Karen Yao

Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I–associated neuroimmunological disease

CD4(+)CD25(+) Tregs are important in the maintenance of immunological self tolerance and in the prevention of autoimmune diseases. As the CD4(+)CD25(+) T cell population in patients with human T cell lymphotropic virus type I-associated (HTLV-I-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) has been shown to be a major reservoir for this virus, it was of interest to determine whether the frequency and function of CD4(+)CD25(+) Tregs in HAM/TSP patients might be affected. In these cells, both mRNA and protein expression of the forkhead transcription factor Foxp3, a specific marker of Tregs, were lower than those in CD4(+)CD25(+) T cells from healthy individuals. The virus-encoded transactivating HTLV-I tax gene was demonstrated to have a direct inhibitory effect on Foxp3 expression and function of CD4(+)CD25(+) T cells. This is the first report to our knowledge demonstrating the role of a specific viral gene product (HTLV-I Tax) on the expression of genes associated with Tregs (in particular, foxp3) resulting in inhibition of Treg function. These results suggest that direct human retroviral infection of CD4(+)CD25(+) T cells may be associated with the pathogenesis of HTLV-I-associated neurologic disease.

Increased Expression of Human T Lymphocyte Virus Type I (HTLV-I) Tax11-19 Peptide–Human Histocompatibility Leukocyte Antigen A*201 Complexes on CD4+ CD25+T Cells Detected by Peptide-specific, Major Histocompatibility Complex–restricted Antibodies in Patients with HTLV-I–associated Neurologic Disease

Human T lymphocyte virus type I (HTLV-I)–associated chronic inflammatory neurological disease (HTLV-I–associated myelopathy/tropical spastic paraparesis [HAM/TSP]) is suggested to be an immunopathologically mediated disorder characterized by large numbers of HTLV-I Tax–specific CD8+ T cells. The frequency of these cells in the peripheral blood and cerebrospinal fluid is proportional to the amount of HTLV-I proviral load and the levels of HTLV-I tax mRNA expression. As the stimulus for these virus-specific T cells are immunodominant peptide–human histocompatibility leukocyte antigen (HLA) complexes expressed on antigen-presenting cells, it was of interest to determine which cells express these complexes and at what frequency. However, until now, it has not been possible to identify and/or quantify these peptide–HLA complexes. Using a recently developed antibody that specifically recognizes Tax11-19 peptide–HLA-A*201 complexes, the level of Tax11-19–HLA-A*201 expression on T cells was demonstrated to be increased in HAM/TSP and correlated with HTLV-I proviral DNA load, HTLV-I tax mRNA load, and HTLV-I Tax–specific CD8+ T cell frequencies. Furthermore, CD4+ CD25+ T cells were demonstrated to be the major reservoir of HTLV-I provirus as well as Tax11-19 peptide–HLA-A*201 complexes. These results indicate that the increased detection and visualization of peptide–HLA complexes in HAM/TSP CD4+ CD25+ T cell subsets that are shown to stimulate and expand HTLV-I Tax–specific CD8+ T cells may play an important role in the pathogenesis of HTLV-I–associated neurological disease.

Foxp3 Represses Retroviral Transcription by Targeting Both NF-κB and CREB Pathways

Forkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells. Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis. Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-κB (NF-κB) activation. Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-κB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells. We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-κB-dependent and NF-κB-independent mechanisms. Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3. Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease. These results suggest an expanded role for Foxp3 in regulating NF-κB- and CREB-dependent cellular and viral gene expression.

Differential tropism of human herpesvirus 6 (HHV-6) variants and induction of latency by HHV-6A in oligodendrocytes

Human herpesvirus 6 (HHV-6) is a ubiquitous β-herpesvirus associated with a number of clinical disorders. Two closely but biologically distinct variants have been described. HHV-6 variant B causes the common childhood disease exhanthem subitum, and although the pathologic characteristics for HHV-6 variant A are less well defined, HHV-6A has been suggested to be more neurotropic. We studied the effect of both HHV-6 variants in an oligodendrocyte cell line (MO3.13). Infection of M03.13 was monitored by cytopathic effect (CPE), quantitative TaqMan PCR for viral DNA in cells and supernatant, reverse transcriptase-polymerase chain reaction (RT-PCR) to detect viral RNA, and indirect immunofluorescence (IFA) to detect viral protein expression. HHV-6A infection induced significantly more CPE than infection with HHV-6B. HHV-6B induced an abortive infection associated with a decrease of the initial viral DNA load over time, early RNA expression, and no expression of viral antigen. In contrast, infection with HHV-6A DNA persisted in cells for at least 62 days. During the acute phase of infection with HHV-6A, intracellular and extracellular viral load increased and cells expressed the viral protein IE-2 and gp116/54/64. No HHV-6A RNA or protein was expressed after 30 days post infection, suggesting that HHV-6A formed a latent infection. These studies provide in vitro support to the hypothesis that HHV-6 can actively infect oligodendrocytes. Our results suggest that HHV-6A and HHV-6B have different tropism in MO3.13 cells and that an initially active HHV-6A infection can develop latency. Differences between HHV-6A and -6B infection in different neural cell types may be associated with different neurological diseases.

Detection of human herpesvirus-6 in cerebrospinal fluid of patients with encephalitis.

Virus infections are the most common causes of encephalitis, a syndrome characterized by acute inflammation of the brain. More than 150 different viruses have been implicated in the pathogenesis of encephalitis; however, because of limitations with diagnostic testing, causative factors of more than half of the cases remain unknown.

Review part 2: Human herpesvirus‐6 in central nervous system diseases

J. Med. Virol. 82:1669–1678, 2010.

Human herpesvirus-6 entry into the central nervous system through the olfactory pathway

Viruses have been implicated in the development of neurodegenerative diseases, such as Alzheimers, Parkinson’s, and multiple sclerosis. Human herpesvirus-6 (HHV-6) is a neurotropic virus that has been associated with a wide variety of neurologic disorders, including encephalitis, mesial temporal lobe epilepsy, and multiple sclerosis. Currently, the route of HHV-6 entry into the CNS is unknown. Using autopsy specimens, we found that the frequency of HHV-6 DNA in the olfactory bulb/tract region was among the highest in the brain regions examined. Given this finding, we investigated whether HHV-6 may infect the CNS via the olfactory pathway. HHV-6 DNA was detected in a total of 52 of 126 (41.3%) nasal mucous samples, showing the nasal cavity is a reservoir for HHV-6. Furthermore, specialized olfactory-ensheathing glial cells located in the nasal cavity were demonstrated to support HHV-6 replication in vitro. Collectively, these results support HHV-6 utilization of the olfactory pathway as a route of entry into the CNS.

Reactivation of human herpesvirus-6 in natalizumab treated multiple sclerosis patients.

The α4 integrin antagonist natalizumab was shown to be effective in patients with immune-mediated disorders but was unexpectedly associated with JC polyomavirus associated progressive multifocal leukoencephalopathy (PML) in two multiple sclerosis (MS) and one Crohns disease patients. Impaired immune surveillance due to natalizumab treatment may have contributed to the JCV reactivation. As HHV-6 has been suggested to play a role in MS, we asked whether this virus could also have been reactivated during natalizumab therapy. Matched sera and CSF from a limited set of MS patients treated with and without natalizumab were examined for evidence of HHV-6. In addition, we also superinfected a persistent JC virus infected glial cell with HHV-6A to determine if JC virus can be increased. Elevated serum HHV6 IgG and HHV-6A DNA was detected in the CSF of a subset of patients but not controls. We confirmed that superinfection with HHV-6 of a JC virus infected glial cells increased expression of JCV. These results support the hypothesis that treatment with natalizumab may be associated with reduced immune surveillance resulting in reactivation of viruses associated with MS pathogenesis.

GLUT1 Is Not the Primary Binding Receptor but Is Associated with Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1

ABSTRACT GLUT1 has recently been suggested to be a binding receptor for human T-cell leukemia virus type 1 (HTLV-1). We used a novel, short-term assay to define the role of GLUT1 in cell-to-cell transmission. Although increasing cell surface levels of GLUT1 enhanced HTLV-I transfer, efficient virus spread correlated largely with heparan sulfate proteoglycan (HSPG) expression on target cells. Moreover, since activated CD4+ T cells and cord blood lymphocytes that are susceptible to HTLV-1 infection expressed undetectable levels of surface GLUT1, these results indicate that GLUT1 and HSPGs are important for efficient cell-to-cell transmission of HTLV-1 but raise concerns on the role of GLUT1 as the HTLV-1 primary binding receptor.

Anti-HTLV antibody profiling reveals an antibody signature for HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP)

BackgroundHTLV-I is the causal agent of adult T cell leukemia (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Biomarkers are needed to diagnose and/or predict patients who are at risk for HAM/TSP or ATLL. Therefore, we investigated using luciferase immunoprecipitation technology (LIPS) antibody responses to seven HTLV-I proteins in non-infected controls, asymptomatic HTLV-I-carriers, ATLL and HAM/TSP sera samples. Antibody profiles were correlated with viral load and examined in longitudinal samples.ResultsAnti-GAG antibody titers detected by LIPS differentiated HTLV-infected subjects from uninfected controls with 100% sensitivity and 100% specificity, but did not differ between HTLV-I infected subgroups. However, anti-Env antibody titers were over 4-fold higher in HAM/TSP compared to both asymptomatic HTLV-I (P < 0.0001) and ATLL patients (P < 0.0005). Anti-Env antibody titers above 100,000 LU had 75% positive predictive value and 79% negative predictive value for identifying the HAM/TSP sub-type. Anti-Tax antibody titers were also higher (P < 0.0005) in the HAM/TSP compared to the asymptomatic HTLV-I carriers. Proviral load correlated with anti-Env antibodies in asymptomatic carriers (R = 0.76), but not in HAM/TSP.ConclusionThese studies indicate that anti-HTLV-I antibody responses detected by LIPS are useful for diagnosis and suggest that elevated anti-Env antibodies are a common feature found in HAM/TSP patients.