Degenerate specificity of HTLV-1-specific CD8+ T cells during viral replication in patients with HTLV-1-associated myelopathy (HAM/TSP)

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-1 infection, in which HTLV-1-infected CD4(+) T cells and HTLV-1-specific CD8(+) T cells may play a role in the disease pathogenesis. Patients with HAM/TSP have high proviral loads despite vigorous virus-specific CD8(+) T-cell responses; however, it is unknown whether the T cells are efficient in eliminating the virus in vivo. To define the dynamics of HTLV-1-specific CD8(+) T-cell responses, we investigated longitudinal alterations in HTLV-1 proviral load, amino acid changes in an immunodominant viral epitope, frequency of HTLV-1-specific T cells, and degeneracy of T-cell recognition in patients with HAM/TSP. We showed that the frequency and the degeneracy of the HTLV-1-specific CD8(+) T cells correlated well with proviral load in the longitudinal study. The proviral load was much higher in a patient with low degeneracy of HTLV-1-specific T cells compared to that in a patient with comparable frequency but higher degeneracy of the T cells. Furthermore, in a larger number of patients divided into 2 groups by the proviral load, those with high proviral load had lower degeneracy of T-cell recognition than those with low proviral load. Sequencing analysis revealed that epitope mutations were remarkably increased in a patient when the frequency and the degeneracy were at the lowest. These data suggest that HTLV-1-specific CD8(+) T cells with degenerate specificity are increased during viral replication and control the viral infection.     

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.     

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.     

Priming of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cell responses by dendritic cells loaded with HIV-1 proteins

Proteins may serve as ideal CD8(+) T cell immunogens for human immunodeficiency virus type 1 (HIV-1) if they can be delivered to and processed through the human leukocyte antigen class I pathway. This study shows that human blood monocyte-derived dendritic cells loaded with liposome-complexed HIV-1 proteins and matured with CD40 ligand can prime CD8(+) T cells to HIV-1 in vitro. Whole HIV-1 protein in liposome may be an effective immunogen for HIV-1 vaccine protocols.     

Human T cell lymphotropic virus (HTLV) type-1-specific CD8+ T cells: frequency and immunodominance hierarchy

Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We used interferon- gamma enzyme-linked immunospot assays with overlapping peptides spanning the entire HTLV-1 proteome to test whether the HTLV-1-specific CD8(+) T cells differed significantly in frequency or immunodominance hierarchy between patients with HAM/TSP and asymptomatic carriers and whether the frequency correlated with provirus load. Tax was the immunodominant target antigen. There was no significant qualitative or quantitative difference in the HTLV-1-specific CD8(+) T cell response between the 2 groups. Virus-specific CD8(+) T cell frequency alone does not indicate the effectiveness of the cytotoxic T lymphocyte response in controlling provirus load at equilibrium.     

Divergent strains of human T-lymphotropic virus type 1 (HTLV-1) within the Cosmopolitan subtype in Argentina

HTLV-1 Cosmopolitan subtype Transcontinental subgroup A has been described among aboriginal communities from the northwest endemic area of Argentina. Moreover, Transcontinental subgroup A and the Japanese subgroup B were reported among blood donors from the nonendemic central region of the country. We carried out the first HTLV-1 phylogenetic study in individuals residing in Buenos Aires capital city. Phylogenetic analysis performed on the LTR region showed that all 44 new strains clustered within the Cosmopolitan subtype, with 42 (95.4%) belonging to Transcontinental subgroup A. Of them, 20 (45.5%) strains grouped in the large Latin American cluster and 4 (9.1%) in the small Latin American cluster. The majority of them belonged to individuals of nonblack origin, grouped with Amerindian strains. Three (6.8%) were closely related to South African references and two monophyletic clusters including only HIV/HTLV-1 coinfected individuals were observed. Interestingly, two (4.5%) new sequences (divergent strains) branched off from all five known Cosmopolitan subgroups in a well-supported clade. In summary, these findings show that HTLV-1 Cosmopolitan subtype Transcontinental subgroup A is infecting residents of Buenos Aires, a nonendemic area of Argentina, and confirm the introduction of divergent strains in the country.     

Impaired response of HIV type 1-specific CD8(+) cells from antiretroviral-treated patients

Impairment of the response of HIV-specific CD8(+) T cells, in spite of the high frequency of occurrence of these cells even in the advanced phase of HIV-1 infection, has been demonstrated. It is also known that new antiretroviral treatments are able to reduce the viral load and partially repair the immunological damage caused by HIV-1, but it is not clear whether the extent of these changes affects the functional profile of HIV-specific CD8(+) T cells. We evaluated, in HIV-1(+) patients undergoing antiretroviral therapy, the HIV-specific CD8(+) subset distribution and their functional capacity as intracellular expression of IFN-gamma, TNF-alpha, and perforin after PMA stimulation. Our results indicate that HIV-1(+)-treated individuals show distributions of HIV-specific CD8 subsets similar to nontreated patients, while the frequency of HIV-specific CD8 cells expressing IFN-gamma and perforin after stimulation is lower in HAART-treated patients. This indicates that HAART, which controls viral replication, may impair the HIV-specific CD8(+) response.     

In vitro spontaneous lymphoproliferation in patients with human T-cell lymphotropic virus type I-associated neurologic disease: predominant expansion of CD8+ T cells

Peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) proliferate spontaneously in vitro. This spontaneous lymphoproliferation (SP) is one of the immunologic hallmarks of HAM/TSP and is considered to be an important factor related to the pathogenesis of HAM/TSP. However, the cell populations involved in this phenomenon have not yet been definitively identified. To address this issue, the study directly evaluated proliferating cell subsets in SP with a flow cytometric method using bromodeoxyuridine and Ki-67. Although both CD4+ and CD8+ T cells proliferated spontaneously, the percentage of proliferating CD8+ T cells was 2 to 5 times higher than that of CD4+ T cells. In addition, more than 40% of HTLV-I Tax11-19-specific CD8+ T cells as detected by an HLA-A*0201/Tax11-19 tetramer proliferated in culture. In spite of this expansion of HTLV-I-specific CD8+ T cells, HTLV-I proviral load did not decrease. This finding will help elucidate the dynamics of in vivo virus-host immunologic interactions that permit the coexistence of high HTLV-I-specific CD8+ cytotoxic T-lymphocyte responses and high HTLV-I proviral load in HAM/TSP.     

Antibody to the human T-lymphotropic virus type 1 (HTLV-1) envelope protein Gp46 in patients co-infected with HCV and HTLV-1

Human T-lymphotropic virus type 1 (HTLV-1) infection is known to affect hepatitis C virus (HCV) clearance and to accelerate the development of hepatocellular carcinoma in HCV-infected patients. In this study, we found the prevalence and titer of an antibody recognizing the central region of the HTLV-1 Gp46 protein to be associated with the severity of chronic liver disease. The antibody prevalence was significantly correlated with the stage of chronic liver disease (P < 0.0001): 3 (14.3%) of 21 patients with minimal-mild chronic hepatitis, 12 (24%) of 50 with moderate-severe chronic hepatitis, 7 (87.5%) of 8 with liver cirrhosis, and 13 (100%) of 13 with hepatocellular carcinoma. These results indicate that the antibody may be a useful marker of the deterioration of liver disease in patients co-infected with HCV and HTLV-1. This antibody may be useful for the diagnosis of liver diseases and the development of more effective treatments.     

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.     

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.     

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.     

Multicentric Castleman disease is associated with polyfunctional effector memory HHV-8-specific CD8+ T cells

Multicentric Castleman disease (MCD) is a devastating human herpesvirus 8 (HHV-8)–related lymphoproliferative disorder that occurs in immunocompromised persons. To determine the role of immune responses in MCD, we studied the frequency, antigenic repertoire, differentiation, and functional profile of HHV-8–specific CD8⁺ T cells in MCD patients and in human immunodeficiency virus–coinfected asymptomatic HHV-8 carriers (AC). Screening CD8⁺ T-cell responses with ELISpot interferon- (IFN-) assays using 56 peptides on 6 latent and lytic HHV-8 proteins showed that MCD and AC patients had responses of similar magnitude and antigenic repertoire and identified a new 10-mer human leukocyte antigen B7 CD8 epitope in K15. Intracellular IFN- staining showed significantly more CD45RA^–CCR7^–CD27^– CD8⁺IFN-⁺ cells (late phenotype) and significantly fewer CCR7^–CD27⁺CD45RA^– cells (early and intermediate phenotype) in MCD than in AC patients. This phenotypic shift was not found for Epstein-Barr virus–specific CD8⁺ T cells tested as controls. HHV-8 viral loads were negatively correlated with early and intermediate effector memory cells. HHV-8–specific T cells were polyfunctional (secretion of IFN-, tumor necrosis factor-, macrophage inflammatory protein-1β, and/or CD107a) in both MCD and AC patients. In conclusion, MCD is not associated with a lack of HHV-8–specific CD8⁺ T cells or limitation of their functional profile. Their differentiation increases with HHV-8 viral load. These results offer new insight into the pathophysiology of MCD.     

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.     

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.