Decrease of suppressor inducer (CD4+2H4+) T cells in multiple sclerosis cerebrospinal fluid

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

Derivation of ganglioside-specific T cell lines of suppressor or helper phenotype from cerebrospinal fluid of multiple sclerosis patients

In order to investigate the specificity of activated T cells in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we have cultured cells in the presence of mitogen-free IL-2 but without any antigen. Two T cell lines have been derived and showed specific reactivity to certain purified gangliosides (GM1, GD1a, GD1b and GQ1b). However, responses to other brain and viral antigens were not seen, and neither were T cell lines from peripheral blood lymphocytes (PBL) of normal, MS or other neurological disease patients stimulated by these gangliosides. Release of IL-2 could be detected after incubation of these CSF lines with specific gangliosides. One line exhibited predominantly helper/inducer (T4+) phenotype whilst the other was suppressor/cytotoxic (T8+), and further analysis indicated it could be of the suppressor phenotype. These data may have implications for T cell-induced demyelination in MS.     

T-cell subsets in the cerebrospinal fluid and blood of patients with multiple sclerosis

The absolute numbers and ratios of helper/inducer (T4) and cytotoxic/suppressor (T8) T-cells were determined in cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) and various other neurologic diseases (OND). In patients with MS, the T4:T8 ratio was higher in both blood and CSF, and the increase was significantly greater in CSF than in blood. These findings were due to an increased proportion of T4-lymphocytes in the CSF and to a decreased proportion of T8-cells in blood. These results indicate the need for additional studies of CSF lymphocytes in patients with MS.     

Chronic progressive multiple sclerosis: changes in phenotype and function of T helper subsets

We have measured pokeweed mitogen-induced IgG secretion by peripheral blood mononuclear cells obtained from two different groups of progressive multiple sclerosis (MS) patients. MS patients with chronic progressive active disease (CPMS-A) have higher IgG secretion than stable (burnt-out) patients (CPMS-S). We have also measured suppressor cell function and phenotyped the T helper cells of some CPMS-A patients. This group differed from CPMS-S and from controls: they had high IgG secretion, low suppression and their T helper phenotypes showed a high ratio of T helper/inducers over T suppressor/inducers.     

Correlation of clinical and immunologic states in multiple sclerosis

Cyclophosphamide was administered to 14 patients with chronic progressive multiple sclerosis on an intermittent escalating dosage schedule adjusted to maintain numbers of peripheral blood B lymphocytes and helper/inducer (CD4) T cells below the fifth percentile of the normal population. Peripheral blood B cells, T cells, suppressor/cytotoxic (CD8) T cells, CD4 cells, and FcR+-bearing cell numbers and percentages were monitored at one-week to two-week intervals. Clinical status was assessed by neurologic examinations at approximately four-week intervals. Regression analysis revealed a statistically significant correlation between changes in immunologic status and changes in clinical state. The immunologic changes preceded the neurologic changes. Increases in percent of CD8 cells and decreases in percent of CD4 cells forecast improved clinical course. These findings, coupled with other studies, strongly suggest a pathogenetic role for helper and suppressor T cells in the production of clinical signs of multiple sclerosis.     

Cerebrospinal fluid cells in multiple sclerosis and other neurological diseases: an immunocytochemical study

Summary Determinations of mononuclear cell subsets in cerebrospinal fluid (CSF), using monoclonal antibodies against surface antigens which identified pan T-cells, helper/inducer T-cells, cytotoxic/suppressor T-cells and Ia-positive cells, were performed in patients with multiple sclerosis (MS), other neuroimmunological diseases (NID), infectious diseases (INF) of the central nervous system and with other neurological diseases. Whereas there was an elevated helper T/suppressor T ratio in CSF of patients with NID (Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, cerebral vasculitis), no other significant differences could be detected between the different groups of patients. Our results suggest that analysis of these mononuclear cell subsets in CSF is not helpful in discriminating between MS and other neurological diseases and that in MS patients changes in disease activity are not clearly indicated by fluctuations in the different CSF cell subsets. Further studies will be needed to confirm our findings in NID patients and to understand the diagnostic and theoretical implications.     

T cell subsets and disease progression after total lymphoid irradiation in chronic progressive multiple sclerosis.

T lymphocyte subset percentages were determined in 16 total lymphoid irradiation (TLI) treated and 18 sham treated control patients with chronic progressive multiple sclerosis. During the first year after treatment, the ratio of T helper/inducer to T suppressor/cytotoxic cells (Th/Ts ratio) was significantly higher in sham treated multiple sclerosis patients who worsened clinically compared with TLI treated and sham treated multiple sclerosis patients who remained clinically stable. TLI caused a fall in the percentage of T helper cells in treated patients, while the percentage of T suppressor cells remained stable during the first year after treatment. In contrast, the percentage of T suppressor cells fell in sham treated multiple sclerosis patients who worsened clinically.     

Administration of monthly-pulse cyclophosphamide in multiple sclerosis patients. Effects of long-term treatment on immunologic parameters

Eleven patients with chronic progressive multiple sclerosis received monthly pulses of cyclophosphamide (CY) for approximately one year. During the final 9 months the monthly dose ranged between 1000 mg/m2 and 2000 mg/m2. This resulted in a marked (47% or greater) reduction in CD4 (T helper/inducer) cells a less striking (22%) decrease in CD8 (T suppressor/cytotoxic) cells and a decline in the CD4/CD8 ratio. The magnitude of the decrease in CD4 cells correlated with the total dose received (r = 0.88, P less than 0.05). B cells were reduced 50% and FcR+ lymphocytes were reduced 48% without comparable reduction in natural killer cells or antibody-dependent cellular cytotoxicity. Proliferative responses to PHA were suppressed. Two patients improved, seven stabilized and two continued to worsen. Monthly pulses of CY can achieve substantial and differential reduction in immune parameters and appear to slow the disease progression in some MS patients.     

Cyclophosphamide modulates CD4+ T cells into a T helper type 2 phenotype and reverses increased IFN-gamma production of CD8+ T cells in secondary progressive multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system considered to be mediated by T helper type-1 cells. Several agents have been found to modify the disease course of MS, including interferon-beta1 (IFN-beta1), glatiramer acetate mitoxantrone. We have employed pulse therapy with cyclophosphamide in a selected group of patients with actively progressive disease. Chemokine receptors have been found to differentiate between polarized T helper type-1 (Th1) and type-2 (Th2) lymphocytes. The chemokine receptors CCR5 and CXCR3 are expressed primarily on Th1 cells and CCR3, CCR4 and CCR8 on Th2 cells. Previous studies of the expression of chemokine receptors in MS have shown that active MS plaques are infiltrated by CCR5(+) and CXCR3(+) T cells. Some of these T cells may express both CCR5 and CXCR3. These T cells are major producers of IFN-gamma, which worsens the clinical condition of patients with MS. We previously found that patients with MS had a high proportion of CXCR3(+) T cells and that those with chronic progressive MS had a high proportion of CCR5(+) T cells in their peripheral blood. We report here that in patients with secondary progressive MS, cyclophosphamide induces a marked increase in the percentage of CCR4(+) T cells that produce high levels of IL-4 and reverses the increase in the percentages of IFN-gamma-producing CCR5(+) and CXCR3(+) CD8(+) T cells. Furthermore, therapy with cyclophosphamide increases IL-4-producing CD4(+) T cells and reverses the increase in IFN-gamma-producing CD8(+) T cells. Our study shows that cyclophosphamide has immunomodulatory properties besides its suppressive effects, and that chemokine receptors can be important tools both for understanding the immune dysregulation in MS and for monitoring response to therapy.     

T lymphocyte populations in multiple sclerosis

Summary In 36 patients representing different clinical stages of multiple sclerosis (MS) (9 patients with acute exacerbations; 21 patients in remission; 5 patients with chronic progressive MS) determinations of T lymphocyte populations using monoclonal antibodies against surface antigens (OKT3 (pan T cells), OKT4 (helper T cells), OKT8 (cytotoxic/suppressor T cells)) were performed. Compared to the control group (40 healthy individuals) a clear elevation of the T4/T8 ratio was found in acute exacerbations and to a lesser degree in patients with inactive phases of MS. Patients with chronic progressive disease did not show increased T4/T8 ratios. Serial determination of lymphocyte populations after corticosteroid therapy in 10 selected patients revealed no significant changes which could be attribted to this therapeutic modality.Pathogenetic and clinical implications of the shifts in surface antigen expression of T lymphocyte populations mirroring the clinical course of MS are discussed.Deceased in July 1985     

Cell count and ratio of helper/inducer to suppressor/cytotoxic T-cells in the cerebrospinal fluid of patients with multiple sclerosis

Summary The cell count and the helper/inducer to suppressor/cytotoxic T-cell ratio (T4:T8) in cerebrospinal fluid (CSF) were compared in patients with multiple sclerosis (MS) and other neurological diseases. The T4:T8 ratio was higher in patients with MS than in patients with other neurological diseases. A low or normal CSF cell count was associated with a more significant increase in the T4:T8 ratio in patients with MS. These findings imply that the increased T4:T8 ratio found in MS is masked by an inflammatory pleocytosis or that the shift appears only when pleocytosis is absent.     

Japanese encephalitis: identification of inflammatory cells in cerebrospinal fluid

Fifteen patients with acute encephalitis were studied during the epidemic season of Japanese encephalitis (JE) in Northern Thailand; 13 of 15 proved to have the encephalitis. In serial cerebrospinal fluids and blood samples, mononuclear cells were identified using monoclonal antibodies. In cerebrospinal fluid from the patients with Japanese encephalitis, T cells predominated with a 4.2:1 ratio of T helper/inducer cells to T suppressor/cytotoxic cells; B cells and macrophages were often present but in small numbers compared to their presence in blood. Distribution of cell types did not vary between the first and fifth day of hospitalization, were similar in fatal and nonfatal cases, and were unaffected by administration of steroids.     

Phenotypic and functional analysis of T cells cloned directly from the blood and cerebrospinal fluid of patients with multiple sclerosis

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

Lymphocyte subpopulations in blood and cerebrospinal fluid from patients with subacute sclerosing panencephalitis

Lymphocyte subpopulations in peripheral blood (PB) and cerebrospinal fluid (CSF) from 26 children affected with subacute sclerosing panencephalitis (SSPE) and from 13 controls with various neurological diseases without any immunological implication were examined for surface markers. SSPE patients were found to have significantly lower %s of E-rosette forming cells (RFC) (T lymphocytes) and of EA-RFC (TG lymphocytes, suppressor T cells) in both CSF and PB. No difference was found in EAC-RFC (B lymphocytes) either in CSF or PB. The low EA-RFC values can be explained by genetic factors, immune complexes or virus infection, and they could account for the hypersynthesis of oligoclonal immunoglobulins in the central nervous system. Longitudinal studies performed in 6 SSPE patients during isoprinosine therapy revealed a time-dependent decrement of the %s of E- and EA-RFC in CSF. It cannot be affirmed whether this is related to the disease or to the therapy.     

CSF cells in multiple sclerosis: monoclonal antibody analysis and relationship to peripheral blood T-cell subsets

Mononuclear cells were analyzed in CSF and blood of 102 patients with MS. In CSF, the majority (78%) of cells were T lymphocytes (T3+), and the ratio of inducer (T4+) to suppressor/cytotoxic (T8+) cells was 2:1. No characteristic alterations in CSF phenotypes could be related to changes in circulating T8 cells or to disease activity. In a group of 75 patients, CSF cell count was higher in patients with low numbers of circulating T8 cells than in those with normal T8 cells. Thus, decreases in suppressor cells in the blood of MS patients are associated with CSF pleocytosis but not with fluctuations in the ratio of different subsets in CSF. Furthermore, large numbers of T8 cells are not sequestered in CSF when these cells are decreased in peripheral blood.     

Identification of the inflammatory cells in the central nervous system of patients with adrenoleukodystrophy

Adrenoleukodystrophy is a disorder of long-chain fatty acid metabolism associated with adrenal cortical insufficiency and central nervous system demyelination. The central nervous system disease is unusual in that it is abrupt in onset and accompanied by a considerable infiltration of mononuclear inflammatory cells. To determine the nature of these inflammatory cells, immunocytochemical staining was carried out on the mononuclear cells in the brain and cerebrospinal fluid of patients with adrenoleukodystrophy. Monoclonal antibodies to T lymphocytes (T11), the helper/inducer (T4) and cytotoxic/suppressor (T8) subsets of T lymphocytes, B lymphocytes (B1), and monocyte/macrophages (M1 or esterase) were used. Mononuclear cells in the perivascular cuffs of autopsy material from 4 patients were, on average, 59% T cells, 34% T4 cells, 16% T8 cells, 24% B cells, and 11% monocyte/macrophages. Cerebrospinal fluid from 8 of 10 patients had increased IgG concentrations. Mononuclear cells in the cerebrospinal fluid of 6 patients with active disease were, on average, 61% T cells, 40% T4 cells, 16% T8 cells, 3% B cells, and 18% monocyte/macrophages. This distribution of cells is similar to that found in the central nervous system during a cellular immune response and suggests the possibility that one component of this disease is immunologically mediated.     

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.     

In vivo labeling of blood T cells: rapid traffic into cerebrospinal fluid in multiple sclerosis

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

Surface markers on lymphocytes from human cerebrospinal fluid : Identification by monoclonal antibodies

Lympocyte subpopulations in cerebrospinal fluid (CSF) and peripheral blood (PB) were studied using monoclonal antibodies and the common membrane markers. The results in three groups of patients were compared: 36 subjects with ‘non-immunological disorders’ (NID), 14 subjects with multiple sclerosis (MS) and 6 with subacute sclerosing panencephalitis (SSPE). It was found that, in patients with NID, (1) 90% of cells were T lymphocytes, reactive with OKT3; (2) the helper/suppressor (T4/T8) ratios were the same in the CSF and the PB; (3) the OKIa1 percentage was lower in the CSF than in the PB; and (4) only a few cells were ‘immature’, reacting with OKT10. Using the membrane markers (E rosettes, Fc IgG receptors and surface immunoglobulins), on the other hand, it was noted that the majority of cells in the CSF were identified as suppressor T lymphocytes and surface immunoglobulin-positive B cells were less common than the Ia1 marker suggested.There were no significant differences between the CSF results in patients with NID and MS but the OKT3 lymphocytes were reduced in CSF samples from patients with SSPE.     

Cyclophosphamide 'pulses' in chronic progressive multiple sclerosis. A preliminary clinical trial

To our knowledge, this is the first clinical trial in multiple sclerosis (MS) demonstrating the feasibility of directing immunomodulating therapy by monitoring immunologic results. Cyclophosphamide was administered at monthly intervals, escalating the dose until there was a significant reduction in both the number of blood B lymphocytes and helper/inducer (CD4) T cells of 14 patients with chronic progressive MS. The frequency and severity of adverse effects led us to conclude that the regimen is too toxic for the long-term treatment of patients with MS.