correlation between CD4-8- T cells and T cell receptor (TCR) gamma delta bearing T cells in blood and CSF. The double-negative T cells exceeded the TCR gamma delta T cells by about 1%. A positive correlation between CD5+ B cells and CD4-8- T cell level in CSF was found in MS and AM, but not in TH, nor in blood of any patient group. HLA-DR expression was lower in CD5+ B cells than in CD5- B cells. We conclude that fetal-type lymphocytes are enriched in CSF compartment of patients with inflammatory diseases of the central nervous system, irrespective of autoimmune mechanisms involved, but the function of CD5+ B cells is mainly to produce the autoantibodies.     

CD95/Fas expression on peripheral blood T lymphocytes in patients with multiple sclerosis: effect of high-dose methylprednisolone therapy

Recent data indicate that the apoptotic process, mediated by the CD95/Fas cell surface receptor, is impaired in activated lymphocytes of patients with relapsing-remitting multiple sclerosis. Using flow cytometric-immunophenotyping, we analyzed the expression of CD95/Fas on peripheral blood CD4+ and CD8+ T lymphocytes (PBL) in 10 MS patients in relapse, and the effect of pulse corticosteroid therapy on the apoptosis of autoreactive lymphocytes. The proportions of CD8+ and CD8+CD95+ T lymphocytes were significantly higher in MS patients in relapse before than after pulse corticosteroid therapy. Conversely, the proportions of CD4+ and CD4+CD95+ T cells were significantly lower before than after therapy, but not significantly different from healthy persons. The different expression of CD95/Fas on peripheral blood CD8+ T lymphocytes in relapsing RRMS and in healthy controls suggests a possible involvement of apoptosis in the pathogenesis of MS. Our results also show that pulse corticosteroid therapy influences the CD95/Fas expression on CD8+ and CD4+ T lymphocytes in patients with RRMS.     

Bystander modulation of chemokine receptor expression on peripheral blood T lymphocytes mediated by glatiramer therapy

BACKGROUND: Glatiramer acetate therapy is thought to be effective for multiple sclerosis (MS) by promoting T(H)2 cytokine deviation, possibly in the brain, but the exact mechanism and site of action are incompletely understood. Determining the site of action and effect of glatiramer on cell trafficking is of major importance in designing rational combination therapy clinical trials. OBJECTIVE: To determine whether glatiramer therapy will also act in the peripheral blood through bystander modulation of chemokine receptor (CKR) expression and cytokine production on T lymphocytes. DESIGN: Before-and-after trial. SETTING: A university MS specialty center. PATIENTS: Ten patients with relapsing-remitting MS. INTERVENTIONS: Treatment with glatiramer for 12 months and serial phlebotomy. MAIN OUTCOME MEASURES: Cytokine production, CKR expression, and cell migration. RESULTS: The glatiramer-reactive T cells were T(H)2 cytokine biased, consistent with previous studies. We found a significant reduction in the expression of the T(H)1 inflammation associated with the CKRs CXCR3, CXCR6, and CCR5 on glatiramer- and myelin-reactive T cells generated from patients with MS receiving glatiramer therapy vs baseline. Conversely, expression of the lymph node-homing CKR, CCR7, was markedly enhanced on the glatiramer-reactive T cells derived from patients with MS undergoing glatiramer therapy. There was a reduction in the percentage of CD4+ glatiramer-reactive T cells and an increase in the number of CD8+ glatiramer-reactive T cells. CONCLUSIONS: Glatiramer may suppress autoreactive CD4+ effector memory T cells and enhance CD8+ regulatory responses, and bystander modulation of CKRs may occur in the periphery.     

Comparison of immunoglobulin G heavy-chain sequences in MS and SSPE brains reveals an antigen-driven response

OBJECTIVE: To better understand B-cell activation in MS by analyzing the immunoglobulin (Ig)G heavy chain variable region (VH) repertoire found in MS brains and comparing it with brain VH sequences in individuals with subacute sclerosing panencephalitis (SSPE)--a chronic encephalitis produced by measles virus (MV)-and characterized by an antigen-driven oligoclonal IgG response to MV antigens. BACKGROUND: The specificity of oligoclonal IgG in MS CSF and plaques, and their relevance to the pathogenesis of MS is unknown. METHODS: Nested PCR was used to amplify and sequence the rearranged IgG heavy-chain VH repertoire in plaques of three acute MS brains and in three SSPE brains. A representative population of VH sequences from each tissue was aligned to the known 51 functional VH germline segments. From this the authors determined the closest VH family germline segment, and the degree and location of somatic mutations for each unique IgG. RESULTS: As expected for an antigen-driven response against MV antigens, most VH sequences from the SSPE brains were mutated extensively compared with their closest germline segments. Furthermore, SSPE VH sequences accumulated replacement mutations preferentially in the complementary-determining regions (CDRs) relative to framework regions-features normally observed during antigen-driven selection. A comparison of VH family and germline usage also demonstrated that each SSPE brain had its own unique IgG response. When the authors compared the VH response in MS plaques with SSPE, MS VH sequences were also mutated extensively, displayed a preferential accumulation of replacement mutations in CDRs, and were unique in each MS brain. CONCLUSION: The presence of an antigen-driven response in MS, rather than a nonconventional mechanism of B-cell activation, warrants additional analysis of the specificity of IgG in MS brain and CSF.     

Time-dependent cytokine deviation toward the Th2 side in Japanese multiple sclerosis patients with interferon beta-1b

To address the immune mechanism sustaining interferon beta (IFNbeta) efficacy in multiple sclerosis (MS), we longitudinally analyzed expressions of IFN-gamma, IL-4, IL-5 and IL-13 in CD4+ T cells and CD8+ T cells in 22 Japanese MS patients (16 patients with conventional MS and 6 with opticospinal MS) undergoing IFNbeta using flow cytometry. During the 48-week observation period, five opticospinal MS patients (83%) relapsed compared to only four conventional MS patients (25%); the frequency of relapsed patients was significantly higher in the former (p=0.046). The effects of IFNbeta on individual cytokines were time-dependent and altered cytokine productions were particularly evident in CD4+ rather than CD8+ T cells. A decreased intracellular IFN-gamma/IL-4 ratio in CD4+ T cells was thus evident soon after the initiation of therapy, and persisted for the entire 1 year follow-up period, regardless of whether or not the patient relapsed (p<0.01). IFNbeta treatment resulted in a rapid increase in the percentage of IFN-gamma- IL-4+ and IL-13+ CD4+ T cells 1 week after the initiation of therapy and high values were sustained for 6 months but declined to the baseline over 1 year. Later, the percentage of IFN-gamma+ IL-4- CD4+ T cells decreased significantly from weeks 24 through 48 of therapy (p<0.01). When comparisons with the pretreatment values were made for each subtype of MS, a significant reduction of IFN-gamma+ IL-4- CD4+ T cell percentages was shown in conventional MS (p<0.0001), but not in opticospinal MS. Moreover, when such a comparison was made by the presence or absence of relapse during therapy, a significant reduction of IFN-gamma+ IL-4- CD4+ T cell percentages was observed in MS patients without relapse (p<0.01). Thus, a reduction of IFN-gamma+ IL-4- CD4+ T cell percentages in the late phase of therapy is considered important for reducing relapse in conventional MS. When the expression patterns of IFN-gamma, IL-4, IL-5 and IL-13 in CD4+ T cells and CD8+ T cells were compared between patients with and without relapse during therapy, the only significant difference was an increase in the IL-13+ CD4+ T cell percentages in patients with relapse compared to those without (p<0.05). The results indicate that in CD4+ T cells IL-4 was preferentially up-regulated in the early course and IFN-gamma was down-regulated in the late phase of IFNbeta therapy. The net effect of IFNbeta on the immune balance was entirely toward type 2 immune deviation, possibly contributing to its beneficial effects on MS.     

Association of Th1/Th2-related chemokine receptors in peripheral T cells with disease activity in patients with multiple sclerosis and neuromyelitis optica

We evaluated 30 patients with clinically definite multiple sclerosis (MS) and 8 patients with neuromyelitis optica (NMO) to investigate correlations between Th1/Th2 balance, disease activity, effects of interferon (IFN)-β treatment, and expressions of chemokine receptors CXCR3 and CCR4 on CD4+ and CD8+ T cells in peripheral blood. MS and NMO patients in the relapsing phase showed a significantly increased CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio compared with respective patients in the remission phase. After IFN-β treatment, the CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio were significantly decreased compared with the relapsing phase and slightly lower than in the remission phase. The CD8+CXCR3+/CD8+CCR4+ ratio showed a more marked change associated with disease activity than CD4+ T cells in MS and NMO patients. Moreover, in patients in the relapsing phase of NMO, the CD4+CXCR3+/CD4+CCR4+ ratio and CD8+CXCR3+/CD8+CCR4+ ratio were significantly higher than in MS patients in the relapsing phase. We confirmed marked changes in the CD8+CXCR3+/CD8+CCR4+ ratio according to disease activity and treatment of MS and NMO. Furthermore, this ratio was more strongly linked to immune and inflammatory activity in NMO patients than in MS patients, and may represent an important factor in differentiating the pathogenesis of MS and NMO.