Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis

We have demonstrated that GA therapy induces a differential upregulation of GA-specific, cytotoxic/suppressor CD8+ T-cell responses in MS patients. We utilized a novel combination of flow sorting and anchored PCR to analyze the evolving clonal composition of GA-specific CD4+ and CD8+ T-cells. TCRβ chain analysis revealed the development of an oligoclonal GA-specific CD8+ repertoire with persistence of dominant clones over long periods. Interestingly, some sequences resembled published oligoclonal CD8+ TCR sequences from MS lesions. In contrast, GA-specific CD4+ responses were polyclonal and showed continual evolution of their repertoire. This clonotypic and functional analysis provides mechanistic insights into GA therapy.     

The role of CD8+ T-cells in lesion formation and axonal dysfunction in multiple sclerosis

The etiology of multiple sclerosis (MS) remains unknown. However, both genetic and environmental factors play important roles in its pathogenesis. While demyelination of axons is a hallmark histological feature of MS, axonal and neuronal dysfunction may correlate better with clinical disability. All major immune cell types have been implicated in the pathogenesis of MS, with the CD4+ T-cells being the most commonly studied. In this review, we discuss the involvement of CD8+ T-cells in MS. In addition, we review the contribution of CD8+ T-cells to the pathogenesis of experimental autoimmune encephalitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) mouse models of MS, including the concept of CD8+ T-cell mediated axonal damage.     

Replacement therapy with plasma-derived factor VIII concentrates induces skew in T-cell receptor usage and clonal expansion of CD8+ T-cell in HIV-seronegative hemophilia patients

Replacement therapy with factor VIII (FVIII) products causes immune abnormalities in human immunodeficiency virus (HIV)-seronegative hemophilia patients. However, the question remains why an absolute increase in the number of CD8+ T-cells and diminished proliferation responses of lymphocytes to antigen stimulation in vitro occurs in HIV-seronegative hemophilia patients. To examine whether the FVIII products induce skewing of T-cell receptor (TCR) repertoires, TCR variable region alpha-chain and beta-chain repertoires were analyzed for peripheral blood mononuclear cells (PBMCs) from 15 hemophilia patients treated with heated and/or non-heated plasma-derived FVIII concentrates and 10 age-matched healthy adults. Also, T-cell clonality was compared between these groups using complementarity-determining region 3 (CDR3) size spectratyping. The skewing of TCR repertoires was significantly greater for hemophilia patients than healthy controls. The extent of T-cell clonality was greater for hemophilia patients than the controls, indicating that clonal T-cells frequently expanded in hemophilia patients. The skew in TCR usage and clonal expansion were primarily observed in patients treated with non-heated plasma-derived products. The spectratyping and sequencing of CDR3 regions revealed that the clonal expansion of T-cells was observed for CD8+ T-cells, but not CD4+ T-cells. These results suggest that extensive expansion of CD8+ T-cells is induced by some viruses other than HIV present in FVIII preparations, and the resulting accumulation of CD8+ T-cells is responsible for changes in peripheral T-cell population in HIV-seronegative hemophilia patients.     

Relapses in multiple sclerosis are associated with increased CD8+ T-cell mediated cytotoxicity in CSF

MS is thought to be mediated by CD4(+) T-helper cells. To investigate the importance of CD8(+) cytotoxic T-cells in MS we analyzed peripheral blood T-cells by DNA microarray, and plasma and CSF levels of granzymes from MS patients and controls. Cytotoxic gene expression was decreased in peripheral T-cells from RRMS patients whereas plasma levels of granzymes were unchanged. However, granzyme levels were elevated in the CSF of RRMS patients at relapse compared with controls and remission. Thus, CD8+ T-cell-mediated cytotoxicity is confined to the CSF/CNS compartment in RRMS patients and may be involved in the immunopathogenesis of clinical relapses.     

Role of perforin secretion from CD8+ T-cells in neuronal cytotoxicity in multiple sclerosis

Objectives: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8⁺ T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions.

Methods: EAE animal models was established by plantar injections of MBP (200 μg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay.

Results: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 μg/mL).

Conclusion: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4⁺ T-cells can enhance CD8⁺ T-cell neuronal cytotoxicity via induction of intense inflammation.     

CD5 B cells and CD48 T cells in neuroimmunological diseases

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

The role of CD4+ T-cells in the development of MS

OBJECTIVE: Multiple sclerosis (MS) is a chronic, progressive central nervous system (CNS) disease with unknown cause. Considerable evidence supports an autoimmune origin with an important role for cellular immune responses in its pathogenesis. METHODS: We have reviewed the current literature dealing with lymphocyte responses and their interactions as it relates to MS and present supporting evidence from animal models. RESULTS: Issues regarding CD4+ T-cell subpopulations, their functional differentiation and regulatory interactions as they relate to their presumed role in MS-related pathology have been updated with references to the current literature. DISCUSSION: The evidence reviewed supports an important role of CD4+ T-cells in the immunopathogenesis of MS. The successful outcome of blocking CD4 cells entry into the CNS of animals with experimental demyelinating disease and humans with MS is a strong support for other evidence of an important role of these cell populations in the pathogenesis of MS. The understanding of the specific roles of CD4+ T-cells in the development of MS is crucial for better disease management and the prevention of neurological disability.     

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

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

Memory and naive CD4+ lymphocytes in multiple sclerosis

Summary Helper-inducer (CD29+CD4+) and suppressor (CD45RACD4+) T-cells have been recently renamed as memory and naive T-cells, respectively. We measured cells expressing these phenotypes in peripheral blood of 46 definite multiple sclerosis (MS) patients [32 relapsing-remitting (RR-MS), 14 secondary progressive (P-MS)] and controls. CD25+ (interleukin-2-receptor-positive) cells were also evaluated in the same groups of patients. RR-MS patients showed increased levels of CD29+CD4+ and CD25+ cells compared with controls. This finding was more evident in RR-MS patients during the attack than during the stable phase of the disease. In P-MS patients we found a reduction of CD45+CD4+ cells compared with either RR-MS patients or control subjects. Our results show that RR-MS and P-MS are characterized by two different T-cell subpopulations. This finding supports the hypothesis that during the evolution from RR-MS and P-MS changes occur in the immunological status of the patients.     

Decrease of CD4+ CD45+ T-cells in chronic-progressive multiple sclerosis

Summary Circulating lymphocyte subpopulations defined by anti-CD45 and other more common T-cell-specific monoclonal antibodies were analysed in 77 patients with multiple sclerosis and 38 healthy controls. A selective decrease of CD4+ CD45+ cell percentages and absolute numbers in chronic-progressive patients was found; in 13 out of 26 patients this subpopulation was less than 11% CD4+ CD45+ cells. Similarly, the whole CD45+ cell subset, as well as CD45+ cells expressed as percentages of CD4+ cells, were significantly reduced in chronic-progressive multiple sclerosis. CD4+ CD45+ cells, commonly termed inducer of suppression T-lymphocytes, did not correlate with percentages or numbers of CD8+ cells. It is concluded that suppressor inducer T-cells act on the CD8+ subset function rather than reducing CD8+ cell numbers. Since CD4+ CD45+ cells represent an early stage of lymphocyte maturation (naive T-cells), an under-representation of this subpopulation in active multiple sclerosis might reflect an increased conversion of naive cells into memory cells. This concept may be relevant for a better understanding of the disease pathogenesis.     

The CD4+ T-cell subset lacking expression of the CD28 costimulatory molecule is expanded and shows a higher activation state in multiple sclerosis

Multiple sclerosis (MS) is a chronic debilitating disease, in which T-cells are considered to play a pivotal role. CD28 is the quintessential costimulatory molecule on T-cells and its expression declines progressively with repeated stimulations, leading to the generation of CD28(-) T-cells. Our aim was to examine whether CD4(+)CD28(-) T-cells were enriched in MS patients, and characterize the phenotype of this subset in MS patients and healthy controls (HC). All these changes could provide these CD4(+)CD28(-) T-cell characteristics that might be involved in the pathogenesis of MS, turning this T-cell subset into a potential target for future therapeutic strategies.     

Analysis of effector CD4 (OX-40+) and CD8 (CD45RA+CD27-) T lymphocytes in active multiple sclerosis

OBJECTIVE: Recently, effector T-cell subpopulations have been identified that can be distinguished by expression of members of the TNF-R family: CD4+OX-40+ cells are CD4 helper-effector cells CD8+CD45RA+CD27 cells are CD8-killer-effector cells. We investigated whether these lymphocyte subsets were increased in the active phase of multiple sclerosis (MS). MATERIAL AND METHODS: Multiple colour immunofluorescence staining was performed on peripheral blood lymphocytes of 28 patients with active MS and of 29 healthy controls, followed by FACS analysis. RESULTS: Frequencies of CD8-killer-effector cells showed a wide interindividual range in both groups and percentages of CD4 helper-effector cells were low. No significant difference between the groups was observed for these subsets, but CD8+CD45RA-CD27 were increased in MS. In healthy individuals, CD4 helper-effector cells correlated with the total percentages of memory cells. Moreover, CD4+ and CD8 memory cells were strongly correlated. CONCLUSION: The here described recently identified effector CD4 and CD8 lymphocyte subpopulations were not increased in clinically active MS. It is however still possible that in MS, myelin-specific encephalitogenic cells reside within these subsets.     

CD8+ T cells in inflammatory demyelinating disease

We review the contribution made by CD8+ T cells to inflammation in the central nervous system (CNS) in Multiple Sclerosis (MS), and discuss their role in the animal model Experimental Autoimmune Encephalomyelitis (EAE). We show that the inflammatory cytokines interferon-gamma and interleukin-17 are differentially regulated in CNS-infiltrating CD4+ and CD8+ T cells in EAE, and that CD8+ T cells regulate disease. In MS, CD8+ T cells appear to play a role in promotion of disease, so cytokine regulation is likely different in CD8+ T cells in MS and EAE.     

Isolation and characterization of CD8+ regulatory T cells in multiple sclerosis

To investigate CD8+ regulatory T cell influence on multiple sclerosis development, peripheral blood and cerebrospinal fluid (CSF) CD8+ T cell clones (TCCs) recognizing MBP_83–102 and MOG_63–87-specific CD4+ T cells were isolated from 20 patients during acute exacerbations, 15 in remission and 15 controls. Blood and CSF CD8+ regulatory TCC cloning frequency decreased more during exacerbations than remissions or controls. Target cell pre-activation significantly enhanced CD8+ T granule-mediated cell killing of CD4+ targets, and was restricted by HLA-E. During exacerbations, killer-inhibitory receptor CD94/NKG2A expression was significantly higher in CD8+ TCCs, limiting their cytotoxic activity. Moreover, IL-15 and IFN-γ significantly increased CD94 and NKG2A expression. These data provide evidence that CD94/NKG2A receptors play an important role in regulating T cell activity during the course of MS.     

Reduced thymic output and peripheral naïve CD4 T-cell alterations in primary progressive multiple sclerosis (PPMS)

We compared na?ve CD4 and CD8 T-cell homeostasis in primary progressive multiple sclerosis (PPMS), relapsing-remitting MS (RRMS) and controls. Quantitation of signal joint T-cell receptor (TCR) excision circles (sjTRECs) and quantitative estimates of daily thymic export confirm our previous report of reduced thymic output in RRMS and demonstrate reduced thymic output in PPMS. In PPMS, the decreasing % CD31+ na?ve CD4 T-cells but constant sjTRECs and constant na?ve CD4 T-cell numbers with age, together with increased Bcl-2 expression suggest increased TCR signaling with increased na?ve T-cell survival. We conclude PPMS patients have peripheral immune alterations related to reduced thymic output.     

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

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

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

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

Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.     

Acute aerobic exercise in humans increases cytokine expression in CD27− but not CD27+ CD8+ T-cells

Exercise alters the percentage of CD8⁺ T-cells in the bloodstream expressing type I and type II cytokines. It is unknown if this reflects a change in cytokine expression within individual cells, or whether these observations result from the exercise-induced shift in the proportions of early/intermediate (CD27⁺) and late (CD27⁻) differentiated cells, which have vastly different cytokine profiles. 16 males cycled for 60 min at 95% maximal steady state. Mononuclear cells isolated from blood collected before, immediately after, and 1 h after exercise were cultured overnight with and without phytohaemagglutinin stimulation. CD8⁺ T-cells were assessed for differentiation markers and intracellular cytokine expression by flow cytometry. The numbers and percentage of CD27⁻CD8⁺ T-cells increased immediately after exercise and fell below pre-exercise values 1 h later. At 1 h after exercise, an increased number and percentage of CD8⁺ T-cells expressing IL-2, IFN-γ, TNF-α, IL-6, IL-4, and IL-10 was observed in both stimulated and unstimulated cells. The cytokine response to exercise was confined to CD27⁻CD8⁺ T-cells, although cytokine expression among CD8⁺ T-cells was highest when the proportion of CD27⁻CD8⁺ T-cells was lowest. Moreover, the cytokine response to exercise could be predicted by the number of late cells in resting blood: cytokine expression was highest among those with low resting proportions of late cells. We conclude that exercise-induced changes in the percentage of CD8⁺ T-cells expressing cytokines are not due to proportional shifts in early/intermediate and late differentiated T-cells. Exercise may prime late-differentiated blood CD8⁺ T-cells to initiate effector functions in preparation for their extravasation into the tissues.     

A potential role for CD8+ T-cells as regulators of CNS vascular permeability

The role of immune cells in promoting central nervous system (CNS) vascular permeability is poorly understood. In recent years, there is a growing body of literature that suggests CD8+ T-cells are potent mediators of vascular permeability in peripheral viral infections as well as in immune mediated neurological diseases. This review outlines the recent advances in tissue culture and animal models used to study vascular permeability. In addition, we put forth our hypothesis that CD8+ T-cells promote the opening of tight junctions between cerebral endothelial cells, enabling the infiltration of white blood cells and in certain models even leading to microhemorrhages in the CNS. Determining the mechanism by which CD8+ T-cells and other immune cells promote CNS vascular permeability in animal models could define new targets for immune mediated neurological conditions characterized by vascular permeability.