High Expression of CD244 and SAP Regulated CD8+ T Cell Responses of Patients with HTLV-I Associated Neurologic Disease

HTLV-I-specific CD8+ T cells have been characterized with high frequencies in peripheral blood and cerebrospinal fluid and production of proinflammatory cytokines, which contribute to central nervous system inflammation in HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, little is known about the differences in CD8+ T cell activation status between asymptomatic carrier (ACs) and patients with HAM/TSP. The expression of CD244, a signaling lymphocyte activation molecule (SLAM) family receptor, was significantly higher on CD8+ T cells in HTLV-I-infected patients, both ACs and patients with HAM/TSP, than those on healthy normal donors (NDs). Blockade of CD244 inhibited degranulation and IFN-γ production in CD8+ T cells of patients with HAM/TSP, suggesting that CD244 is associated with effector functions of CD8+ T cells in patients with HAM/TSP. Moreover, SLAM-associated protein (SAP) was overexpressed in patients with HAM/TSP compared to ACs and NDs. SAP expression in Tax-specific CTLs was correlated in the HTLV-I proviral DNA loads and the frequency of the cells in HTLV-I-infected patients. SAP knockdown by siRNA also inhibited IFN-γ production in CD8+ T cells of patients with HAM/TSP. Thus, the CD244/SAP pathway was involved in the active regulation of CD8+ T cells of patients with HAM/TSP, and may play roles in promoting inflammatory neurological disease

Minocycline modulates antigen-specific CTL activity through inactivation of mononuclear phagocytes in patients with HTLV-I associated neurologic disease

<p>Abstract</p> <p>Background</p> <p>The activation of mononuclear phagocytes (MPs), including monocytes, macrophages and dendritic cells, contributes to central nervous system inflammation in various neurological diseases. In HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), MPs are reservoirs of HTLV-I, and induce proinflammatory cytokines and excess T cell responses. The virus-infected or activated MPs may play a role in immuneregulation and disease progression in patients with HTLV-I-associated neurological diseases.</p> <p>Results</p> <p>Phenotypic analysis of CD14⁺monocytes in HAM/TSP patients demonstrated high expression of CX₃CR1 and HLA-DR in CD14^lowCD16⁺monocytes, compared to healthy normal donors (NDs) and asymptomatic carriers (ACs), and the production of TNF-α and IL-1β in cultured CD14⁺cells of HAM/TSP patients. CD14⁺cells of HAM/TSP patients also showed acceleration of HTLV-I Tax expression in CD4⁺T cells. Minocycline, an inhibitor of activated MPs, decreased TNF-α expression in CD14⁺cells and IL-1β release in PBMCs of HAM/TSP patients. Minocycline significantly inhibited spontaneous lymphoproliferation and degranulation/IFN-γ expression in CD8⁺T cells of HAM/TSP patients. Treatment of minocycline also inhibited IFN-γ expression in CD8⁺T cells of HAM/TSP patients after Tax11-19 stimulation and downregulated MHC class I expression in CD14⁺cells.</p> <p>Conclusion</p> <p>These results demonstrate that minocycline directly inhibits the activated MPs and that the downregulation of MP function can modulate CD8⁺T cells function in HAM/TSP patients. It is suggested that activated MPs may be a therapeutic target for clinical intervention in HAM/TSP.</p

Viral Immune signatures from cerebrospinal fluid extracellular vesicles and particles in HAM and other chronic neurological diseases

Background and objectivesExtracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease.MethodsWe analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease.ResultsSignificant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p &lt; 0.0001), CD2+ (p &lt; 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without.DiscussionThese data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals

Deep Phenotyping of Post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention

Role of HTLV-1 Tax and HBZ in the Pathogenesis of HAM/TSP

Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Understanding the interaction between host and HTLV-1 and the molecular mechanisms associated with disease pathogenesis is critical for development efficient therapies. Two HTLV-1 genes, tax and HTLV-1 basic leucine zipper factor (HBZ), have been demonstrated to play important roles in HTLV-1 infectivity and the growth and survival of leukemic cells. Increased HTLV-1 Tax expression induces the expression of various cellular genes such as IL-2 and IL-15, which directly contributes to lymphocyte activation and immunopathogenesis in HAM/TSP patients. However, little is known about the molecular and cellular mechanism of HBZ in development of HAM/TSP. It has been reported that HBZ mRNA expression was detected in HAM/TSP patients higher than in asymptomatic carriers and correlated with proviral load and disease severity. Unlike HTLV-1 tax, HBZ escapes efficient anti-viral immune responses and therefore these reactivities are difficult to detect. Thus, it is important to focus on understanding the function and the role of HTLV-1 tax and HBZ in disease development of HAM/TSP and discuss the potential use of these HTLV-1 viral gene products as biomarkers and therapeutic targets for HAM/TSP

Expression of SLAM-associated proteins in CD8⁺ T cells of HTLV-I-infected patients.

<p>(A) Representative histograms of SAP expression in CD8⁺ T cells of a ND (opened histogram), an AC (grayed histogram), and a patient with HAM/TSP (closed histogram). (B) Comparison of SAP expression in CD8⁺ T cells of NDs (n = 11), ACs (n = 8) and patients with HAM/TSP (n = 10). The horizontal line represents the median. (C) Comparison of EAT-2 expression in CD8⁺ T cells of NDs (n = 10), ACs (n = 5) and patients with HAM/TSP (n = 10). (D) SAP expression in Tax11-19-specific CTLs of HTLV-I-infected patients. SAP expressions were shown in Tax11-19-specific CD8⁺ T cells of AC and patient with HAM/TSP. (E) Correlation of the HTLV-I-proviral DNA loads (circles; R² = 0.4746, P = 0.0401) and the frequency of Tax11-19-specific CD8⁺ T cells (squares R² = 0.6811, P = 0.0062) with SAP expression in the cells of AC (n = 4, opened circles and squares) and patients with HAM/TSP (n = 5, closed circles and squares).</p

SAP and CD244 expressions in CD8⁺ T cells of NDs after stimulation with IL-2 or IL-15.

<p>(A) SAP expressions in CD8⁺ T cells isolated from ND PBMCs were compared after the culture with rhIL-2 (opened circle) or rhIL-15 (closed circle) for 7 days. The graphs were prepared from data obtained from two NDs (left and right). (B) CD244 expressions in CD8⁺ T cells isolated from ND PBMCs were compared after the culture with rhIL-2 (opened circle) or rhIL-15 (closed circle) for 7 days. The graphs were prepared from data obtained from two NDs (left and right).</p

Inhibition of degranulation and IFN-γ expression in CD8⁺ T cells of patients with HAM/TSP by SAP siRNA.

<p>(A) SAP and EAT-2 expression in transfected CD8⁺ T cells with either control, SAP or EAT-2 siRNA was determined after the culture for 6 hours. The cell lysates were prepared from transfected CD8⁺ T cells, and each 10µg of the cell lysates was loaded on the gel. (B) Inhibitory effects of SAP or EAT-2 siRNA on degranulation and IFN-γ expression in transfected CD8⁺ T cells. Isolated CD8⁺ T cells from patients with HAM/TSP were transfected with control, SAP or EAT-2 siRNA, and then cocultured with autologous CD14⁺ cells. The amounts of CD107a/IFN-γ expressions in CD8⁺ T cells transfected with control siRNA were normalized to 100%, and then, those in CD8⁺ T cells transfected with SAP or EAT-2 siRNA were calculated. The graph was prepared from data obtained from three patients with HAM/TSP. Error bars represent SD.</p

Involvement of CD244 in CD8⁺ T cell degranulation and IFN-γ expression of patients with HAM/TSP.

<p>(A) Representative dot plot of spontaneous degranulation and IFN-γ expression in CD8⁺ T cells of a ND and a patient with HAM/TSP after culture for 24 hours. The PBMCs were cultured for 24 hours without any exogenous stimulators. (B) Dose-dependent inhibition of spontaneous degranulation and IFN-γ expression in CD8⁺ T cells of a patient with HAM/TSP by anti-CD244 (closed circle) and anti-CD48 (opened circle). The PBMCs were cultured with each antibody for 24 hours. (C) Inhibitory effects of anti-CD244 and anti-CD48 on degranulation and IFN-γ production in CD8⁺ T cells of patients with HAM/TSP. The PBMCs were cultured with 1 µg/ml of control IgG, anti-CD244 or anti-CD48 for 24 hours. The amount of CD107a/IFN-γ expressions of CD8⁺ T cells cultured with control IgG were normalized to 100%, and then, those in PBMCs cultured with each antibody were calculated. The graph was prepared from data obtained from 7 patients with HAM/TSP. Error bars represent SD. (D) CD48 and Tax expression in HTLV-I-infected cells of a patient with HAM/TSP. The top panels show CD48 and Tax expression in CD4⁺ T cells before (left) and after (right) the culture for 20 hours. The bottom panels show CD48 and Tax expression in CD14⁺ cells before (left) and after (right) the culture for 20 hours.</p

Distribution of CD244 in PBMCs of patients with HAM/TSP.

<p>After the culture for 8 hours, PBMCs were stained with antibodies against perforin, CD244 and CD48, and visualized through microscope. Two representative 3D images were shown in A and B. The image shows DAPI (blue), perforin (green), CD244 (orange) and CD48 (purple). In addition to DAPI and perforin (left), CD244 (middle) and CD48 (right) are merged. The white arrows indicate CD244 clustering at the cell contact area.</p