Increased program cell death-1 expression on T lymphocytes of patients with progressive multifocal leukoencephalopathy

The cellullar immune response is important in the containment of progressive multifocal leukoencephalopathy (PML). We examined program cell death-1 (PD-1) expression, a marker of cellular immune exhaustion, on T lymphocytes in PML. PD-1 expression was elevated on total CD4(+) and CD8(+) T cells (medians 36% and 24%) in PML patients compared with healthy control subjects (medians 14% and 18%; P = 0.0015 and P = 0.033). In PML patients, JC virus (JCV)-specific CD8(+) cytotoxic T lymphocytes expressed PD-1 more frequently than total CD8 T lymphocytes (means 39% and 78%, P = 0.0004). Blocking the PD-1 receptor increased JCV-specific T-cell immune response in a subgroup of PML patients.     

Efficient in vitro expansion of JC virus-specific CD8 T-cell responses by JCV peptide-stimulated dendritic cells from patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the brain caused by JC virus (JCV) for which there is no cure. PML patients who have JCV-specific CD8⁺ cytotoxic T lymphocytes (CTL) in their blood have a better clinical outcome. We compared JCV-specific CTL responses in vitro elicited either by JCV peptide-loaded dendritic cells (DC) or by direct peptide stimulation of lymphocytes from 20 HLA-A?0201⁺ healthy controls, HIV⁺ and PML patients. JCV peptide-loaded DC elicited a stronger CTL expansion in 13/15 responders. DC can induce a potent JCV-specific CTL response in vitro, and may constitute a promising approach for PML immunotherapy.     

Dual expression of CD45RA and CD45RO isoforms on myelin basic protein-specific CD4+ T-cell lines in multiple sclerosis

Myelin basic protein (MBP)-specific T-cell lines from patients with multiple sclerosis (MS) and healthy controls were analyzed for the expression of CD45 isoforms and adhesion molecules. In the multiple sclerosis group, 22 of 24 MBP-specific T-cell lines were CD4+. Two distinct patterns were observed with regard to CD45 isoform expression. Pattern I showed dual expression of CD45 isoforms (CD4+CD45RA+CD45RO+CD29+) and Pattern II included cells with a single CD45 isoform (CD4+CD45RA–CD45RO+CD29+). All 10 cell lines from healthy controls were CD4+ and displayed Pattern II (CD4+CD45RA–CD45RO+CD29+). The dual expression of CD45 isoform in T-cell lines from MS was stable, did not represent a transition stage from CD45RA to CD45RO, and was cell-cycle independent. All cell lines from MS and controls expressed increased levels of LFA-1 (CD11a), LFA-2 (CD2), LFA-3 (CD58), ICAM-1 (CD54), and VLA-4 (CDw49d). These data show the presence of unique MBP-specific T cells (CD4+CD45RA+CD45RO+CD29+) that might play a role in the pathogenesis of MS.     

Extensive subpial cortical demyelination is specific to multiple sclerosis

Cortical demyelinated lesions are frequent and widespread in chronic multiple sclerosis (MS) patients, and may contribute to disease progression. Inflammation and related oxidative stress have been proposed as central mediators of cortical damage, yet meningeal and cortical inflammation is not specific to MS, but also occurs in other diseases. The first aim of this study was to test whether cortical demyelination was specific for demyelinating CNS diseases compared to other CNS disorders with prominent meningeal and cortical inflammation. The second aim was to assess whether oxidative tissue damage was associated with the extent of neuroaxonal damage. We studied a large cohort of patients diagnosed with demyelinating CNS diseases and non‐demyelinating diseases of autoimmune, infectious, neoplastic or metabolic origin affecting the meninges and the cortex. Included were patients with MS, acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), viral and bacterial meningoencephalitis, progressive multifocal leukoencephalopathy (PML), subacute sclerosing panencephalitis (SSPE), carcinomatous and lymphomatous meningitis and metabolic disorders such as extrapontine myelinolysis, thus encompassing a wide range of adaptive and innate cytokine signatures. Using myelin protein immunohistochemistry, we found cortical demyelination in MS, ADEM, PML and extrapontine myelinolysis, whereby each condition showed a disease‐specific histopathological pattern. Remarkably, extensive ribbon‐like subpial demyelination was only observed in MS, thus providing an important pathogenetic and diagnostic cue. Cortical oxidative injury was detected in both demyelinating and non‐demyelinating CNS disorders. Our data demonstrate that meningeal and cortical inflammation alone accompanied by oxidative stress are not sufficient to generate the extensive subpial cortical demyelination found in MS, but require other MS‐specific factors.     

CSF enrichment of highly differentiated CD8+ T cells in early multiple sclerosis

CD8+ T cells may play an important role in multiple sclerosis (MS). Whether these cells would be involved in early stages of MS is unclear. We enrolled 52 patients with suspected MS, determined the recruitment of their highly differentiated (CCR7-/CD45RA+ or -) T cells (T(HD)) in the CSF as compared to peripheral blood and followed them for 12+/-7.3 months. A ROC curve showed that a CD8+/CD4+ T(HD) cells ratio of 0.94 helped to distinguish relapsing-remitting (RR-MS) and possible MS (Po-MS) from primary-progressive MS (PP-MS) and other neurological diseases patients (OND) patients (p=0.039), risk ratio of 2.29 (95% CI: 1.13-4.66; p=0.006). The CSF enrichment in CD8+ T(HD) cells was greater than in CD4+ T(HD) cells in RR/Po-MS patients (p=0.024) and than in CD8+ T(HD) cells in PP-MS/OND patients (p=0.006). These data suggest that CD8+ T(HD) cells play a role in the early stages of RR-MS.     

BKV and JCV large T antigen-specific CD8+ T cell response in HLA A*0201+ kidney transplant recipients with polyomavirus nephropathy and patients with progressive multifocal leukoencephalopathy.

BACKGROUND: BK virus (BKV), which causes polyomavirus-associated nephropathy (PVN) in kidney transplant recipients (KTx), has 75% homology with JC virus (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML). The large T-antigen (T-ag) is the main regulatory protein of polyomaviruses that is expressed early in the viral cycle. OBJECTIVES: To characterize epitopes of BKV and JCV T-ag recognized by CD8+ T-cells and explore the role of these cells in containing polyomavirus infection. STUDY DESIGN: We tested peripheral blood mononuclear cells of HLA A*0201+ BKV- and JCV-seropositive individuals, including patients with active BKV or JCV infection and healthy control subjects in a cross-sectional study. RESULTS: CD8+ T-cells that recognized the nonamer BKV Tp579, which is identical to JCV Tp578, were detected by tetramer staining in 10/13 (77%) healthy individuals, 3/10 (30%) KTx/PVN, and 4/9 (44%) patients with PML and/or HIV-infection. Conversely, BKV Tp398- and Tp410-specific CD8+ T cells were detected in 3/13 (23%) and 1/13 (8%) healthy individuals only. CONCLUSION: These data suggest that, as it is the case for the VP1 protein, the same population of CD8+ T-cells may recognize epitopes located on the BKV and JCV T protein. The overall cellular immune response against polyomavirus T-ag, however, is lower than against the VP1 protein and is more frequently detected in healthy individuals than in patients with active BKV or JCV infection.     

Neuroantigen-specific CD8+ regulatory T-cell function is deficient during acute exacerbation of multiple sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). MS is thought to be T-cell-mediated, with prior research predominantly focusing on CD4+ T-cells. There is a high prevalence of CNS-specific CD8+ T-cell responses in MS patients and healthy subjects. However, the role of neuroantigen-specific CD8+ T-cells in MS is poorly understood, with the prevalent notion that these may represent pathogenic T-cells. We show here that healthy subjects and MS patients demonstrate similar magnitudes of CD8+ and CD4+ T-cell responses to various antigenic stimuli. Interestingly, CD8+ T-cells specific for CNS autoantigens, but not those specific for control foreign antigens, exhibit immune regulatory ability, suppressing proliferation of CD4+CD25? T-cells when stimulated by their cognate antigen. While CD8+ T-cell-mediated immune suppression is similar between healthy subjects and clinically quiescent treatment-naïve MS patients, it is significantly deficient during acute exacerbation of MS. Of note, the recovery of neuroantigen-specific CD8+ T-cell suppression correlates with disease recovery post-relapse. These studies reveal a novel immune suppressor function for neuroantigen-specific CD8+ T-cells that is clinically relevant in the maintenance of peripheral tolerance and the intrinsic regulation of MS immune pathology.     

Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28- T cells and inhibit both T-cell proliferation and CTL function

Nonantigen specific CD8+ suppressor T lymphocytes (CD8+ Ts) inhibit T-cell proliferation of antigen-specific T lymphocytes. The impossibility to generate in vitro these cells has been correlated with the appearance of relapses in patients affected by autoimmune diseases, suggesting the involvement of these cells in immune regulation. This study was aimed to identify circulating precursors and to characterize the phenotype and mechanism of action of CD8+ Ts. We found that CD8+ Ts can be generated in vitro from CD8+CD28- T lymphocytes, but not from CD8+CD28+ T cells. A key role in their generation is played by monocytes that secrete interleukin-10 (IL-10) after granulocyte macrophage-colony-stimulating factor (GM-CSF) stimulation. Cell-to-cell direct interaction between CD8+CD28- T cells and monocytes does not play a role in the generation of CD8+ Ts. CD8+ Ts have a CD45RA+, CD27-, CCR7-, IL-10Ralpha+ phenotype and a TCR Vbeta chain repertoire overlapping that of autologous circulating CD8+ T cells. This phenotype is typical of T lymphocytes previously expanded due to antigen stimulation. Their suppressive effect on T-cell proliferation targets both antigen presenting cells, such as dendritic cells, and antigen-specific T lymphocytes, and is mediated by IL-10. CD8+ Ts suppress also the antigen-specific cytotoxic activity of CTL decreasing the expression of HLA class I molecules on target cells through IL-10 secretion. These findings can be helpful for the better understanding of immune regulatory circuits and for the definition of new pathogenic aspects in autoimmunity and tumor immunology.     

Progressive multifocal leukoencephalopathy in a patient with common variable immunodeficiency and abnormal CD8+ T-cell subset distribution.

BACKGROUND: Patients with primary hypogammaglobulinemia have been reported to have encephalopathy, but progressive multifocal leukoencephalopathy (PML) due to JC virus reactivation is a rare cause. OBJECTIVE: To provide the clinical details and case discussion of a patient diagnosed as having common variable immunodeficiency (CVID) who has progressive neurodegenerative symptoms and was found to have PML and an abnormal CD8+ T-cell subset distribution. METHODS: A detailed case report providing the patient's immunodeficiency history, diagnostic evaluation, and medical management and a review of related literature. RESULTS: Before his neurodegenerative illness, the patient was found to have hypogammaglobulinemia, poor specific antibody responses, low circulating B-cell levels, and abnormal delayed-type hypersensitivity responses; there was no Bruton tyrosine kinase (BTK) mutation. The PML was diagnosed using brain biopsy and was confirmed using a DNA probe specific for JC virus. Peripheral blood flow cytometry at the time of PML diagnosis revealed an accumulation of naive CD8+ T cells (CD3+CD8+CD45RA+) and a deficiency of memory CD8+ T-cell subsets (CD3+CD8+CD45RA- or CD3+CD8+CD45RO+). Despite aggressive treatment with interleukin 2, interferon-gamma, and intravenous cidofovir, the patient died. CONCLUSIONS: JC virus infection should be considered in the differential diagnosis of the patient with CVID and signs and symptoms of encephalopathy. The role of this patient's abnormal CD8' T-cell subset distribution in the development or control of this rare infection is worthy of consideration and has encouraged us to enumerate naive and memory CD4+ and CD8+ T-cell subsets in patients diagnosed as having CVID, even in the absence of neurodegenerative symptoms.     

Production of IL-16 correlates with CD4+ Th1 inflammation and phosphorylation of axonal cytoskeleton in multiple sclerosis lesions

Background  

Multiple sclerosis (MS) is a central nervous system-specific autoimmune, demyelinating and neurodegenerative disease. Infiltration of lesions by autoaggressive, myelin-specific CD4+Th1 cells correlates with clinical manifestations of disease. The cytokine IL-16 is a CD4+ T cell-specific chemoattractant that is biased towards CD4+ Th1 cells. IL-16 precursor is constitutively expressed in lymphocytes and during CD4+ T cell activation; active caspase-3 cleaves and releases C-terminal bioactive IL-16. Previously, we used an animal model of MS to demonstrate an important role for IL-16 in regulation of autoimmune inflammation and subsequent axonal damage. This role of IL-16 in MS is largely unexplored. Here we examine the regulation of IL-16 in relation to CD4+ Th1 infiltration and inflammation-related changes of axonal cytoskeleton in MS lesions.     

重型乙型病毒性肝炎肝组织内T淋巴细胞亚群的观察

本文用抗CD_3,CD_4,CD_8McAb和ABC法检测21例重型乙肝患者肝大片,亚大片坏死区炎性浸润细胞中的T细胞亚群,CD_3~+细胞>70％,其中主要为CD_8~+细胞,CD_4~+细胞减少,CD_4~+/CD_8~+比值显著下降,与急黄肝和慢活肝比较差异显著。相反,非HBV感染性肝病患者非T细胞和CD_4~+细胞增加,CD_4~+/CD_8~+比值>1。提示重肝时T细胞可能参与了肝损伤,CD_8细胞亚群可能是介导肝细胞坏死的重要因素之一。还观察到相当数量的淋巴细胞和肝细胞HLA—DR抗原阳性,淋巴细胞与膜型HBAg(+)肝细胞密切接触。     

Progressive multifocal leucoencephalopathy in a patient with idiopathic CD4+ lymphocytopenia

Progressive multifocal leucoencephalopathy (PML) is an opportunistic, demyelinating neurological disease caused by reactivation of the JC polyomavirus. PML occurs almost exclusively in immunosuppressed individuals, with only isolated case reports of PML occurring in patients without apparent immunosuppression. Idiopathic CD4+ lymohocytopenia (ICL) is a syndrome defined by the Centre for Disease Control and Prevention as a CD4+ count <300 cells/uL or <20% of total T cell count on >1 occasion, with no evidence of human immunodeficiency virus (HIV) infection, and the absence of other known immunodeficiency or therapy associated with lymphocytopenia. We describe a case of PML occurring in a patient with idiopathic CD4+ lymphocytopenia.     

Human anti-idiotypic T cells induced by TCR peptides corresponding to a common CDR3 sequence motif in myelin basic protein-reactive T cells

T cells recognizing myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In vivo clonal expansion of MBP-reactive T cells in MS may relate in part to dysfunction of peripheral regulatory mechanisms, including the anti-idiotypic immune network. In this study, we examined anti-idiotypic immune responses and the functional properties of anti-idiotypic T cells in patients with MS and healthy controls using TCR peptides corresponding to a CDR3 sequence motif preferentially expressed among T cells recognizing the 83-99 immunodominant peptide of MBP in some patients with MS. The study demonstrated that anti-idiotypic T cells could be induced in vitro by 8mer and 15mer peptides containing the CDR3 motif in MS patients and healthy controls respectively. The estimated precursor frequency of the anti-idiotypic T cells was slightly reduced in MS patients compared to control subjects. The obtained anti-idiotypic T cells recognizing the 15mer TCR peptide were found to express the CD4 phenotype, produce predominantly IL-10 and inhibit the proliferation of autologous T cells recognizing the immunodominant peptide of MBP. Anti-idiotypic T cells induced by the 8mer TCR peptide were predominantly CD8+ cytotoxic T cells and exhibited cytotoxic activity against autologous MBP-specific T cells expressing the CDR3 sequence. When added in primary culture, both TCR peptides had a significant inhibitory effect on the T cell responses to the immunodominant peptide of MBP. The findings suggest that anti-idiotypic immune responses can be activated by selected TCR peptides and may play an important role in the in vivo regulation of MBP-reactive T cells.     

Elevated CD40 ligand expressing blood T-cell levels in multiple sclerosis are reversed by interferon-beta treatment

Myelin protein reactive CD4+ T cells are considered to be involved in the proposed immunopathogenesis of multiple sclerosis (MS). One particularly important molecule for T-cell activation is the CD40L (gp39) that is expressed on the surface of T cells. This study focuses on the CD40 and the CD40L expression on mononuclear cells prepared from blood from patients with MS, other neurological diseases (OND) and healthy subjects. Immunostaining followed by a three channel flow cytometry was adopted. Patients with MS had higher levels of CD3+CD40L+, CD4+CD40L+ and CD8+CD40L+ T cells compared to patients with OND and healthy subjects. Cross-sectional comparisons revealed that the elevation of CD40L+ T cell subtypes was confined to the patients with untreated MS and not observed in the patients with MS treated with interferon-beta (IFN-beta). Follow up studies showed that levels of CD3+CD40L+ and CD4+CD40L+ T cells decreased in individual patients after the initiation of the IFN-beta treatment. The enhanced expression of CD40L on CD3+, CD4+ and CD8+ T cells in patients with MS may implicate a role for this molecule in disease immunopathogenesis.     

Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis

Recent findings in the animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis, implicate a novel CD4+ T-cell subset (TH17), characterized by the secretion of interleukin-17 (IL-17), in disease pathogenesis. To elucidate its role in MS, brain tissues from patients with MS were compared to controls. We detected expression of IL-17 mRNA (by in situ hybridization) and protein (by immunohistochemistry) in perivascular lymphocytes as well as in astrocytes and oligodendrocytes located in the active areas of MS lesions. Further, we found a significant increase in the number of IL-17+ T cells in active rather than inactive areas of MS lesions. Specifically, double immunofluorescence showed that IL-17 immunoreactivity was detected in 79% of T cells in acute lesions, 73% in active areas of chronic active lesions, but in only 17% of those in inactive lesions and 7% in lymph node control tissue. CD8+, as well as CD4+, T cells were equally immunostained for IL-17 in MS tissues. Interestingly, and in contrast to lymph node T cells, no perivascular T cells showed FoxP3 expression, a marker of regulatory T cells, at any stage of MS lesions. These observations suggest an enrichment of both IL-17+CD4+ and CD8+ T cells in active MS lesions as well as an important role for IL-17 in MS pathogenesis, with some remarkable differences from the experimental autoimmune encephalomyelitis model.     

Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

Immunoregulatory T cells of (CD4+)CD25+ phenotype suppress T cell function and protect rodents from organ-specific autoimmune disease. The human counterpart of this subset of T cells expresses high levels of CD25 and its role in human autoimmune disorders is currently under intense investigation. In multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), the activation of circulating self-reactive T cells with specificity for myelin components is considered to be an important disease initiating event. Here, we investigated whether MS is associated with an altered ability of (CD4+)CD25high regulatory T cells (Treg) to confer suppression of myelin-specific immune responses. Whereas Treg frequencies were equally distributed in blood and cerebrospinal fluid of MS patients and did not differ compared to healthy controls, the suppressive potency of patient-derived (CD4+)CD25high T lymphocytes was impaired. Their inhibitory effect on antigen-specific T cell proliferation induced by human recombinant myelin oligodendrocyte protein as well as on immune responses elicited by polyclonal and allogeneic stimuli was significantly reduced compared to healthy individuals. The effect was persistent and not due to responder cell resistance or altered survival of Treg, suggesting that a defective immunoregulation of peripheral T cells mediated by (CD4+)CD25high T lymphocytes promotes CNS autoimmunity in MS.     

Circulating CD3+ CD4+ CD+ T lymphocytes in multiple sclerosis

Triple-antibody flow cytometry was used to search for distinctive populations of peripheral blood lymphocyte immunophenotypes in multiple sclerosis (MS). Using monoclonal antibodies to the cell surface markers CD3, CD4, and CD8, T cell subsets were quantified on a cohort of 31 MS patients (not treated with corticosteroids for at least 6 months), 30 healthy donors, and 14 patients with other autoimmune diseases (also corticosteroid treatment-free for at least 6 months). Untreated MS patients displayed a significantly greater population of CD3+CD4+CD8+ circulating T cells than healthy donors (P = 0.023). Patients with other autoimmune diseases displayed mean populations of CD3+CD4+CD8+ cells greater than normal donors and less than MS, but not significantly different from either. An additional 45 MS patients who had received corticosteroid therapy within the previous 6 months were phenotyped. Treatment of symptomatic MS with corticosteroids was associated with a smaller population of circulating CD3+CD4+CD8+ cells. Some MS patients have significantly greater numbers of peripheral blood T lymphocytes simultaneously expressing CD3, CD4, and CD8 surface markers than healthy donors and this population of cells may be reduced by corticosteroids treatment. This triple positive phenotype may be a manifestation of a systemic immune abnormality in MS.     

Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). I. Cell detachment and lysis induced by encephalitogenic CD4+ T lymphocytes.

Myelin basic protein (MBP) reactive CD4+ T lymphocytes, capable of inducing experimental autoimmune encephalomyelitis (EAE), were examined for their ability to damage target cells of central nervous system (CNS) origin. Damage was assessed by monitoring detachment of adherent astrocytes from substratum and astrocyte lysis. MBP-specific, but non-encephalitogenic CD4+ T cells mediated astrocyte detachment but not lysis. However, encephalitogenic CD4+ T cell lines were more efficient in causing astrocyte detachment and could also cause astrocyte lysis. The detachment and lytic activities of the MBP-reactive T cell lines tested were often independent of the presence of specific antigen, and were not restricted to syngeneic major histocompatibility (MHC) antigens. MBP often augmented the detaching and, if lytic, lytic activities of these T cells. The encephalitogenic CD4+ T cells also detached and lysed allogeneic 'bystander' fibroblasts in the presence of unlabelled syngeneic astrocytes, suggesting the involvement of a soluble mediator(s). Although MBP is essential for the initiation of EAE, the presence of MBP on cells of CNS origin, such as astrocytes and oligodendrocytes, does not appear to be necessary for their damage by MBP-specific CD4+ T cells. Immune CD4+ T cells, which penetrate the CNS, may disorganize brain tissue structure by lysing astrocytes directly and by damaging other brain cells indirectly by soluble mediators. Thus cellular detachment, in addition to cell lysis, mediated by MBP-specific CD4+ cells may contribute to EAE pathogenesis.     

Interleukin-10-modulated immature dendritic cells control the proinflammatory environment in multiple sclerosis

Multiple sclerosis (MS) is a disabling, inflammatory, demyelinating disease of the central nervous system considered to be mediated by autoreactive T cells. Dendritic cells (DC), being professional antigen‐presenting cells, play a pivotal role in the decision between T‐cell activation and anergy. It has been suggested that mature DC (mDC) induce immunity, whereas immature DC (imDC) have the potential to induce tolerance. In this study, we investigated the effects of autologous imDC versus autologous mDC on lymphocytes with respect to the expression of functionally important cell‐surface molecules and production of cytokines. Our aims were to investigate whether the maturation status of DC differs between MS and healthy controls (HC) and to explore whether the effects of DC on T‐cell responses differ between MS and HC. DC were generated from adherent blood mononuclear cells from patients with MS and HC. imDC were obtained by culture with either granulocyte–macrophage colony‐stimulating factor (GM‐CSF) + interleukin‐4 (IL‐4) or GM‐CSF + IL‐4 + IL‐10. mDC were obtained by adding lipopolysaccharide to DC cultures. Upon coculture with autologous lymphocytes, mDC activated the autologous T cells as reflected by increased CD25 and cytotoxic T‐lymphocyte antigen‐4 expression on CD4⁺ T cells together with the increased production of both T helper 1 (Th1) (IL‐2 and interferon‐γ) and Th2 (IL‐10 and IL‐4) cytokines. Unmodulated naïve imDC induced the production of only IL‐4. An exposure of imDC to IL‐10 induced the production of IL‐4 as well as IL‐10 by autologous lymphocytes. We hypothesize that such imDC are important in controlling the proinflammatory environment in vivo in patients with MS.