Yoshihisa Yamano

Increased HTLV-I proviral load and preferential expansion of HTLV-I tax-specific CD8+ T cells in cerebrospinal fluid from patients with HAM/TSP

To date, high human T‐cell lymphotropic virus type I proviral load in patients with human T‐cell lymphotropic virus type I‐associated myelopathy/tropical spastic paraparesis has been reported and is thought to be related to the pathogenesis of human T‐cell lymphotropic virus type I‐associated myelopathy/tropical spastic paraparesis. However, the proviral load in cerebrospinal fluid has not been well investigated. We measured human T‐cell lymphotropic virus type I proviral load in cerebrospinal fluid cells from human T‐cell lymphotropic virus type I‐associated myelopathy/tropical spastic paraparesis patients using real‐time quantitative polymerase chain reaction (TaqMan). Human T‐cell lymphotropic virus type I proviral load in cerebrospinal fluid cells were significantly higher than that of the matched peripheral blood mononuclear cells, and a high ratio of human T‐cell lymphotropic virus type I proviral load in cerebrospinal fluid cells to peripheral blood mononuclear cells were observed in patients with short duration of illness. Human T‐cell lymphotropic virus type I Tax‐specific CD8+ T cells, as detected by peptide‐loaded HLA tetramers, accumulated in cerebrospinal fluid compared with that in peripheral blood mononuclear cells, while the frequency of cytomegalovirus‐specific CD8+ T cells in cerebrospinal fluid was reduced. These observations suggest that accumulation of both human T‐cell lymphotropic virus type I‐infected cells and preferential expansion of human T‐cell lymphotropic virus type I‐specific CD8+ cells in cerebrospinal fluid may play a role in the pathogenesis of human T‐cell lymphotropic virus type I‐associated myelopathy/tropical spastic paraparesis.

Usefulness of proviral load measurement for monitoring of disease activity in individual patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis

High human T-lymphotropic virus type I (HTLV-I) proviral load in peripheral blood mononuclear cells (PBMCs) has been reported in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the proviral load has been reported to fluctuate in individual patients during the course of the disease. Clinical symptoms usually became stable after a prolonged period of symptom progression. However, the authors have experienced having some patients whose clinical manifestations suddenly became worse during the course of the disease. To clarify the role of high proviral load and its fluctuation in the pathogenesis of HAM/TSP, the authors measured the proviral load of serially taken PBMCs as well as of cerebrospinal fluid (CSF) cells from patients with HAM/TSP on long-term follow-up and compared these with their clinical manifestations. There was a wide distribution of proviral load, from 0.3 to 37.8 copies/100 PBMCs; however, the proviral load in individual patients was relatively stable during the course of the disease. Eighty-three percent of the patients with clinical worsening showed an increase in proviral load at the time point when clinical worsening was recorded, or at the preceding time point. The proviral loads in CSF cells were higher than those in PBMCs in individual patients. The ratio of proviral loads in CSF cells/in PBMCs, but not the absolute load, in either compartment, was significantly associated with clinically progressive disease and with recent onset of HAM/TSP. These findings indicate that clinical progression of HAM/TSP is associated with increased proliferation or immigration of HTLV-I-infected lymphocytes in the central nervous system.

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Detection of virus-specific T cells and CD8+ T-cell epitopes by acquisition of peptide-HLA-GFP complexes: analysis of T-cell phenotype and function in chronic viral infections.

Antigen-specific CD8+ T cells acquire peptide–major histocompatibility complex (MHC) clusters through T-cell receptor (TCR)–mediated endocytosis after specific antigen stimulation. We generated an antigen-presenting cell (APC) expressing human leukocyte antigen (HLA)-A*201 coupled to the enhanced green fluorescent protein (GFP), which delivered GFP to an antigen-specific T cell when pulsed with antigenic peptide. We quantitatively identified human T-cell lymphotropic virus type I (HTLV-I) Tax(11–19) peptide–specific T-cell populations in peripheral blood mononuclear cells (PBMCs) from patients with HTLV-I–associated neurologic disease and defined a new CD8+ T-cell epitope in the HTLV-I envelope region. Acquisition of peptide–HLA-GFP complexes by antigen-specific T cells could distinguish, with respect to phenotype and perforin production, T cells from the chronic viral infections cytomegalovirus and HTLV-I. This approach will be a powerful tool in understanding the role of antigen-specific T-cell responses in health and disease.

Human T Lymphotropic Virus Types I and II Western Blot Seroindeterminate Status and Its Association with Exposure to Prototype HTLV-I

Human T lymphotropic virus types I and II (HTLV-I/II) Western blot (WB) seroindeterminate status, which is defined as an incomplete banding pattern of HTLV protein Gag (p19 or p24) or Env (GD21 or rgp46), is commonly observed. To investigate the significance of this finding, we examined HTLV-I/II serostatus and HTLV-I proviral load in 2 groups of individuals with WB seroindeterminate status. Low proviral loads were detected in 42% of patients with neurologic symptoms and 44% of voluntary blood donors. These data suggest that a subset of WB seroindeterminate individuals may be infected with prototype HTLV-I. To confirm this hypothesis, we evaluated HTLV-I/II serostatus and proviral load in prospectively collected specimens from 66 WB seronegative patients who had received HTLV-I-infected blood products by transfusion. Eight individuals developed WB seroindeterminate profiles after the transfusion. In addition, using a human leukocyte antigen type A*201-restricted HTLV-I Tax11-19 tetramer, we detected virus-specific CD8(+) T cells in peripheral blood mononuclear cells from WB seroindeterminate patients. These CD8(+) T cells were effective at targeting HTLV-I-infected cells. Collectively, the results suggest that HTLV-I/II WB seroindeterminate status may reflect a history of HTLV-I exposure. Our findings warrant further investigation of the possible clinical outcomes associated with WB seroindeterminate status.

Human CD4+ T lymphocytes recognize a highly conserved epitope of human T lymphotropic virus type 1 (HTLV‐1) env gp21 restricted by HLA DRB1*0101

HTLV‐1 causes two distinct human diseases, HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukaemia/lymphoma (ATL). Persistently infected individuals carry a risk of < 1% of developing either disease. These basic epidemiological data imply that virus–host interactions, especially immunogenetic factors, influence the outcome of infection. Several studies showed that the HLA class II DR1 DQ5 haplotype is over‐represented in HAM/TSP, but rare in ATL. Therefore, we selected four patients with HAM/TSP and one seronegative control who all carried the HLA DR1 DQ5 haplotype. We analysed the CD4+ T lymphocyte response against eight synthetic peptides of HTLV‐1 envelope (env) glycoprotein gp21, a crucial target antigen in HAM/TSP. The first of two immunodominant epitopes corresponded to a domain of the HTLV‐1 envelope protein which had previously been shown to be essential for HTLV‐1 envelope function. The second immunodominant epitope overlapped a highly conserved sequence of the retroviral transmembrane envelope protein. DR1 (DRB1*0101)‐restricted T lymphocytes were activated by the conserved peptide sequence in nanomolar concentrations. In contrast, this conserved sequence can also induce non‐specific, cAMP‐mediated immunosuppressive effects on T cells when added in micromolar concentrations to culture media, as shown by Haraguchi S, Good RA, James‐Yarish M, Cianciolo GJ, Day NK, Proc Natl Acad Sci USA 1995; 92:5568–71. Hence, HTLV‐1 env gp21 might exert either stimulating immunological or immunosuppressive effects in HTLV‐1‐infected individuals, depending on the level of its expression and the presence of HLA DRB1*0101.

Preferential recognition of synthetic peptides from HTLV-I gp21 envelope protein by HLA-DRB1 alleles associated with HAM/TSP (HTLV-I-associated myelopathy/tropical spastic paraparesis)

To determine CD4+ T-cell epitopes of HTLV-I-envelope protein recognized by the HLA alleles associated with HAM/TSP, we established 20 CD4+ T-cell lines from peripheral blood mononuclear cells (PBMCs) of naive healthy donors using a panel of synthetic peptides spanning the entire length of HTLV-I-envelope proteins, gp46 and gp21. We quantitated the precursor frequencies of HTLV-1-envelope specific CD4+ T-cells and analyzed epitope specificity in the context of HLA alleles. The precursor frequencies ranged from 3.0 to 10.6 per 10(7) PBMCs in the naive healthy donors. The CD4+ T-cell epitopes of HTLV-I-envelope protein were clustered in amino acids 76 to 90, 136 to 160, 171 to 185 and 196 to 210 of gp46, and in amino acids 366 to 400 and 436 to 485 of gp21. The CD4+ T-cell epitopes of gp21 were preferentially recognized by HLA-DRB1 0101 and 1502 which were known to be associated with HAM/TSP. Thus, it was suggested that HTLV-I gp21 might contain the major CD4+ T-cell epitopes recognized by HLA-DRB1 alleles of HAM/TSP.

Natural History of Viral Markers in Children Infected with Human T Lymphotropic Virus Type I in Jamaica

PURPOSE We conducted a longitudinal analysis of human T lymphotropic virus type I (HTLV-I) viral markers in 28 Jamaican mothers and their children, who were monitored for a median of 6.2 years after the birth of the children. METHODS The HTLV-I provirus DNA load was measured using the Taqman system (PE Applied Biosystems). The HTLV-I antibody titer was determined using the Vironstika HTLV-I/II Microelisa System (Organon Teknika). The HTLV-I Tax-specific antibody titers were measured using an enzyme-linked immunosorbent assay. Generalized estimating equations were used to describe the associations of exposure variables with sequentially measured levels of HTLV-I viral markers in children. RESULTS The HTLV-I antibody titer increased significantly up to 1 year after infection, reaching equilibrium at a median titer of 1 : 7,786. The prevalence of Tax-specific antibody reached 80% at 2 years after infection. The provirus load increased up to 2 years after infection, reaching equilibrium at a median of 6,695 copies/100,000 peripheral blood mononuclear cells. The increase in the provirus load was significant only among children with eczema, but not among children without eczema. CONCLUSIONS The provirus loads in children increased for an additional year after their antibody titers had stabilized, possibly as a result of the expansion of HTLV-I-infected clones. This effect was significant only for children with eczema. Among HTLV-I-infected children, eczema may be a cutaneous marker of the risk of HTLV-I-associated diseases developing in adulthood.

In vitro virus propagation and high cellular responsiveness to the infected cells in patients with HTLV-I-associated myelopathy (HAM/TSP)

The reasons for the development of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in certain infected individuals remain poorly understood, but the susceptibility should involve both viral factors and host conditions. To assess simultaneously both virus-induced activation of infected cells and the cellular response to virus producing cells, an analysis of fractionated peripheral blood lymphocytes obtained from patients with HAM/TSP (n = 15) were compared with those of asymptomatic HTLV-I carriers (n = 9) in an age-matched manner. The in vitro propagation of HTLV-I infection was evaluated as the spontaneous thymidine incorporation into CD4+ cells, and proliferative response of CD8+ cells against cultured and irradiated autologous CD4+ cells was employed to analyze the HTLV-I-induced cellular response. The comparative analysis using these two parameters demonstrated that HAM/TSP patients were characterized by the concomitance of a high inducibility of HTLV-I propagation and a high cellular responsiveness against HTLV-I as compared with asymptomatic HTLV-I carriers, suggesting the involvement of both of these factors in disease susceptibility. In addition, the coupled evaluation of these two in vitro phenomena may offer a better diagnostic hallmark for HTLV-I seropositive myelopathy cases with other known cause of myelopathy.

Alteration of cytokine levels by fosfomycin and prednisolone in spontaneous proliferation of cultured lymphocytes from patients with HTLV-I-associated myelopathy (HAM/TSP)

Fosfomycin has recently been reported as an antibiotic with immunomodulatory activities. To evaluate the possibility of clinical administration of fosfomycin in patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), the effects of this agent on the HTLV-I-induced in vitro phenomenon were studied. The influence of fosfomycin on in vitro spontaneous proliferation (SP) of peripheral blood mononuclear cells (PBMCs) from four patients with HAM/TSP was measured by thymidine incorporation into the cells, and the concentration of several cytokines in the culture supernatants was examined in three HAM/TSP patients. Enzyme-linked immunosorbent assays (ELISAs) were employed to detect the concentrations of interleukin-4 (IL-4), IL-6, IL-10, interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and macrophage inflammatory protein-1alpha (MIP-1alpha). The data were compared to the changes by prednisolone which is known to regulate the HTLV-I-associated in vitro phenomenon and to have a therapeutic benefit in patients with HAM/TSP. Production of IL-6, IFN-gamma and MIP-1alpha from the spontaneously proliferating cells were demonstrated. Fosfomycin could not suppress the HTLV-I-associated SP, but had the properties to decrease the levels of TGF-beta1 and MIP-1alpha. It was also demonstrated that the concentrations of IFN-gamma and MIP-1alpha in the cultures in the presence of prednisolone were apparently decreased, suggesting a possible involvement of these cytokines in the pathogenesis of HAM/TSP. These findings support the hypothesis that fosfomycin may have immunomodulatory potentials in HTLV-I-related cellular interactions in a different manner from ordinary immunomodulatory agents.