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.

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

Background and objectives: Extracellular 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. Methods: We 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. Results: Significant 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 < 0.0001), CD2+ (p < 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. Discussion: These 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 Neurologic Postacute Sequelae of SARS-CoV-2 Infection.

BACKGROUND AND OBJECTIVES: SARS-CoV-2 infection has been associated with a syndrome of long-term neurologic sequelae that is poorly characterized. We aimed to describe and characterize in-depth features of neurologic postacute sequelae of SARS-CoV-2 infection (neuro-PASC). METHODS: Between October 2020 and April 2021, 12 participants were seen at the NIH Clinical Center under an observational study to characterize ongoing neurologic abnormalities after SARS-CoV-2 infection. Autonomic function and CSF immunophenotypic analysis were compared with healthy volunteers (HVs) without prior SARS-CoV-2 infection tested using the same methodology. RESULTS: Participants were mostly female (83%), with a mean age of 45 ± 11 years. The median time of evaluation was 9 months after COVID-19 (range 3-12 months), and most (11/12, 92%) had a history of only a mild infection. The most common neuro-PASC symptoms were cognitive difficulties and fatigue, and there was evidence for mild cognitive impairment in half of the patients (MoCA score <26). The majority (83%) had a very disabling disease, with Karnofsky Performance Status ≤80. Smell testing demonstrated different degrees of microsmia in 8 participants (66%). Brain MRI scans were normal, except 1 patient with bilateral olfactory bulb hypoplasia that was likely congenital. CSF analysis showed evidence of unique intrathecal oligoclonal bands in 3 cases (25%). Immunophenotyping of CSF compared with HVs showed that patients with neuro-PASC had lower frequencies of effector memory phenotype both for CD4+ T cells (p < 0.0001) and for CD8+ T cells (p = 0.002), an increased frequency of antibody-secreting B cells (p = 0.009), and increased frequency of cells expressing immune checkpoint molecules. On autonomic testing, there was evidence for decreased baroreflex-cardiovagal gain (p = 0.009) and an increased peripheral resistance during tilt-table testing (p < 0.0001) compared with HVs, without excessive plasma catecholamine responses. DISCUSSION: CSF immune dysregulation and neurocirculatory abnormalities after SARS-CoV-2 infection in the setting of disabling neuro-PASC call for further evaluation to confirm these changes and explore immunomodulatory treatments in the context of clinical trials.

Identification and tracking of HTLV-1-infected T cell clones in virus-associated neurologic disease.

Human T lymphotropic virus type 1-assoicated (HTLV-1-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease caused by the persistent proliferation of HTLV-1-infected T cells. Here, we performed a T cell receptor (TCR) repertoire analysis focused on HTLV-1-infected cells to identify and track the infected T cell clones that are preserved in patients with HAM/TSP and migrate to the CNS. TCRß repertoire analysis revealed higher clonal expansion in HTLV-1-infected cells compared with noninfected cells from patients with HAM/TSP and asymptomatic carriers (ACs). TCR clonality in HTLV-1-infected cells was similar in patients with HAM/TSP and ACs. Longitudinal analysis showed that the TCR repertoire signature in HTLV-1-infected cells remained stable, and highly expanded infected clones were preserved within each patient with HAM/TSP over years. Expanded HTLV-1-infected clones revealed different distributions between cerebrospinal fluid (CSF) and peripheral blood and were enriched in the CSF of patients with HAM/TSP. Cluster analysis showed similarity in TCRß sequences in HTLV-1-infected cells, suggesting that they proliferate after common antigen stimulation. Our results indicate that exploring TCR repertoires of HTLV-1-infected cells can elucidate individual clonal dynamics and identify potential pathogenic clones expanded in the CNS.

The repertoire of CSF antiviral antibodies in patients with neuroinflammatory diseases.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although various viruses have been proposed to contribute to MS pathology, the etiology of MS remains unknown. Since intrathecal antibody synthesis is well documented in chronic viral infection and neuroinflammatory diseases, we hypothesized whether the patterns of antigen-specific antibody responses associated with various viral exposures may define patients with CNS chronic immune dysregulation. The pan-viral antibody profiling in cerebrospinal fluid (CSF) and serum of patients with MS showed significant differences from those in healthy volunteers and a pattern of antibody responses against multiple viruses, including the previously identified Epstein-Barr virus. These findings demonstrate that virus-specific antibody signatures might be able to reflect disease-associated inflammatory milieu in CSF of subjects with neuroinflammatory diseases.

T cell receptor repertoire analysis in HTLV-1-associated diseases.

Human T lymphotropic virus 1 (HTLV-1) is a human retrovirus identified as the causative agent in adult T-cell leukemia/lymphoma (ATL) and chronic-progressive neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 is estimated to infect between 5-20 million people worldwide, although most infected individuals remain asymptomatic. HTLV-1 infected persons carry an estimated lifetime risk of approximately 5% of developing ATL, and between 0.25% and 1.8% of developing HAM/TSP. Most HTLV-1 infection is detected in CD4+ T cells in vivo which causes the aggressive malignancy in ATL. In HAM/TSP, the increase of HTLV-1 provirus induces immune dysregulation to alter inflammatory milieu, such as expansion of HTLV-1-specific CD8+ T cells, in the central nervous system of the infected subjects, which have been suggested to underlie the pathogenesis of HAM/TSP. Factors contributing to the conversion from asymptomatic carrier to disease state remain poorly understood. As such, the identification and tracking of HTLV-1-specific T cell biomarkers that may be used to monitor the progression from primary infection to immune dysfunction and disease are of great interest. T cell receptor (TCR) repertoires have been extensively investigated as a mechanism of monitoring adaptive T cell immune response to viruses and tumors. Breakthrough technologies such as single-cell RNA sequencing have increased the specificity with which T cell clones may be characterized and continue to improve our understanding of TCR signatures in viral infection, cancer, and associated treatments. In HTLV-1-associated disease, sequencing of TCR repertoires has been used to reveal repertoire patterns, diversity, and clonal expansions of HTLV-1-specific T cells capable of immune evasion and dysregulation in ATL as well as in HAM/TSP. Conserved sequence analysis has further been used to identify CDR3 motif sequences and exploit disease- or patient-specificity and commonality in HTLV-1-associated disease. In this article we review current research on TCR repertoires and HTLV-1-specific clonotypes in HTLV-1-associated diseases ATL and HAM/TSP and discuss the implications of TCR clonal expansions on HTLV-1-associated disease course and treatments.

Clinical trial of raltegravir, an integrase inhibitor, in HAM/TSP.

OBJECTIVE: Human T-cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive myelopathy. A high proviral load (PVL) is one of the main risk factors for HAM/TSP. Recently, it was shown that raltegravir could inhibit cell-free and cell-to-cell transmission of HTLV-1 in vitro. Given the substantial clinical experience in human immunodeficiency virus infection and its excellent safety profile, this agent may be an attractive therapeutic option for HAM/TSP patients. METHODS: Sixteen subjects with HAM/TSP received raltegravir 400 mg orally twice daily in an initial 6-month treatment phase, followed by a 9-month post-treatment phase. HTLV-1 PVLs were assessed using droplet digital PCR from the PBMCs every 3 months, and from the CSF at baseline, month 6, and month 15. We also evaluated the ability of raltegravir to regulate abnormal immune responses in HAM/TSP patients. RESULTS: While a downward trend was observed in PBMC and/or CSF PVLs of some patients, raltegravir overall did not have any impact on the PVL in this HAM/TSP patient cohort. Clinically, all patients' neurological scores and objective measurements remained relatively stable, with some expected variability. Immunologic studies showed alterations in the immune profiles of a subset of patients including decreased CD4+ CD25+ T cells and spontaneous lymphoproliferation. INTERPRETATION: Raltegravir was generally well tolerated in this HAM/TSP patient cohort. A subset of patients exhibited a mild decrease in PVL as well as variations in their immune profiles after taking raltegravir. These findings suggest that raltegravir may be a therapeutic option in select HAM/TSP patients. CLINICAL TRIAL REGISTRATION NUMBER: NCT01867320.

BK virus-specific T cells for immunotherapy of progressive multifocal leukoencephalopathy: an open-label, single-cohort pilot study.

BACKGROUND: Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. METHODS: We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1 × 106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2 × 106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov, NCT02694783. FINDINGS: Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23-699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. INTERPRETATION: We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. FUNDING: Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.

Effect of Teriflunomide on Cells From Patients With Human T-cell Lymphotropic Virus Type 1-Associated Neurologic Disease.

OBJECTIVE: To test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHODS: PBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 tax and HTLV-1 hbz messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression. RESULTS: In culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 µM (38.3% inhibition), 50 µM (65.8% inhibition), and 100 µM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8+ and CD4+ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or tax mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4+ T cells obtained from a subset of patients with HAM/TSP. CONCLUSIONS: These results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.

Immunopathogenic CSF TCR repertoire signatures in virus-associated neurologic disease.

In this study, we examined and characterized disease-specific TCR signatures in cerebrospinal fluid (CSF) of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). TCR ß libraries using unique molecular identifier-based methodologies were sequenced in paired peripheral blood mononuclear cells (PBMCs) and CSF cells from HAM/TSP patients and normal healthy donors (NDs). The sequence analysis demonstrated that TCR ß repertoires in CSF of HAM/TSP patients were highly expanded and contained both TCR clonotypes shared with PBMCs and uniquely enriched within the CSF. In addition, we analyzed TCR ß repertoires of highly expanded and potentially immunopathologic HTLV-1 Tax11-19-specific CD8+ T cells from PBMCs of HLA-A*0201+ HAM/TSP and identified a conserved motif (PGLAG) in the CDR3 region. Importantly, TCR ß clonotypes of expanded clones in HTLV-1 Tax11-19-specific CD8+ T cells were also expanded and enriched in the CSF of the same patient. These results suggest that exploring TCR repertoires of CSF and antigen-specific T cells may provide a TCR repertoire signature in virus-associated neurologic disorders.

Haploinsufficiency of immune checkpoint receptor CTLA4 induces a distinct neuroinflammatory disorder.

BACKGROUNDCytotoxic T lymphocyte antigen 4 (CTLA4) is essential for immune homeostasis. Genetic mutations causing haploinsufficiency (CTLA4h) lead to a phenotypically heterogenous, immune-mediated disease that can include neuroinflammation. The neurological manifestations of CTLA4h are poorly characterized.METHODSWe performed an observational natural history study of 50 patients with CTLA4h who were followed at the NIH. We analyzed clinical, radiological, immunological, and histopathological data.RESULTSEvidence for neuroinflammation was observed in 32% (n = 16 of 50) of patients in this cohort by magnetic resonance imaging (MRI) and/or by cerebrospinal fluid analysis. Clinical symptoms were commonly absent or mild in severity, with headaches as the leading complaint (n = 13 of 16). The most striking findings were relapsing, large, contrast-enhancing focal lesions in the brain and spinal cord observed on MRI. We detected inflammation in the cerebrospinal fluid and leptomeninges before the parenchyma. Brain biopsies of inflammatory lesions from 10 patients showed perivascular and intraparenchymal mixed cellular infiltrates with little accompanying demyelination or neuronal injury.CONCLUSIONSNeuroinflammation due to CTLA4h is mediated primarily by an infiltrative process with a distinct and striking dissociation between clinical symptoms and radiological findings in the majority of patients.FUNDINGNIAID, NIH, Division of Intramural Research, NINDS, NIH, Division of Intramural Research, and the National Multiple Sclerosis Society-American Brain Foundation.TRIAL REGISTRATIONClinicalTrials.gov NCT00001355.

Immunovirological markers in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Human T cell lymphotropic virus 1 (HTLV-1) is a human retrovirus and infects approximately 10-20 million people worldwide. While the majority of infected people are asymptomatic carriers of HTLV-1, only 4% of infected people develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic, progressive, neurological disease which usually progresses slowly without remission, and is characterized by perivascular inflammatory infiltrates in chronic inflammatory lesions of the central nervous system (CNS), primarily affecting the spinal cord. A high HTLV-1 proviral load, high levels of antibodies against HTLV-1 antigens, and elevated concentration of proteins are detected in cerebrospinal fluid (CSF) of HAM/TSP patients. These chronically activated immune responses against HTLV-1 and infiltration of inflammatory cells including HTLV-1 infected cells into the CNS contribute to clinical disability and underlie the pathogenesis of HAM/TSP. Since the disease development of HAM/TSP mainly occurs in adults, with a mean age at onset of 40-50 years, it is important for HTLV-1-infected carriers and HAM/TSP patients to be monitored throughout the disease process. Recent advances in technologies and findings provide new insights to virological and immunological aspects in both the CNS as well as in peripheral blood. In this review, we focus on understanding the inflammatory milieu in the CNS and discuss the immunopathogenic process in HTLV-1-associated neurologic diseases.

Clinical trial of a humanized anti-IL-2/IL-15 receptor ß chain in HAM/TSP.

OBJECTIVE: Human T cell lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive, neurological disease. Chronic activation of CD8+ T cells, as evidenced by increased spontaneous lymphoproliferation and HTLV-1-specific cytotoxic T cells, has been demonstrated in HAM/TSP patients. Since IL-2 and IL-15 stimulate memory CD8+ T cell activity, these cytokines have been implicated in the immunopathogenesis of HAM/TSP. In this phase I trial, we evaluated the safety, pharmacokinetics, and ability of Hu-Mikß1, a humanized monoclonal antibody directed toward the IL-2/IL-15 receptor ß-chain (IL-2/IL-15Rß: CD122), to saturate CD122 and regulate abnormal immune responses in patients with HAM/TSP by inhibition of IL-15 action. METHODS: Hu-Mikß1 was administered intravenously at doses of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg in a total of nine HAM/TSP patients. Five doses of Hu-Mikß1 were administered at 3-week intervals. The clinical response was evaluated using standardized scales. Viral and immunologic outcome measures were examined including HTLV-1 proviral load, T cell phenotypic analysis and spontaneous lymphoproliferation in HAM/TSP patients. RESULTS: There was no significant toxicity associated with Hu-Mikß1 administration in HAM/TSP patients. Saturation of CD122 by Hu-Mikß1 was achieved in five out of nine HAM/TSP patients. Administration of Hu-Mikß1 was associated with inhibition of aberrant CD8+ T cell function including spontaneous lymphoproliferation and degranulation and IFN-γ expression, especially in HAM/TSP patients that achieved CD122 saturation. INTERPRETATION: The treatment with Hu-Mikß1 had a number of immunological effects on HAM/TSP patients although no clinical efficacy was observed. We also did not see any dose-related toxicity.

Pembrolizumab Treatment for Progressive Multifocal Leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is an opportunistic brain infection that is caused by the JC virus and is typically fatal unless immune function can be restored. Programmed cell death protein 1 (PD-1) is a negative regulator of the immune response that may contribute to impaired viral clearance. Whether PD-1 blockade with pembrolizumab could reinvigorate anti-JC virus immune activity in patients with PML was unknown. METHODS: We administered pembrolizumab at a dose of 2 mg per kilogram of body weight every 4 to 6 weeks to eight adults with PML, each with a different underlying predisposing condition. Each patient received at least one dose but no more than three doses. RESULTS: Pembrolizumab induced down-regulation of PD-1 expression on lymphocytes in peripheral blood and in cerebrospinal fluid (CSF) in all eight patients. Five patients had clinical improvement or stabilization of PML accompanied by a reduction in the JC viral load in the CSF and an increase in in vitro CD4+ and CD8+ anti-JC virus activity. In the other three patients, no meaningful change was observed in the viral load or in the magnitude of antiviral cellular immune response, and there was no clinical improvement. CONCLUSIONS: Our findings are consistent with the hypothesis that in some patients with PML, pembrolizumab reduces JC viral load and increases CD4+ and CD8+ activity against the JC virus; clinical improvement or stabilization occurred in five of the eight patients who received pembrolizumab. Further study of immune checkpoint inhibitors in the treatment of PML is warranted. (Funded by the National Institutes of Health.).

Viral antigens detectable in CSF exosomes from patients with retrovirus associated neurologic disease: functional role of exosomes.

BACKGROUND: HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences. RESULTS: Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12), (Fisher exact test, p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis. CONCLUSION: Cumulatively, these results show that there are HTLV-1 proteins present in exosomes found in virus-free CSF. HAM/TSP PBMCs, particularly CD4+CD25+ T cells, can excrete these exosomes containing HTLV-1 Tax and may be a source of the exosomes found in patient CSF. Importantly, these exosomes are capable of sensitizing an HTLV-1 specific immune response, suggesting that they may play a role in the immunopathology observed in HAM/TSP. Given the infiltration of HTLV-1 Tax-specific CTLs into the CNS of HAM/TSP patients, it is likely that exosomes may also contribute to the continuous activation and inflammation observed in HAM/TSP, and may suggest future targeted therapies in this disorder.

Immunophenotypic characterization of CSF B cells in virus-associated neuroinflammatory diseases.

Intrathecal antibody synthesis is a well-documented phenomenon in infectious neurological diseases as well as in demyelinating diseases, but little is known about the role of B cells in the central nervous systems. We examined B cell and T cell immunophenotypes in CSF of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) compared to healthy normal donors and subjects with the other chronic virus infection and/or neuroinflammatory diseases including HIV infection, multiple sclerosis (MS) and progressive multifocal leukoencephalopathy. Antibody secreting B cells (ASCs) were elevated in HAM/TSP patients, which was significantly correlated with intrathecal HTLV-1-specific antibody responses. High frequency of ASCs was also detected in patients with relapsing-remitting multiple sclerosis (RRMS). While RRMS patients showed significant correlations between ASCs and memory follicular helper CD4+ T cells, CD4+CD25+ T cells were elevated in HAM/TSP patients, which were significantly correlated with ASCs and HTLV-1 proviral load. These results highlight the importance of the B cell compartment and the associated inflammatory milieu in HAM/TSP patients where virus-specific antibody production may be required to control viral persistence and/or may be associated with disease development.

Imaging spinal cord atrophy in progressive myelopathies: HTLV-I-associated neurological disease (HAM/TSP) and multiple sclerosis (MS).

OBJECTIVE: Previous work measures spinal cord thinning in chronic progressive myelopathies, including human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and multiple sclerosis (MS). Quantitative measurements of spinal cord atrophy are important in fully characterizing these and other spinal cord diseases. We aimed to investigate patterns of spinal cord atrophy and correlations with clinical markers. METHODS: Spinal cord cross-sectional area was measured in individuals (24 healthy controls [HCs], 17 asymptomatic carriers of HTLV-1 (AC), 47 HAM/TSP, 74 relapsing-remitting MS [RRMS], 17 secondary progressive MS [SPMS], and 40 primary progressive MS [PPMS]) from C1 to T10. Clinical disability scores, viral markers, and immunological parameters were obtained for patients and correlated with representative spinal cord cross-sectional area regions at the C2 to C3, C4 to C5, and T4 to T9 levels. In 2 HAM/TSP patients, spinal cord cross-sectional area was measured over 3 years. RESULTS: All spinal cord regions are thinner in HAM/TSP (56 mm2 [standard deviation, 10], 59 [10], 23 [5]) than in HC (76 [7], 83 [8], 38 [4]) and AC (71 [7], 78 [9], 36 [7]). SPMS (62 [9], 66 [9], 32 [6]) and PPMS (65 [11], 68 [10], 35 [7]) have thinner cervical cords than HC and RRMS (73 [9], 77 [10], 37 [6]). Clinical disability scores (Expanded Disability Status Scale [p = 0.009] and Instituto de Pesquisas de Cananeia [p = 0.03]) and CD8+ T-cell frequency (p = 0.04) correlate with T4 to T9 spinal cord cross-sectional area in HAM/TSP. Higher cerebrospinal fluid HTLV-1 proviral load (p = 0.01) was associated with thinner spinal cord cross-sectional area. Both HAM/TSP patients followed longitudinally showed thoracic thinning followed by cervical thinning. INTERPRETATION: Group average spinal cord cross-sectional area in HAM/TSP and progressive MS show spinal cord atrophy. We further hypothesize in HAM/TSP that is possible that neuroglial loss from a thoracic inflammatory process results in anterograde and retrograde degeneration of axons, leading to the temporal progression of thoracic to cervical atrophy described here. Ann Neurol 2017;82:719-728.

Dynamic acquisition of HTLV-1 tax protein by mononuclear phagocytes: Role in neurologic disease.

Pathology of HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP) is believed to be the result of "bystander damage" involving effector CD8 (+) T lymphocytes (CTLs) killing of virus infected cells. But the specific cellular events leading up to tissue injury are still unclear. Here, we developed the Microscopy Imaging of Cytotoxic T lymphocyte assay with Fluorescence emission (MI-CaFé), an optimized visualization analysis to explore the interactions between CTLs and virus infected or viral antigen presenting target cells. Various cell-to-cell formations can be observed and our results demonstrate elevated frequencies of CTL-target cell conjugates in HAM/TSP patient PBMCs compared to control PBMCs. Furthermore, HTLV-1 Tax protein expression can be localized at the cell-cell junctions and also tracked moving from an infected cell to a CD14 (+) mononuclear phagocyte (MP). Activation of CD14 (+) MPs in HAM/TSP patient PBMCs and antigenic presentation of HTLV-1 Tax by MPs can be inferred by their spontaneous cytotoxicity after 18h of in vitro culture. Given that CD4 (+) T lymphocytes are the primary reservoirs of HTLV-1 and MPs are scavenger cells responsible for pathogen clearance, spontaneous cytotoxicity against MPs in HAM/TSP PBMCs suggests a mechanism of chronic inflammation, secondary to low level of persistent virus infection within the central nervous system.

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.

Development of neurologic diseases in a patient with primate T lymphotropic virus type 1 (PTLV-1).

BACKGROUND: Virus transmission from various wild and domestic animals contributes to an increased risk of emerging infectious diseases in human populations. HTLV-1 is a human retrovirus associated with acute T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 originated from ancient zoonotic transmission from nonhuman primates, although cases of zoonotic infections continue to occur. Similar to HTLV-1, the simian counterpart, STLV-1, causes chronic infection and leukemia and lymphoma in naturally infected monkeys, and combined are called primate T-lymphotropic viruses (PTLV-1). However, other clinical syndromes typically seen in humans such as a chronic progressive myelopathy have not been observed in nonhuman primates. Little is known about the development of neurologic and inflammatory diseases in human populations infected with STLV-1-like viruses following nonhuman primate exposure. RESULTS: We performed detailed laboratory analyses on an HTLV-1 seropositive patient with typical HAM/TSP who was born in Liberia and now resides in the United States. Using a novel droplet digital PCR for the detection of the HTLV-1 tax gene, the proviral load in PBMC and cerebrospinal fluid cells was 12.98 and 51.68 %, respectively; however, we observed a distinct difference in fluorescence amplitude of the positive droplet population suggesting possible mutations in proviral DNA. A complete PTLV-1 proviral genome was amplified from the patient's PBMC DNA using an overlapping PCR strategy. Phylogenetic analysis of the envelope and LTR sequences showed the virus was highly related to PTLV-1 from sooty mangabey monkeys (smm) and humans exposed via nonhuman primates in West Africa. CONCLUSIONS: These results demonstrate the patient is infected with a simian variant of PTLV-1, suggesting for the first time that PTLV-1smm infection in humans may be associated with a chronic progressive neurologic disease.