Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.This Work was partially supported by an IRCCS Current Research Grant 1994.     

Circulating natural killer cells but not cytotoxic T lymphocytes are reduced in patients with active relapsing multiple sclerosis and little clinical disability as compared to controls

Triple antibody flow cytometry was used to compare the populations of CD56⁺ effector cells in the peripheral circulation of 29 patients with relapsing multiple sclerosis (MS) and little disability who were exacerbation-free for over 2 months and 29 healthy control subjects. Populations were characterized by two panels of antibodies (CD8, CD16, CD56 and CD3, CD8, CD56), as well as by size or granularity. In the MS patients, mature natural killer (NK) cells (CD3⁻CD8⁻CD56⁺) of small size and low granularity were significantly reduced compared to normals (P 〈 0.0003). The quantities of other effector cells (cytotoxic T lymphocytes, large granular lymphocytes and monocytes) were not different in MS patients compared to the control subjects. Also, we identified a previously unrecognized population of CD56⁺ monocytes (CD3⁻CD14⁺CD56⁺) in both the normal control subjects and the MS patients which would have been misclassified as NK cells using one or two antibody cytometry employed in previous studies.     

Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression in patients with multiple sclerosis

Objectives – To determine whether percentages of CD4⁺CD25^high T cells (a group of regulatory T cells, Treg) differ in patients with multiple sclerosis (MS) in relapse vs remission after glucocorticoid treatment and whether treatment for relapses changes Treg population and the expression of Foxp3, a key Treg‐associated molecule. Materials and methods – Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with MS during relapse, just before and 2 days after starting steroid treatment (i.v. methylprednisolone 1 g/day for 3 days) and then 6 weeks after treatment. CD4⁺CD25^hi cells were analysed by using flow cytometry. Cytokines were measured by using an ELISA and Foxp3, CD3 and CD25 expression by using quantitative real‐time PCR. Results – The percentage of CD4⁺CD25^hi cells, plasma IL‐10 and Foxp3/CD3 ratio increased 48 h after methylprednisolone initiation and returned to baseline values by 6 weeks post‐treatment. Conclusions – Results suggest that glucocorticoids increase Treg cell functional molecules and percentages. This may be a mechanism whereby steroids expedite recovery from MS relapses.     

Functional defects in peripheral blood T cells of multiple sclerosis patients: Diminished in vitro responsiveness in accessory cell dependent activation systems

Function and phenotype of peripheral blood (PB) T cells in multiple sclerosis (MS) patients were analyzed. In whole blood cultures, T cell proliferation of multiple sclerosis (MS) patients, using soluble CD3 mAB and CD2 mAb as stimulants, was reduced in comparison to healthy controls. A similar difference was seen when isolated PBMC were tested after stimulation with soluble CD3 mAb. However, in accessory cell-independent activation systems, i.e. after stimulation of PBMC with immobilized CD3 mAb or after co-stimulation with CD28 mAb, both patients and controls responded equally well. Phenotypical analysis of the circulating T cell population showed that there were no differences in the percentage of CD26⁺, ‘memory’ (CD45R0⁺) or ‘effector’ (CD4⁺CD45R0⁺CD27⁻) cells between MS patients and healthy controls. Finally, although MS patients did show an enhanced proportion of ‘naive’ (CD4⁺CD45RA⁺) T cells, this did not correlate with the observed functional defects.     

Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.     

Human CD8 TCR-αβ and TCR-γδ cells modulate autologous autoreactive neuroantigen-specific CD4 T-cells by different mechanisms

To investigate the regulatory interactions among autologous T-cells during the course of multiple sclerosis (MS), proteolipid protein peptide-specific CD4⁺ T-cell clones (TCCs) were irradiated and used as immunogens to stimulate purified populations of autologous CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ T-cells isolated from the peripheral blood of MS patients, patients with other non-inflammatory neurological diseases, and healthy blood donors. The resulting blasts were expanded in the presence of hIL-2 and then cloned by limiting dilution. Two different groups of CD8⁺ TCCs were revealed. A first group of CD8⁺ TCCs recognized autologous CD4⁺ T-cells based in their TCRVβ structures (anti-idiotypic responsiveness). A second group of CD8⁺ TCCs recognized Ag activated autologous CD4⁺ TCCs irrespective of their Ag specificity or TCRVβ expression (anti-ergotypic responsiveness). Both groups showed MHC class I restricted cytotoxicity against CD4⁺ T-cells and were able to secrete IFN-γ, TNFα/β and TGF-β. TCR-γδ⁺ TCCs isolated in response to stimulation with autologous peptide-specific CD4⁺ TCCs showed only anti-ergotypic cytotoxicity, which was not inhibited by anti-MHC class Ia monoclonal antibodies. Moreover, they were able to secrete IFN-γ and TNFα/β, but not TGF-β. These data demonstrate that regulatory mechanisms among human autologous T-cells can be mediated by cytolytic interactions or by the release of specific cytokines. Furthermore, they provide evidence that CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ cells differ in their patterns of recognition and in their abilities to modulate the immune response mediated by autologous autoreactive CD4⁺ T-cells.     

Leukocyte chemotactic factor,a natural ligand to CD4, is expressed by lymphocytes and microglial cells of the MS plaque

The leukocyte chemotactic factor (LCF) is a proinflammatory cytokine and natural soluble ligand to the human CD4 molecule. LCF is produced by CD4⁺ and CD8⁺ T lymphocytes and is considered essential to the influx of CD4⁺ T lymphocytes and macrophages into an inflammatory lesion. In order to investigate the role of LCF in the multiple sclerosis (MS) lesion, we have used a synthetic gene to express LCF in E. coli and have produced monoclonal antibodies against LCF. Monoclonal antibodies are suited to demonstrate LCF in ELISAs, Western blots and paraffin-embedded tissue sections. In the MS lesion, immunopositive lymphocytes and microglial cells, notably, have been found. This is the first demonstration that LCF is present in MS lesions. Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance and like other cells of the monocytic lineage express CD4 molecules. Thus, LCF might be a paracrine factor regulating T-lymphocyte chemoattraction and an autocrine molecule regulating microglial cell immune reactivity. © 1996 Wiley-Liss, Inc.     

The immune profile of multiple sclerosis: T-lymphocyte effects predominate over all other factors in cyclophosphamide-treated patients

It is widely believed that multiple sclerosis is a T-cell mediated autoimmune disease associated with abnormalities in immunoregulation. This large, prospective study evaluated the lymphocyte immunophenotypic profile of 246 MS patients, divided clinically into a remitting/relapsing group (n = 176) and a progressive group (n = 70), and compared their results to those of 117 healthy controls. All patients were found to have significantly elevated percentage and absolute numbers of IL2R⁺CD3⁺ cells as well as depressed percentages of CD45RA⁺CD4⁺ cells. However, when the factor of treatment with cyclophosphamide (CY) versus no treatment or treatment with other agents was used to group patients, dramatic declines in both percentages and absolute numbers of CD45RA ⁺ CD4⁺ cells were discovered. These declines were associated specifically with CY and could be explained by this factor independent of the clinical state of the patient. The effects were seen in patients undergoing current treatment or in those exposed to CY in the near or remote past. These findings highlight the confounding effect of specific treatments on the immune profile of MS patient groups and suggest that there may be important implications for cellular function and clinical outcome in these and other patient groups.     

Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis

The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3⁺Tim3⁺, CD4⁺Tim3⁺, and CD4⁺CD25⁺Tim3⁺ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4⁺ and CD4⁺CD25⁺ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4⁺CD25(high) T cell subset. The proportions of CD4⁺Tim3⁺ and CD4⁺CD25⁺Tim3⁺ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3⁺Tim3⁺ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS.     

Analysis of CD27 surface expression on T cell subsets in MS patients and control individuals

Within the peripheral blood, CD4⁺CD27⁻ T cells only reside within the CD45RAT ⁻(memory or primed) T cell subset. Cells with this phenotype have characteristics of specialized effector T cells according to their cytokine secretion profiles and the expression of tissue-specific adhesion molecules. This subset was previously found to be increased in certain diseases that are associated with immune activation. Therefore we analyzed CD27 expression of peripheral blood and CSF T cells in MS patients. Within the CD4⁺ T cell subset no differences were seen between MS patients and controls in proportions of CD45RA⁻CD27⁻ cells. However, when the CD3⁺ T cell compartment was analyzed, CD27⁻ cells were also found within the CD45RA⁺ subset. These cells, most likely CD8⁺, are significantly reduced in PBL and CSF of MS patients as compared with OND patients. In MS and OND groups the level of CD27⁻ cells in peripheral blood correlated significantly with that in CSF, indicating a balanced migration of CD27⁻ cells between the two compartments. In OIND patients, however, this equilibrium was lost. The correlation of the level of CD27⁺ cells with the amount of intrathecally produced IgG in MS patients may suggest that CD27⁺ cells are responsible for B cell help in this disease.     

Effect of IFN-ß therapy on the frequency and function of CD4+CD25+ regulatory T cells and Foxp3 gene expression in relapsing–remitting multiple sclerosis (RRMS): A preliminary study

Interferon-ß (IFN-ß) is an immunomodulatory drug of choice to control relapsing–remitting multiple sclerosis (RR-MS), although its function is still unclear. A reduced suppressive function of CD4⁺CD25⁺ regulatory T cells (T_reg) has been shown in RR-MS patients. In this study, to understand the effect of IFN-ß on CD4⁺CD25⁺ regulatory T cells, we analyzed the frequency and function of these cells and Foxp3 gene expression before and after treatment.We evaluated the frequency and function of CD4⁺CD25⁺Foxp3⁺ regulatory T cells by flow cytometry and co-culture inhibition test respectively and gene expression of Foxp3 by real-time PCR in a longitudinal follow-up study in 18 relapsing–remitting MS patients. Our data revealed that IFN-ß significantly improved frequency and suppressive function of T_reg cells (P < 0.05) without any significant effect on gene expression of Foxp3 after 6 months. The results of the present study indicate that IFN-ß therapy in some of patients with RR-MS may restore function of regulatory T cells and control the unchecked immune cascade activity. Larger longitudinal studies on more MS patients are required to confirm our findings.     

Long-term immunological changes in azathioprine-treated MS patients

The long-term immunological effects of azathioprine treatment have been investigated in 8 multiple sclerosis patients with different course of disease, chronic progressive (CP) or relapsing progressive (RP). We studied fluctuations in peripheral blood mononuclear cell subsets, IgG, IgM and soluble vascular cell adhesion molecule-1 (sVCAM-1), before and after 2 (T24) and 3 (T36) years of therapy. We observed a significant decrease in CD8⁺ cells over time and a trend to lower percentage of CD3⁻CD56⁺ cells at T24 and T36. CD4⁺CD45RA⁺ cells in MS patients were lower than in healthy controls before therapy and reached values similar to those of healthy controls at T24 and T36. The remaining immunological parameters did not show any significant fluctuations. Received: 24 November 1999 / Accepted in revised form: 8 February 2000     

Interferon-beta-1a treatment increases CD56 natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis

Disease modifying effects of interferon (IFN)-β therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56^bright subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-β-1a(IM) treatment of relapsing–remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56^bright NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-β-1a in MS include control of pernicious autoimmunity.     

Presence of T-cell subset abnormalities in newly diagnosed cases of multiple sclerosis and relationship with short-term clinical activity

Abnormalities of T-cell subsets in patients with multiple sclerosis are well known; in order to assess whether immunological abnormalities are relevant in the pathogenesis of the disease after its clinical onset, peripheral blood lymphocyte subsets (CD3⁺, CD4⁺, CD4⁺ CD45RA⁺, CD4⁺CD45RA^–, CD8⁺, CD8⁺CD57⁺, CD57⁺, CD25⁺) were analysed serially in 25 patients at the first clinical episode suggestive of inflammatory demyelinating disease and in an equal number of age- and sex-matched controls. During the follow-up period (12–18 months, mean 14) 6 of 25 patients presented new relapses: in this subgroup of patients, significant changes in CD4⁺ ratio (% CD4⁺CD45RA^–/%CD4⁺CD45RA^–) were detected in comparison both with healthy controls and with clinically stable patients. Patients clinically stable at follow-up did not display immunological abnormalities, regardless of the presence or absence of cerebrospinal fluid and/or magnetic resonance imaging alterations consistent with multiple sclerosis. These findings suggest a possible prognostic role of early T-cell subset imbalance in multiple sclerosis.     

Immunological effects of in vivo interferon-β1b treatment in ten patients with multiple sclerosis: a 1-year follow-up

Ten patients with multiple sclerosis and treated with interferon-β_1b (IFN-β_1b) were followed-up for 1 year with quantitation of serum VCAM-1 and ICAM-1 levels, mean fluorescence intensity of HLA-DR, VLA-4, CD11a, and CD18 on peripheral blood monocytes and lymphocytes, and adhesion of peripheral blood monocytes and CD45⁺ cells on endothelial cell monolayers. Adhesion molecule expression and adhesion of peripheral blood monocytes to endothelium were also monitored in healthy controls. No differences in adhesion were detected between MS patients before treatment and healthy controls, while after 1 year a marked decrease in the number of monocytes and mononuclear cells adhering to human umbilical vein endothelial cell monolayers was observed in patients treated with IFN-β_1b. After 1 year of treatment a significant increase in HLA-DR on peripheral blood monocytes was also detected. Our findings regarding lowered adhesion add information to available evidence of the mechanisms of action of IFN-β_1b in MS. Received: 10 August 1998 Received in revised form: 9 December 1998 Accepted: 23 December 1998     

Interleukin-17-secreting T cells in neuromyelitis optica and multiple sclerosis during relapse

Growing evidence suggests that interleukin (IL)-17 and IL-17-secreting CD4⁺T (Th17) cells are involved in the pathogenic mechanisms of multiple sclerosis (MS). IL-17-secreting CD8⁺T cells were recently identified as a novel subset of CD8⁺T cells. We aimed to analyze the role of Th17 and IL-17 secreting CD8⁺T cells in the pathogenesis of neuromyelitis optica (NMO) as well as MS. Fourteen patients with NMO, 20 with MS and 16 control participants (CTL) were enrolled between November 2008 and December 2009. The proportion of Th17 cells and IL-17 secreting CD8⁺T cells were counted using flow cytometry, and serum levels of IL-6, IL-17, IL-21, IL-23, and transforming growth factor-beta (TGF-β) were measured by enzyme-linked immunosorbent assay. Patients with NMO had a larger proportion of Th17 cells than patients with MS (3.72% versus [vs.] 2.58%, p = 0.02) and CTL (3.72% vs. 1.36%, p < 0.001). The proportion of Th17 cells in patients with MS was also markedly higher than in the CTL (2.58% vs. 1.36%, p < 0.001). IL-17-secreting CD8⁺T cell counts in NMO patients were markedly higher than in MS patients (1.61% vs. 1.09%, p = 0.036) and CTLs (1.61% vs. 0.58%, p < 0.001). The proportion of IL-17-secreting CD8⁺T cells in MS patients was also higher than in CTLs (1.09% vs. 0.58%, p = 0.002). Serum IL-17 and IL-23 levels were increased in patients with NMO and MS, while serum IL-21 concentration was higher only in NMO patients compared to CTL. We concluded that Th17 cells were highly activated in patients with NMO. IL-17-secreting CD8⁺T cells were increased in patients with NMO and MS during relapse and have an important role in the pathological mechanism of NMO and MS.     

Evaluation of interleukin 10 and interferon-α in the cerebrospinal fluid and serum of patients with multiple sclerosis

The activities of IL-10, a cytokine produced by TH2 cells, monocytes and B-cells, and IFN-α, a product of activated macrophages/monocytes, were investigated in the CSF and serum samples of 25 subjects with clinically definite multiple sclerosis (MS), of whom 14 were in the active and nine in the stable phase, and of 15 controls with other noninflammatory neurological diseases (OND). Elevated CSF IL-10 and IFN-α levels were found in MS patients in the stable phase with respect to patients in the active phase (p > 0.001), while no significant differences were observed in the mean serum levels between MS patients (both in the active and stable phase) and controls. Finally, a significant correlation was detected between IL-10 and IFN-α in the CSF of MS patients in remission. This study suggests that IL-10 may downregulate MS progression.     

Dopamine favors expansion of glucocorticoid-resistant IL-17-producing T cells in multiple sclerosis

Dopamine (DA) is a neurotransmitter produced mainly in the central nervous system (CNS) that has immunomodulatory actions on T cells. As the multiple sclerosis (MS) has long been regarded as an autoimmune disease of CNS mediated by T cells, the objective of this study was to evaluate the impact of DA on in vitro functional status of T cells from relapsing–remitting (RR)–MS patients. Peripheral T-cells from RR–MS patients were activated by mitogens and cell proliferation and cytokine production were assayed by [³H]-thymidine uptake and ELISA, respectively. Our results demonstrated that DA enhanced in vitro T cell proliferation and Th17-related cytokines in MS-derived cell cultures. In addition, this catecholamine reduced Treg-related cytokines (IL-10 and TGF-β) release by activated CD4⁺ T cells. These DA-induced effects on T cells were mainly dependent on IL-6 production by both polyclonally-activated CD4⁺ T cells and LPS-stimulated monocytes. Furthermore, the production of IL-17 and IL-6 by MS-derived T cells was directly related with neurological disability (EDSS score), and the release of these cytokines was less sensitive to glucocorticoid inhibition in MS patients than in control group, mainly after DA addition. In conclusion, our data suggest that DA amplifies glucocorticoid-resistant Th17 phenotype in MS patients, and this phenomenon could be, at least in part, due to its ability to induce IL-6 production by monocytes and CD4⁺ T cells.     

Activated suppressor cell function in multiple sclerosis — clinical correlations

Activated suppressor cell function mediated by either freshly isolated peripheral blood mononuclear cells (MNCs), freshly isolated CD8⁺ lymphocytes or by CD8⁺ cell lines, has previously been found to be reduced compared to controls in multiple sclerosis (MS) patients with progressive disease (MS-P). In this study, we found that suppressor activity mediated by CD8⁺ cell lines, derived from MS patients with stable disease (MS-S) patients and maintained in culture for 14 days, was significantly greater (45 ± 6%) compared to that mediated by MS-P patients' CD8⁺ cells (11 ± 4%, P < 0.005). The MS-S suppressor values were, however, suggestively reduced compared to controls (60 ± 6%, P < 0.05). MNC-mediated suppressor values for the MS-S group (61 ± 5%) did not differ from the control group (67 ± 6%). Values for the MS-P group (7 ± 6%) were significantly reduced compared to MS-S and control groups. Cytotoxic activity mediated by CD8⁺ cell lines showing defective suppressor function did not differ from control values. The cell lines in MS and control did not differ with respect to their rate of proliferation in the presence of IL-2 and OKT3. Suppressor function in this assay was ablated if exogenous IL-2 was removed from the culture media. These data suggest that defective activated suppressor function is characteristic of the progressive form of MS, although a suppressor defect is also partially expressed in stable MS patients when CD8⁺ cell lines are studied.