B cells in multiple sclerosis therapy—A comprehensive review

For decades, B cells were ignored in multiple sclerosis (MS) pathogenesis, and the disease was always regarded as a T cell‐mediated disorder. Recent evidence shows that there is an antigen‐driven B‐cell response in the central nervous system of patients with MS, and memory B cells/plasma cells are detectable in MS lesions. The striking efficacy of B cell‐depleting therapies in reducing the inflammatory activity of the disease highlights that B cells may play more pathogenetic roles than expected. B cells express several unique characteristic markers on their surface, for example, CD19, CD20 molecules, that provide selective targets for monoclonal antibodies. In this respect, several B cell‐targeted therapies emerged, including anti‐CD20 antibodies (rituximab, ocrelizumab, and ofatumumab), anti‐CD19 antibody (inebilizumab), and agents targeting the BAFF/APRIL signaling pathway (atacicept, belimumab, and LY2127399). In this review, we discuss, in detail, the immunobiology of B cells and their protective and destructive roles in MS pathogenesis. In the second part, we list the completed and ongoing clinical trials investigating the safety and efficacy of B cell‐related monoclonal antibodies in MS.     

A Milestone in Multiple Sclerosis Therapy: Monoclonal Antibodies Against CD20—Yet Progress Continues

Multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system, still represents one of the most common causes of persisting disability with an early disease onset. Growing evidence suggests B cells to play a crucial role in its pathogenesis and progression. Over the last decades, monoclonal antibodies (mabs) against the surface protein CD20 have been intensively studied as a B cell targeting therapy in relapsing MS (RMS) as well as primary progressive MS (PPMS). Pivotal studies on anti-CD20 therapy in RMS showed remarkable clinical and radiological effects, especially on acute inflammation and relapse biology. These results paved the way for further research on the implication of B cells in the pathogenesis of MS. Besides controlling relapse development in RMS, ocrelizumab (OCR) also showed clinical benefits in patients with PPMS and became the first approved drug for this disease course. In this review, we provide an overview of the current anti-CD20 mabs used or tested for the treatment of MS—namely rituximab (RTX), OCR, ofatumumab (OFA), and ublituximab (UB). Besides their effectiveness, we also discuss possible limitations and safety concerns especially in regard to long-term treatment, both for this class of drugs overall as well as for each anti-CD20 mab individually. Additionally, we elucidate to what extent anti-CD20 therapy may alter the function of other immune cells, both directly or indirectly. Finally, we cover the current knowledge on repopulation of CD20⁺ cells after cessation of anti-CD20 treatment and discuss future aspirations towards alternative, further developed B cell silencing therapies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01048-z.Key Words: Anti-CD20 treatment, B cells in MS, Ocrelizumab, Rituximab, Ublituximab, Ofatumumab     

Role of B Cells in Multiple Sclerosis and Related Disorders

The success of clinical trials of selective B‐cell depletion in patients with relapsing multiple sclerosis (MS) and primary progressive MS has led to a conceptual shift in the understanding of MS pathogenesis, away from the classical model in which T cells were the sole central actors and toward a more complex paradigm with B cells having an essential role in both the inflammatory and neurodegenerative components of the disease process. The role of B cells in MS was selected as the topic of the 27th Annual Meeting of the European Charcot Foundation. Results of the meeting are presented in this concise review, which recaps current concepts underlying the biology and therapeutic rationale behind B‐cell–directed therapeutics in MS, and proposes strategies to optimize the use of existing anti–B‐cell treatments and provide future directions for research in this area. ANN NEUROL 2021;89:13–23     

Natalizumab treatment during pregnancy – effects on the neonatal immune system

Background

Previous studies on Natalizumab (NAT) have shown increased circulation of most white blood cells (WBC) in multiple sclerosis (MS) patients shortly after its introduction.

Aim

To describe peripheral immune cell phenotypes after more than 2 years of continuous NAT therapy and test for associations with clinical response to therapy.

Methods

Peripheral immune cell subsets were analyzed in 44 NAT‐MS patients receiving NAT for over 24 months, and in 22 NAT‐free control‐MS patients.

Results

NAT‐MS patients displayed significantly higher numbers of all WBC when compared with controls. B lymphocytes exhibited a more pronounced increase when compared with CD4+, CD8+ and NK T‐cells (P = 0.011). CD4/CD8 ratio was significantly decreased in NAT‐MS patients (P = 0.018) and showed no correlation with the number of NAT doses. The reduced CD4/CD8 ratio was attributable to the ‘EDSS improvement’ group only, irrespective of age, sex and disease severity.

Conclusions

The study suggests that there is no desensitization effect after prolonged NAT exposure. A reduced CD4/CD8 ratio was associated with long‐term response to therapy; thus, those patients who most benefitted from the drug might be at greater risk for opportunistic infections like progressive multifocal leucoencephalopathy (PML). We provide implications for future research for the CD4/CD8 ratio as a possible contributor to the recently developed risk stratification scheme for PML.     

Myasthenia gravis,castleman disease,pemphigus, and anti‐phospholipid syndrome

Introduction: Myasthenia gravis is an autoimmune disease marked by neuromuscular transmission failure at the neuromuscular junction. Castleman disease is a rare lymphoproliferative disease characterized by non‐cancerous angiofolicular hyperplasia of lymphatic tissue. Methods and results: We describe a young man with rapid, successive manifestations of myasthenia gravis, a solitary form of Castleman disease, pemphigus vulgaris, and anti‐phospholipid syndrome, which resulted in 2 ischemic cerebrovascular events that caused a severe central neurological deficit. Discussion: We were unable to find a similar case in the literature, but we hypothesize that the temporal concidence of these clinical entities may be related to a common immunological pathway, such as B‐cell activation. Therefore, we treated the patient with an immunosuppressant and anticoagulant treatment, as well as rituximab, a monoclonal antibody therapy against CD20+. Muscle Nerve 47:447‐451, 2013     

Immune surveillance in multiple sclerosis patients treated with natalizumab

OBJECTIVE: Our objective was to test whether natalizumab, an antibody against very late activating antigen (VLA)-4, interferes with central nervous system immune surveillance as assessed by leukocyte cell numbers and cellular phenotypes in cerebrospinal fluid (CSF) and peripheral blood. METHODS: Cell numbers and cellular phenotypes in CSF and peripheral blood were analyzed in multiple sclerosis (MS) patients treated with natalizumab, untreated MS patients, and patients with other neurological disease (OND). JC virus DNA in the CSF and peripheral blood was quantified by kinetic polymerase chain reaction. RESULTS: CSF leukocyte counts, CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD138(+) plasma cells were significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. JC virus DNA was not detected in CSF or peripheral blood from natalizumab-treated patients. Six months after cessation of natalizumab therapy, low lymphocyte counts in the CSF persisted. The patient with the highest total leukocyte and CD4(+) and CD8(+)T-cell counts in the CSF experienced a clinical relapse. INTERPRETATION: These data suggest that natalizumab treatment results in a prolonged decrease of lymphocytes in the CSF and are consistent with the hypothesis that natalizumab impairs immune surveillance of the central nervous system.     

CD19 as a molecular target in CNS autoimmunity

Multiple sclerosis (MS) and neuromyelitis optica (NMO) are the most prevalent neuroinflammatory diseases of the central nervous system (CNS). The immunological cascade of these disorders is complex, and the exact spatial and temporal role of different immune cells is not fully understood. Although MS has been considered for many years to be primarily T cell driven, it is well established that B cells and the humoral immune response play an important role in its pathogenesis. This has long been evident from laboratory findings that include the presence of oligoclonal bands in the CSF. In NMO, the importance of the humoral immune system appears even more obvious as evidenced by pathogenic antibodies against aquaporin 4 (AQP4). Besides their capacity to mature into antibody-producing plasma cells, B cells are potent antigen-presenting cells to T lymphocytes and they can provide soluble factors for cell activation and differentiation to other immune-competent cells. In MS and NMO, there are substantial data from clinical trials that B cell depletion with CD20-directed agents is effective and relatively safe. Plasma cells, which produce antibodies against molecular targets expressed by the host, but which also provide humoral immune responses against pathogens, are not targeted by anti-CD20 therapies. Therefore, the depletion of CD19-expressing cells would offer potential advantages with regard to efficacy, but potentially higher risks with regard to infectious complications. This review will outline the rationale for CD19 as a molecular target in CNS autoimmunity. The current stage of drug development is illustrated. Potential safety concerns will be discussed.     

B cells as a target of immune modulation

B cells have recently been identified as an integral component of the immune system; they play a part in autoimmunity through antigen presentation, antibody secretion, and complement activation. Animal models of multiple sclerosis (MS) suggest that myelin destruction is partly mediated through B cell activation (and plasmablasts). MS patients with evidence of B cell involvement, as compared to those without, tend to have a worse prognosis. Finally, the significant decrease in new gadolinium-enhancing lesions, new T2 lesions, and relapses in MS patients treated with rituximab (a monoclonal antibody against CD20 on B cells) leads us to the conclusion that B cells play an important role in MS and that immune modulation of these cells may ameliorate the disease. This article will explore the role of B cells in MS and the rationale for the development of B cell–targeted therapeutics. MS is an immune-mediated disease that affects over 2 million people worldwide and is the number one cause of disability in young patients. Most therapeutic targets have focused on T cells; however, recently, the focus has shifted to the role of B cells in the pathogenesis of MS and the potential of B cells as a therapeutic target.     

B-cell depletion abrogates T cell-mediated demyelination in an antibody-nondependent common marmoset experimental autoimmune encephalomyelitis model

CD20-positive B-cell depletion is a highly promising treatment for multiple sclerosis (MS), but the mechanisms underlying therapeutic effects are poorly understood. B cells are thought to contribute to MS pathogenesis by producing autoantibodies that amplify demyelination via opsonization of myelin. To analyze autoantibody-nondependent functions of B cells in an animal model of MS, we used a novel T cell-driven experimental autoimmune encephalomyelitis (EAE) model in marmoset monkeys (Callithrix jacchus). In this model, demyelination of brain and spinal cord white and gray matter and the ensuing neurological deficits are induced by immunization with peptide 34 to 56 of myelin/oligodendrocyte glycoprotein (MOG34-56) in incomplete Freund's adjuvant. Although autoantibodies do not have a detectable pathogeniccontribution in the model, depletion of B cells with monoclonal antibody 7D8, a human IgG1κ monoclonal antibody against human CD20, suppressed clinical and pathological EAE. In B cell-depleted monkeys, the activation of peptide-specific Th17-producing and cytotoxic T cells, which in previous studies were found to play an essential role in disease induction, was impaired. Thus, we demonstrate a critical antibody-nondependent role for B cells in EAE, that is, the activation of pathogenic T cells.     

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.     

Mitoxantrone does not restore the impaired suppressive function of natural regulatory T cells in patients suffering from multiple sclerosis. A longitudinal ex vivo and in vitro study

CD4+CD25+ regulatory T (T(reg)) cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system, where T cells are of major importance. T(reg) cell frequency and function are potential therapeutic targets of MS treatment. Mitoxantrone (MX) is a potent immunosuppressant for the treatment of active MS. We investigated the long-term effects of MX on the suppressive function of T(reg) after mitogen and myelin basic protein (MBP) stimulation in 20 MX-treated patients. MX therapy did not restore the reduced suppressive activity of a mixture of CD25(intermediate) or CD25(high) expressing T(reg) (healthy controls, MBP: 37.3%; MS patients, MBP: -1.9 vs. 6.6% suppression after MX treatment for 6 months, p > 0.2). The frequency of T(reg) cells was not affected by MX. A single infusion of MX (10 mg/m(2) body surface) induced a selective and persistent reduction of approximately 30% of absolute and relative counts of B lymphocytes (p < 0.05). MX therapy does not influence T(reg) cell frequency or function. MX seems to exhibit its efficacy in MS mainly by a suppression of humoral immunity.     

Failed B cell survival factor trials support the importance of memory B cells in multiple sclerosis

Clinical trials are probably the most informative experiments to help an understanding of multiple sclerosis (MS) biology. Recent successes with CD20‐depleting antibodies have focused attention towards B cell subsets as important mediators in MS. The trial of tabalumab (NTC00882999), which inhibits B cell activation factor (BAFF), is reported and reviewed and this trial is contrasted with the trial on the inhibition of a proliferation‐inducing ligand (APRIL) and BAFF using atacicept (NCT00642902). Both tabalumab and atacicept induce depletion of mature B cells and inhibit antibody formation, but they fail to deplete memory B cells and do not inhibit relapsing MS. Atacicept is reported to augment memory B cell responses and may precipitate relapse, suggesting the importance of APRIL. However, BAFF inhibition can enhance peripheral blood memory B cell responses, which was not associated with augmented relapse. Although other interpretations are possible, these data further support the hypothesis that memory B cells may be of central importance in relapsing MS, as they are the major CD20+ B cell subset expressing APRIL receptors. They also suggest that quantitative and/or qualitative differences in B cell responses or other factors, such as an immune‐regulatory effect associated with APRIL, may be important in determining whether MS reactivates following neutralization of peripheral B cell maturation and survival factors.     

Beneficial effect of rituximab monotherapy in multifocal motor neuropathy

Multifocal motor neuropathy (MMN) is a chronic, pure motor, asymmetric neuropathy for which intravenous immunoglobulin (IVIg) is widely regarded as first-line treatment. Rituximab is a monoclonal antibody against CD20+ cells that causes prolonged B cell depletion, a well-tolerated therapy currently explored in several immune-mediated neurologic disorders. We report three patients with MMN, who had become increasingly less responsive to IVIg but showed sustained clinical improvement following rituximab monotherapy. We provide a review of the literature on rituximab for MMN and conclude that rituximab may represent an efficacious, well-tolerated and cost-effective therapeutic option for MMN patients with declining response to IVIg.     

Autoantibodies recognizing native MOG are closely associated with active demyelination but not with neuroinflammation in chronic EAE

It is important to find biomarkers for autoimmune inflammation and demyelination in the CNS to monitor disease status in patients with multiple sclerosis (MS). For this purpose, we determined the titers of antibodies (Ab) reacting with native myelin oligodendrocyte glycoprotein (MOG)‐expressing cells to evaluate the disease activity of chronic experimental autoimmune encephalomyelitis (EAE) in rats and the relationship between anti‐MOGcme (cell membrane‐expressed MOG), Ab titers and clinical and pathological parameters were evaluated. Consequently, we found that elevation of anti‐MOGcme Ab titers was associated with clinical severity, except for some cases in very late stages and with severe and widespread demyelination but with dominant inflammation. In contrast, antibodies detected by standard ELISA using recombinant MOG were elevated in both symptomatic and asymptomatic rats and were not associated with parameters such as inflammation and demyelination. Longitudinal examination of anti‐MOGcme Ab titers in individual rats revealed that Ab titers accurately reflect disease activity. Furthermore, anti‐MOGcme Ab titer was not elevated in acute EAE without demyelination. These findings suggest that autoantibodies reacting with native and glycosylated MOG play an important role in the progression of demyelinating diseases and could be biomarkers for monitoring the status of patients with MS.     

地黄合剂在多发性硬化患者中抗炎性反应及调节免疫平衡机制探讨

目的 观察地黄合剂(DHHJ)在多发性硬化(MS)患者中发挥的双向治疗作用并探讨其机制. 方法 将40例MS患者采用随机数字表法分为激素治疗组(n=20)与激素治疗+DHHJ组(n=20),根据组名采取相应治疗.另设20例排除免疫系统疾病及感染类疾病的外科手术患者作为对照组.分别用EUSA法和流式细胞仪检测两组观察对象脑脊液(CSF)和外周血中胶质原纤维酸性蛋I~(GFAP)和S100B的含量及CD4+细胞,CD8+细胞数目.用下肢功能状态的评分(AD、延展残疾状态评分(EDSS)、上肢功能状态评分(9HPT)对MS患者进行临床评分并分析其与GFAP、S100B之间的关系.随访MS患者的复发情况. 结果 与对照组比较.MS组CSF中GFAP和S100B表达明显增强,差异有统计学意义(P<0.05),并且与MS患者的AI、9HPT评分存在相关关系.DHHJ+激素治疗组与激素治疗组患者CSF中GFAP和S100B含量差异也有统计学意义(P<0.05).同时DHHJ+激素治疗组MS的复发次数与激素治疗组比较差异也有统计学意义(P<0.05).MS患者的外周血和CSF中出现CD4+细胞明显增多,CD8+细胞明显减少;CSF中更明显.给予不同的治疗后.CD4+细胞数目减少.CD8+细胞数目增多,DHHJ+激素治疗组与激素治疗组之间差异有统计学意义(p<0.05). 结论 DHHJ 一方面能够影响MS患者CSF中GFAP和S100B的表达,抑制胶质细胞的激活,达到抗炎性反应作用;另一方面DHHJ还可以通过上调免疫抑制性CD8+细胞的数目,下调免疫辅助性CD4+细胞,发挥调节免疫平衡作用,最终达到双向治疗MS的作用,减少MS患者复发的次数.     

Invited article: inhibition of B cell functions: implications for neurology

B cells are involved in the pathophysiology of many neurologic diseases, either in a causative or contributory role, via production of autoantibodies, cytokine secretion, or by acting as antigen-presenting cells leading to T cell activation. B cells are clonally expanded in various CNS disorders, such as multiple sclerosis (MS), paraneoplastic CNS disorders, or stiff-person syndrome, and are activated to produce pathogenic autoantibodies in demyelinating neuropathies and myasthenia. B cell activating factor (BAFF) and a proliferating inducing ligand (APRIL), key cytokines for B cell survival, are strongly unregulated in MS brain and in muscles of inflammatory myopathies. Modulation of B cell functions using a series of monoclonal antibodies against CD20+ B cells or the molecules that increase B cell survival, such as BAFF/APRIL and their receptors BAFF-R, TACI, and BCMA, provide a rational approach to the treatment of the aforementioned neurologic disorders. In controlled studies, rituximab, a B cell-depleting monoclonal antibody, has been encouraging in MS and paraproteinemic anti-MAG demyelinating neuropathy, exerting long-lasting remissions. In uncontrolled series, benefit has been reported in several disorders. B cell depletion is a well-tolerated therapeutic option currently explored in the treatment of several autoimmune neurologic disorders.     

Prognostic significance of microvessel density determined by an anti‐CD105/endoglin monoclonal antibody in astrocytic tumors: Comparison with an anti‐CD31 monoclonal antibody

There are conflicting reports as to whether the degree of angiogenesis as measured by microvessel density (MVD) has a prognostic value in astrocytic tumors. This may be due to the use of different antibodies against endothelial cells to highlight microvessels. It has been reported that unlike pan‐endothelial antibodies, such as CD31, anti‐CD105 antibodies preferentially react with endothelial cells in angiogenic tissues. To clarify the validity of anti‐CD105 antibody in the evaluation of angiogenesis, we assessed MVD using an anti‐CD105 monoclonal antibody (mAb) (CD105‐MVD) and an anti‐CD31 mAb (CD31‐MVD) in a series of 50 astrocytic tumors, and correlated MVD with expression of the key angiogenic factor vascular endothelial growth factor (VEGF) and prognosis. The mean CD31‐MVD and CD105‐MVD was 36.7 and 24.8 for low‐grade astrocytoma (LGA), 48.0 and 42.7 for anaplastic astrocytoma, 55.3 and 51.9 for glioblastoma multiforme (GBM), respectively. CD105‐MVD was more closely correlated with VEGF expression than CD31‐MVD. Patients with LGA and GBM showing higher CD105‐MVD had a significantly shorter mean survival time (MST) than those with lower CD105‐MVD tumors (P = 0.0381 and 0.0131, respectively). Whereas the MST of patients with higher CD31‐MVD tumors seemed to be shorter than that of lower CD31‐MVD patients within each tumor grade, the differences were not statistically significant. These findings suggest that anti‐CD105 mAb may be a better marker than anti‐CD31 mAb in evaluation of angiogenesis and prediction of prognosis in astrocytic tumors.     

A case of intravascular large B cell lymphoma: New clinical and immunohistochemical findings

Intravascular large B cell lymphoma (IVLBCL) is a rare extranodal non‐Hodgkin lymphoma characterized by proliferation of malignant cells within the lumen of small vessels, with a predilection for the CNS and the skin. IVLBCL clinical course is highly aggressive, clinical signs and symptoms are not specific and may consist of neurological and cognitive impairment, fever of unknown origin and cutaneous lesions, lacking of a typical neuroimaging pattern. For all these reasons the diagnosis is commonly missed and the exitus is frequent, therefore post mortem evaluation is necessary to clarify the clinical history. We present a case of IVLBCL in a 62‐year‐old woman with unusual symptomatology, mimicking a vascular, multi‐infarctual cerebropathy. Hachinski Ischemic Score was 7 suggesting a vascular dementia. Autopsy was unable to define the nature of the disease. Immunohistochemical analysis for cluster of differentiation 20 (CD20) revealed the ubiquitous presence of malignant lymphoid B‐cells into the vessel of all organs analyzed, allowing the definitive diagnosis of IVLBCL. The atypical cells expressed high levels of anti‐apoptotic proteins B‐cell lymphoma 2 (Bcl‐2) and Galectin‐3, and showed cellular myelocytomatosis (c‐Myc) staining in <50% of tumor nuclei. Conversely, cells were immunonegative for multiple myeloma‐1 (MUM1), CD3, CD44, CD30, CD34 and CD133. Fluorescent in situ hybridization analysis for MYC rearrangements was negative. The high expression of Galectin‐3 provides new insights in the understanding of molecular pathogenesis of IVLBCL; indeed, such a finding represents a prognostic factor for other types of lymphoma and should, in the same way, be taken into account in IVLBCL.     

Brain immunohistopathological study in a patient with anti‐NMDAR encephalitis

Background and purpose: Anti‐N‐methyl‐d ‐asparate (NMDA) receptor encephalitis is thought to be antibody‐mediated. To perform an immunohistopathological study of the inflammatory reaction in a brain biopsy performed before immunomodulatory treatments in a patient with anti‐NMDA receptor encephalitis. Methods: An immunohistochemical study was performed using CD3, CD68, CD20, CD138 and CD1a antibodies. Results: Prominent B‐cell cuffing was present around brain vessels accompanied by some plasma cells, while macrophages and T cells were scattered throughout the brain parenchyma. Conclusion: These findings suggest that the B cells interact with the T cells and are involved in antibody secretion by the plasma cells.     

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.