Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+ T cells in HTLV-1-associated neurologic disease

Despite abundant activated virus-specific cytotoxic T lymphocytes (CTLs), patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) showed a significantly higher frequency of infected T cells than did healthy virus carriers (HVCs). Here, we demonstrate that at a given proviral load, the frequency of CD8(+) T cells that are negative for specific costimulatory molecules was significantly higher in HAM/TSP than in age-matched HVCs and uninfected healthy controls (HCs), whereas the frequency of intracellular perforin-positive CD8(+) T cells was significantly lower in both HAM/TSP and HVCs than in HCs. An inverse correlation between HTLV-1 proviral load (PVL) and percent perforin-positive CD8(+) T cells were observed only in disease-protective allele HLA-A*02-positive HVCs, but not in HAM/TSP patients, whether HLA-A*02 positive or negative, nor in HLA-A*02-negative HVCs. Significantly lower perforin expression was observed in HTLV-1-specific than in cytomegalovirus-specific CD8(+) T cells. Majority of HTLV-1-specific CD8(+) T cells in HVCs showed a CD28(-)CD27(+) phenotype, whereas HAM/TSP showed a CD28(-)CD27(-) phenotype. HTLV-1-specific CD8(+) T cells from HAM/TSP patients showed significantly lower degranulation than HVCs by CD107a mobilization assay. These findings suggest that an impaired function of HTLV-1-specific CTLs is associated with failing antiviral control and disease HAM/TSP.     

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", S?o Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm(3) and 89 (53-196) cells/mm(3) for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.     

Tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM): treatment with an anabolic steroid danazol.

Tropical spastic paraparesis or HTLV-I-associated myelopathy is a progressive spastic disorder associated with the human T-lymphotropic virus type I. Some cases have responded to prednisone. Danazol is an attenuated androgen with minimal virilizing effects. It is used in the treatment of endometriosis and various autoimmune hematologic diseases shown to be responsive to prednisone. Because danazol is anabolic, useful in prednisone-responsive diseases, and less toxic than prednisone, we gave danazol to 6 patients with TSP and 1 with HIV, HTLV-I-associated myelopathy. Five patients had a favorable response. Two became ambulatory after having been confined to a wheelchair. Three were able to ambulate greater distances (in walkers) than prior to danazol. Three had noticeable decreases in spasticity. Urinary incontinence resolved in two. Physical therapy was variably employed in all except one patient. Two patients who had not responded to physical therapy responded to physical therapy and danazol. One patient did not tolerate danazol and one patient did not improve. Toxicities noted were mild elevations in liver enzymes in 4 patients; these responded to a decrease in dose of danazol; amenorrhea in one and mild fluid retention in one. We conclude that danazol is a useful agent in the management of TSP.     

Proviral load and the balance of serum cytocines in HTLV-1-asymptomatic infection and in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

This study compared the proviral load and the plasma cytokine profiles (interleukin-IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ) in 87 HTLV-1-infected individuals, including 28 with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 32 with possible pHAM/TSP and 27 asymptomatic carriers (AC). The control group was composed by 21 HTLV-1-seronegative individuals. Our finding demonstrated that HAM/TSP group presented higher proviral load as compared to all other HTLV-1 groups (p < 0.0001). The HAM/TSP group showed higher serum concentration of IL-6 (p = 0.0009) as compared to all other groups. Moreover, higher serum concentration of IFN-γ (p = 0.0118) and IL-4 (p = 0.0166) were observed in HAM/TSP group as compared to the healthy controls. Additionally, the HAM/TSP group also showed higher serum concentration of TNF-α (p = 0.0239) and IFN-γ (p = 0.0118) as compared to AC. No differences in the serum concentration of IL-2 and IL-10 were observed among the groups. The analysis of cytokine balance demonstrated that HAM/TSP presented higher pro-inflammatory profile with enhanced IFN-γ/IL-10 and IFN-γ/IL-4 ratio as compared to AC and pHAM/TSP. Further analysis pointed out to a positive correlation between the IFN-γ response and the proviral load in AC. Conversely, a negative association between TNF-α and IL-2 with the proviral load was the hallmark of HAM/TSP group. These findings suggested that the proviral load and the pro-inflammatory cytokine profile may be independent events in the peripheral blood of HAM/TSP individuals. The knowledge about the existence of individual virological/immunological behavior upon HTLV-1 infection, may guide to the establishment of more effective therapeutic interventions.     

Bronchopneumonopathy in HTLV-1 associated myelopathy (HAM) and non-HAM HTLV-1 carriers]

Human T-cell leukemia virus type 1 (HTLV-1) causes not only adult T-cell leukemia (ATL), but also HTLV-1 associated myelopathy, a recently described slowly progressive spastic paraparesis, in its carrier state. Pulmonary involvement has been reported in HAM patients. Based on bronchoalveolar lavage or histological examination, the pulmonary involvement has been characterized by accumulation of T-cells, especially activated T-cells, in the lung. We reviewed our data on pulmonary involvement in HAM, which suggested that the characteristic pulmonary involvement observed in HAM was not restricted to HAM patients, but was also observed in non-HAM HTLV-1 carriers. Based on the data, we report that HAM is a systemic disease and that HTLV-1 causes characteristic pulmonary involvement, which we termed HTLV-1 associated bronchopneumonopathy (HAB).     

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.     

Frequent clonal proliferation of human T-cell leukemia virus type 1 (HTLV-1)-infected T cells in HTLV-1-associated myelopathy (HAM-TSP).

Human T-cell leukemia virus type 1 (HTLV-1) integrates its proviruses into random sites in host chromosomal DNA. Random integration of the proviruses was observed in asymptomatic HTLV-1 carriers and patients with HTLV-1-associated myelopathy (HAM/TSP). However, clonal integration has been reported in patients with adult T-cell leukemia (ATL), including that in the smoldering, chronic, and acute states, indicating clonal expansion of infected cells. In this study, we found that about 20% of HAM/TSP patients and their seropositive family members harbored subpopulation(s) of clonally proliferated cells infected with HTLV-1, although they still maintained randomly infected cells as a major population. These clones were stable during examination periods of 4 months to 3 years. However, these carriers or HAM/TSP patients did not show any significant indication of ATL. This extremely high frequency of clonal expansion of HTLV-1-infected cells indicates that some clones of HTLV-1-infected cells have a tendency to proliferate more efficiently than the other population without malignant transformation.     

HIV-1 specific CD8+ T cells with an effector phenotype and control of viral replication

Most people infected with HIV-1 cannot control viral replication despite the presence of virus-specific CD8+ T cells. It has been postulated that this inability is related to the failure of these cells to mature into fully differentiated effector cells. We tested this hypothesis by comparing the maturation phenotype of virus-specific CD8+ T cells in people who could control viral replication off anti-retroviral therapy with those who could not. In five patients with treated acute HIV-1-infection, structured treatment interruption (STI) induced control of viral replication was associated with expansion of virus-specific CD8+ T cells with a fully differentiated effector phenotype. These effector cells were also expanded in treatment-naive chronically infected individuals who spontaneously controlled viral replication, and augmented expression of perforin was noted in both settings. Our data show that full maturation of virus-specific CD8+ T cells is possible in the context of HIV-1-infection, and suggest that such maturation might be important in viral control.     

HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells

Functional and phenotypic characterization of virus-specific CD8 T cells against cytomegalovirus, Epstein-Barr virus, influenza (flu), and HIV-1 were performed on the basis of the ability of CD8 T cells to secrete IFN-gamma and IL-2, to proliferate, and to express CD45RA and CCR7. Two functional distinct populations of CD8 T cells were identified: (i) dual IFN-gamma/IL-2-secreting cells and (ii) single IFN-gamma-secreting cells. Virus-specific IFN-gamma/IL-2-secreting CD8 T cells were CD45RA-CCR7-, whereas single IFN-gamma CD8 T cells were either CD45RA-CCR7- or CD45RA+CCR7-. The proportion of virus-specific IFN-gamma/IL-2-secreting CD8 T cells correlated with that of proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell proliferation. Substantial proliferation of virus-specific CD8 T cells (including HIV-1-specific CD8 T cells) was also observed in CD4 T cell-depleted populations or after stimulation with MHC class I tetramer-peptide complexes. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation. These results indicate that IFN-gamma/IL-2-secreting CD8 T cells may promote antigen-specific proliferation of CD8 T cells even in the absence of helper CD4 T cells.     

Effects of valproate on Tax and HBZ expression in HTLV-1 and HAM/TSP T lymphocytes

A determinant of human T-lymphotropic virus-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is the HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the sense and antisense strands of the pX region, respectively, play key roles in HTLV-1 persistence. Tax drives CD4(+)-T cell clonal expansion and is the immunodominant viral antigen recognized by the immune response. Valproate (2-n-propylpentanoic acid, VPA), a histone deacetylase inhibitor, was thought to trigger Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA on Tax, Gag, and HBZ expressions in cultured lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Approximately one-fifth of provirus-positive CD4(+) T cells spontaneously became Tax-positive, but this fraction rose to two-thirds of Tax-positive-infected cells when cultured with VPA. Valproate enhanced Gag-p19 release. Tax- and Gag-mRNA levels peaked spontaneously, before declining concomitantly to HBZ-mRNA increase. VPA enhanced and prolonged Tax-mRNA expression, whereas it blocked HBZ expression. Our findings suggest that, in addition to modulating Tax expression, another mechanism involving HBZ repression might determine the outcome of VPA treatment on HTLV-1-infected-cell proliferation and survival.     

Common human T cell leukemia virus type 1 (HTLV-1) integration sites in cerebrospinal fluid and blood lymphocytes of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis indicate that HTLV-1 crosses the blood-brain barrier via clonal HTLV-1-infected cells

In the spinal cord of patients with human T cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), infiltrating CD4(+) lymphocytes seem to be the major reservoir for the virus. Little, however, is known about the mechanisms by which HTLV-1 crosses the blood-brain barrier. An oligoclonal proliferation of HTLV-1-infected CD4 lymphoid T cells is present in the peripheral blood of all HTLV-1-infected individuals. Here, such oligoclonal distribution of HTLV-1-infected cells is evidenced in the cerebrospinal fluid (CSF) derived from 5 patients with HAM/TSP. Furthermore, clonal populations of HTLV-1-infected lymphocytes sharing the same HTLV-1 proviral flanking sequences (i.e. , integration sites in the cellular DNA), and thus derived from a single HTLV-1-infected progenitor, were found, for a given patient, in both the CSF and the peripheral blood. These data demonstrate that HTLV-1 crosses the blood-brain barrier by migration of HTLV-1-infected lymphocytes in vivo.     

High frequency of CD4+FoxP3+ cells in HTLV-1 infection: inverse correlation with HTLV-1-specific CTL response

Evidence from population genetics, gene expression microarrays, and assays of ex vivo T-cell function indicates that the cytotoxic T lymphocyte (CTL) response to human T-lymphotropic virus type 1 (HTLV-1) controls the level of HTLV-1 expression and the proviral load. The rate at which CTLs kill autologous HTLV-1–infected lymphocytes differs significantly among infected people, but the reasons for such variation are unknown. Here, we demonstrate a strong negative cor-relation between the frequency of CD4⁺FoxP3⁺ Tax^– regulatory T cells (T_regs) in the circulation and the rate of CTL-mediated lysis of autologous HTLV-1–infected cells ex vivo. We propose that the frequency of CD4⁺FoxP3⁺ Tax^– T_regs is one of the chief determinants of the efficiency of T cell–mediated immune control of HTLV-1.      

Quantitative analysis of Epstein-Barr virus (EBV)-specific CD8+ T cells in patients with chronic active EBV infection

To clarify the pathogenesis of chronic active Epstein-Barr virus (EBV) infection, EBV-specific CD8+ T cells were enumerated, by use of human leukocyte antigen (HLA)-A*2402-restricted tetramers, in 8 patients with chronic active EBV infection, 10 patients with infectious mononucleosis, and 16 EBV-seropositive healthy control subjects. In most of the patients with chronic active EBV infection, EBV-specific CD8+ T cells were not detected. Of note, latent membrane protein 2-specific CD8+ T cells were not detectable in any patients with chronic active EBV infection. In contrast, EBV-specific CD8+ T cells were detected in patients with infectious mononucleosis and in healthy control subjects. Low frequencies of EBV-specific CD8+ T cells may be one of the immunological features of chronic active EBV infection.     

Quiescence and functional reprogramming of Epstein-Barr virus (EBV)-specific CD8+ T cells during persistent infection

After acute infection Epstein-Barr virus (EBV)-specific memory CD8+ T cells exit cell cycle, and a proportion of these antigen-experienced cells re-express CD45RA (CD45 which predominantly express exon A). However, the signals involved are not known. We investigated the roles of interleukin 15 (IL-15) and interferon-alpha/beta (IFN-I) in these processes, since these mediators have a crucial but undefined role in the maintenance of CD8+ T-cell memory. We show that IFN-I (but not IL-15) allows activated EBV-specific CD8+ T cells to leave cell cycle without entering apoptosis. This was associated with up-regulation of the cyclin inhibitor p27, but not of CD45RA. In contrast, IL-15 (but not IFN-I) induced "homeostatic" proliferation and CD45RA re-expression by these cells in vitro. Different signals, therefore, induce quiescence and CD45RA re-expression in activated EBV-specific CD8+ T cells. After T-cell receptor (TCR) activation freshly isolated CD45RA+ antigen-experienced CD8+ T cells show poor proliferative activity but are highly cytotoxic and secrete IFN-gamma efficiently. This suggests functional reprogramming toward effector function but away from proliferation. The induction of quiescence and the generation of proliferation-independent effector CD8+ T cells that re-express CD45RA may minimize the impact of replicative senescence in virus-specific populations that would otherwise occur during decades of persistent infection.     

Helminthic infection down-regulates type 1 immune responses in human T cell lymphotropic virus type 1 (HTLV-1) carriers and is more prevalent in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

Human T cell lymphotropic virus type 1 (HTLV-1) infection is associated with an exacerbated type 1 immune response and secretion of high levels of proinflammatory cytokines. In contrast, helminthic infection induces a type 2 immune response. In the present study, the cytokine profile in HTLV-1 carriers coinfected with helminths (Strongyloides stercoralis and/or Schistosoma mansoni) was compared with that in HTLV-1 carriers not coinfected with helminths. Levels of interferon (IFN)- gamma were higher in HTLV-1 carriers not coinfected with helminths than in HTLV-1 carriers coinfected with helminths (P<.05). The overall frequency of IFN- gamma -expressing CD8+ and CD4+ cells was decreased in HTLV-1 carriers coinfected with helminths (P<.05). The percentage of interleukin (IL)-5- and IL-10-expressing T cells in HTLV-1 carriers coinfected with helminths was higher than that in HTLV-1 carriers not coinfected with helminths (P<.05). Moreover, we found that the prevalence of helminthic infection was 7-fold higher in HTLV-1 carriers than in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (P<.05). These data show that helminthic infection decreases activation of type 1 cells, which may influence the clinical outcome of HTLV-1 infection.