Multiple sclerosis: relation of in vitro IgG secretion to T suppressor cell number and function

The proportion of MS patients whose pokeweed mitogen-stimulated mononuclear cells (MNCs) secreted greater than 1,000 ng/ml IgG per 10(6) cells (ie, "high responders") was increased compared with controls. The suppressor effect mediated by a constant number of T8+ cells from high responders, both MS and control, on IgG secretion by standard T helper (T4+) plus B-cell cultures was significantly lower than that for the same number of T8+ cells from "low responders." The proportion of T8+ cells within MNCs from MS patients did not correlate with levels of IgG secretion. Our results indicate that high levels of IgG secretion by MNCs, an occurrence overrepresented in the MS population, is significantly influenced by functional properties of T suppressor cells.     

Analysis of T regulator cell surface markers and functional properties in multiple sclerosis

The relative proportions as well as cell surface and functional properties of T suppressor (T8⁺) and T helper (T4⁺) cells in peripheral blood mononuclear cells (MNCs) of MS patients were analyzed. The proportion of T8 cells compared to normal controls was suggestively lower in patients during relapses and significantly lower in those with progressive disease. The density of T8⁺ antigen on cells of MS patients with active disease as measured by median fluorescence intensity (MFI) was also decreased compared to controls and stable MS patients. Using OKT8-mAb modulated MNCs as a model, we found that reduction of T8 antigen density results in substantial discrepancies between FACS and microscope methods for enumeration of T8⁺ cells. Levels of pokeweed mitogen-induced IgG secretion by MNCs of MS patients did not correlate with proportion of T8⁺ cells within the MNCs, but rather with the functional activity of the T8⁺ cells of given individuals, as tested in an in vitro suppressor assay using constant numbers of T8⁺ cells.     

The effect of cyclophosphamide on T lymphocytes and T lymphocyte subsets in patients with chronic progressive multiple sclerosis

ABSTRACT— The lymphocytes in peripheral blood and cerebrospinal fluid of patients with chronic progressive multiple sclerosis (MS) were characterized with monoclonal antibodies to surface antigens of T cells, helper/inducer T cells and suppressor/cytotoxic T cells. The influence of cyclophosphamide treatment on these immune parameters was investigated.
Compared to healthy persons, the mononuclear cell fraction of the peripheral blood of patients with chronic progressive MS consisted of normal %s of T cells and helper/inducer T cells, but decreased %s of suppressor/cytotoxic T lymphocytes. Intensive as well as chronic treatment of MS patients with cyclophosphamide resulted in a decline in the %s of T cells and helper/inducer T cells, whereas the %s of suppressor/cytotoxic T cells returned to normal. In cerebrospinal fluid, cyclophosphamide also induced a relative decrease in the % of helper/inducer T cells and an increase in the % of suppressor/cytotoxic T cells compared to untreated MS patients. Intensive as well as chronic therapy with cyclophosphamide both led to a significant decrease in the absolute number of T cells and T cell subsets in the blood of the patients.     

Abnormal regulation of IgG production in multiple sclerosis

After stimulation with pokeweed mitogen (PWM), peripheral blood mononuclear cells (MNC) from patients with active multiple sclerosis (MS) produced significantly more IgG (8595 ng per milliliter, p less than 0.01) then MNC from normal age-matched controls (5477 ng per milliliter), whereas those tested during stable periods produced less IgG (4076 ng per milliliter, p less than 0.01). Treatment of MNC with sodium periodate (SP) generated suppressor cells for PWM-driven IgG production in normal controls and in most of the stable MS patients but in only 26% of those during active disease, in whom an increase in IgG production was often seen. This suggests a deficiency of inducible suppressor T cells associated with a supranormal B-cell response to polyclonal activation; T lymphocytes obtained from MS patients during active episodes strongly suppressed IgG production by normal B lymphocytes, whereas their B cells often produced more IgG in the presence of normal T cells. In active MS, a relative B-cell unresponsiveness to activated suppressor T cells would leave helper signals unbalanced, thus leading to increased B-cell activation, which might deplete the pool of inducible suppressor cells for IgG production.     

Comparison of T8 cell-mediated suppressor and cytotoxic functions in multiple sclerosis

Patients with progressive multiple sclerosis (MS) and controls were compared with regard to: (a) in vitro pokeweed mitogen (pwm)-induced IgG secretion, as an indirect measure of T8+ cell-mediated suppressor function; (b) alloantigen-directed cytotoxic activity, a predominantly T8+ cell-mediated function. The MS group had increased IgG secretion (4790 +/- 372 ng/ml vs. 1866 +/- 233 ng/ml, P less than 0.001) compared to controls. In contrast, alloantigen-directed cytotoxic activity did not differ between MS and control groups. These results suggest a selective defect of suppressor cell function in MS rather than a generalized dysfunction of T8+ cells. Defective immunoregulatory control coupled with preserved effector functions may contribute to the autoimmune process, suspected to underlie the pathogenesis of MS.     

Influence of azathioprine (imuran) on in vitro immune function in multiple sclerosis

In vitro immune function was assessed in patients with multiple sclerosis (MS) who were receiving Imuran therapy, in untreated MS patients, and in controls. In untreated stable MS patients, concanavalin A (Con A)-driven mitogenic reactivity (T effector function) and Con A-induced suppressor activity were modestly reduced compared to controls; pokeweed mitogen-induced immunoglobulin G (IgG) secretion was increased. Untreated patients with active MS demonstrated high levels of IgG secretion and marked decreases in suppressor activity. In Imuran-treated patients, Con A mitogenic responses and suppressor activity were comparable to those observed in untreated stable patients, and IgG secretion was reduced. The results in the treated patients likely reflect a direct effect of Imuran on B cell function rather than an indirect effect mediated via suppressor cells.     

T-lymphocyte subpopulations in peripheral blood of patients with multiple sclerosis, patients with other neurological diseases and healthy controls

We report our results in profiling peripheral blood lymphocyte subpopulations with monoclonal antibodies in 17 multiple sclerosis (MS) patients, 22 patients with other neurological diseases (OND), and 11 healthy controls, using a blind experiment. Untreated patients with a chronic progressive MS have higher T-helper cell (OKT4+) counts and a higher ratio OKT4+/OKT8+ than other MS patients, OND or healthy controls. Two weeks after the onset of a relapse of MS there is a decreased T-helper and an increased T-suppressor cell percentage. Treatment with ACTH results in a significant increase of helper cells after 4 weeks of therapy. Patients with the lowest helper cell counts and the lowest helper/suppressor ratio show the best clinical improvement by ACTH. High OKT4+ cell percentages make a chronic progressive course of MS more probable.     

Progressive multiple sclerosis: Abnormal immune functions in vitro and aberrant correlation with enumeration of lymphocyte subpopulations

In a series of 27 consecutive progressive multiple sclerosis (MS) patients under age 50 we have simultaneously measured 3 in vitro immune functions and 6 markers and compared their results to a group of 21 controls. We have confirmed a reduction of concanavalin A (Con A) -induced suppression and NK function contrasting with increased IgG secretion in response to pokeweed mitogen (PWM). Among 6 monoclonal antibody-recognized subpopulation (Leu 1, Leu 2, OKT8, Leu 3, Leu 7 and Leu 11) only Leu 2+ lymphocytes were statistically reduced. OKT8+ were slightly reduced, Leu 3+ were slightly increased. Discriminant analysis revealed that the 3 immune functions together with the results of OKT8 and Leu 3 enumeration were sufficient to appropriately classify most of the individuals. Only 3 MS and 4 controls were misclassified. Correlation analysis suggested disappearance of the doubly labelled OKT8/Leu 7 population in MS patients. In MS as opposed to controls Con A-induced suppression did not correlate with suppressor cell markers but correlated with NK cell markers suggesting that in MS this population mediates Con A-induced suppression. IgG secretion and Con A suppressor cell function were inversely correlated in MS patients but not in controls, suggesting that in chronic progressive multiple sclerosis a common abnormality underlies both increased response to PWM and decreased induction of suppression by Con A.     

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     

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.     

Increased lymphocyte beta-adrenergic receptor density in progressive multiple sclerosis is specific for the CD8+, CD28- suppressor cell.

Beta-adrenergic receptor density on T cells from healthy humans is greatest on suppressor cells (CD8+, CD28-) and the effect of catecholamines, secreted by the sympathetic nervous system, predominates on this subset. The sympathetic skin response, a measure of sympathetic nervous system function, is absent in most patients with chronic progressive multiple sclerosis (MS). We measured beta-adrenergic receptor density on suppressor cells, cytotoxic cells, and monocytes from patients with chronic progressive MS and healthy control subjects. Control receptor density on suppressor cells was 2.8 +/- 0.3 fmol/10(6) cells versus a density of 5.1 +/- 0.7 fmol/10(6) cells for patients. Cytotoxic cell (CD8+, CD28+) receptor density was 1.4 +/- 0.4 fmol/10(6) cells in control subjects and 0.9 +/- 0.3 fmol/10(6) cells in the patients. Monocytes displayed beta-adrenergic receptor densities of 2.6 +/- 0.4 fmol/10(6) cells in normal individuals and 2.7 +/- 0.4 fmol/10(6) cells in the patient group. CD8 lymphocyte beta-adrenergic receptor densities in patients with relapsing-remitting and those with stable MS were not different from control values, yet were significantly less than the values for patients with chronic progressive MS. We find that mononuclear cells from healthy control subjects and patients with chronic progressive MS proliferate in response to 200 units/ml of recombinant human interleukin-2 (IL-2) similarly. However, IL-2 treatment increased beta-adrenergic receptor density on normal mononuclear cells, but failed to increase it on mononuclear cells from patients with chronic progressive MS.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-cell subsets in spinal fluid of multiple sclerosis patients

CSF T-cells and T-cell subsets were characterized by monoclonal antibodies in 15 untreated multiple sclerosis (MS) patients, 17 immunosuppressed chronic progressive MS patients and 9 patients with other neurological diseases. A negative correlation was found between total cell numbers and T suppressor cell percentages in untreated and treated MS patients. A negative correlation (r = -0.71) was found between intrathecal IgG levels and T suppressor cell percentages in untreated MS patients. In peripheral blood, no correlation between T-cells and T-cell subsets with IgG levels was found. It is discussed that T-cell subsets and intrathecal IgG levels may be indicators of the activity of the inflammatory process in the brains of chronic progressive MS patients.     

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.     

Loss of functional suppression is linked to decreases in circulating suppressor inducer (CD4+ 2H4+) T cells in multiple sclerosis

A consistent immunological finding in patients with progressive multiple sclerosis is a loss of functional suppression. We have recently found decreases in suppressor inducer T cells in progressive multiple sclerosis as measured by two-color immunofluorescence using differentiation markers CD4 and 2H4. In the present study, we examined the relationship between functional suppression and circulating CD4+ 2H4+ T cells using a two-stage assay. (1) T cells were stimulated for 7 days with irradiated non-T cells (autologous mixed lymphocyte reaction [AMLR]) and harvested. It has previously been shown that suppressor T cells are generated during the course of the AMLR. (2) The AMLR-generated suppressor T cells were then incubated with mononuclear cells plus pokeweed mitogen, and immunoglobulin (Ig) synthesis was measured. There was less AMLR-induced suppression of IgG synthesis in patients with progressive multiple sclerosis as compared with normal subjects and patients with other neurological diseases. More importantly, there were significant correlations between decreases in circulating CD4+ 2H4+ cells and the AMLR (p = 0.009). Thus, the decreases in functional suppression and the decreases in the AMLR in multiple sclerosis appear tightly linked to CD4+ 2H4+ cells, and their measurement provides a means to monitor suppressor function phenotypically. Decreases in suppressor inducer T cells may in part explain immunoregulatory abnormalities observed in multiple sclerosis.     

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.     

Abnormal regulation of in vitro IgG production in multiple sclerosis

A pokeweed mitogen-stimulated IgG synthesis assay was used to evaluate T suppressor cell function in peripheral blood lymphocytes (PBL) obtained from multiple sclerosis (MS) patients. Autochthonous cultures of fractionated and recombined MS B and T cells exhibited less suppression of IgG synthesis than similar cultures of PBL from control subjects. No difference occurred in the levels of suppression among MS patients grouped according to disease states. When allogenic cultures of MS B and normal T cells or normal B and MS T cells were compared to autochthonous cultures of control cells, no differences were observed. The results suggest that the lower levels of suppression observed in autologous MS cell cultures are not a result of T suppressor cell dysfunction alone.     

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.     

T cell response to myelin basic protein before and after treatment with interferon beta in multiple sclerosis.

Studies on the in vivo effects of interferon-beta (IFNbeta) therapy on autoreactive T cells have never been carried out in multiple sclerosis (MS). We investigated the T cell response to myelin basic protein (MBP), before and after IFN-beta therapy, raising MBP-specific T cell lines (TCL) from the peripheral blood of six MS patients with a satisfactory response to the treatment. IFNbeta did not affect the relative frequency and epitope specificity of the TCL. After IFNbeta therapy, the production of interleukin-4 was decreased in MBP-stimulated TCL while the secretion of interferon-gamma was increased in unstimulated TCL. Interleukin-10 and tumor necrosis factor-alpha did not show significant variations. This finding supports recent suggestions about the complexity of the T helper 1/T helper 2 paradigm in MS and other organ-specific autoimmune diseases. In fact, the beneficial effects of IFNbeta do not exclude an immunostimulatory action that may involve potentially autoreactive T cells. This has implications for future treatment options, including combination therapies.     

Peripheral blood lymphocyte subsets in acute relapsing multiple sclerosis

Anti-Leu series of monoclonal antibodies were used to evaluate lymphocyte subsets with ABC staining method. The peripheral blood total T cells (Tt), helper T cells (Th), suppressor T cells (Ts) and B cells were counted in 10 cases of acute relapsing MS, in which 7 cases had been investigated consecutively. Controls included 12 cases of remitting MS, 18 patients with other neurological diseases and 56 healthy subjects. The results showed that the peripheral blood Ts decreased in number and the Th/Ts ratio elevated during acute relapsing stage of MS, and they tended to return to normal when the disease showed a gradual recovery (The patients with longer periods of high Th/Ts ratio had more serious manifestations of the illness more severe disability) and the relapsing stage would be longer too.     

Defective immunoreguktion in multiple sclerosis

Imbalances in T cell subpopulations have been reported in multiple sclerosis (MS). In the present study of 31 MS patients, the percentage of T cells with Fc receptors for IgG (T_G) was found to be increased in patients with chronic progressive diseare, and another T cell subset binding to the Raji B lymphoid cell line was decreased. An inverse correlation (r = ?0.675; >95% confidence limit) was found between these two subsets, suggesting that they vary inversely in MS. The mitogenic responses of MS mononuclear cells, isolated T cells, and recombined T and non-T cells to the lectins phytohemagglutimn and pokeweed mitogen (PWM) did not differ from those of normal cells. However, more immunoglobulin (Ig)-producing cells were generated in a PWM-driven system with cells from MS patients than with cells from age-matched controls (p < 0.05). Autologous recombination of separated T and non-T cells did not significantly modify these results. T cells from MS patients added to B cells from normal controls exerted an effect that was related to their percentage of T_G cells; that is, values above 15% were associated with a suppression of Ig production, whereas for T_G values below 12%, a helper effect or no modification was observed. These results suggest that changes in T cell subsets in MS are related to changes in functional ability to modulate Ig production by normal B cells. However, MS B cells partly escape regulation by their own T cells, suggesting an associated B cell hyperactivity.