Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens

Five T-cell clones reactive to autologous HTLV-I-infected T-cells (KODA-TV) were established from peripheral blood lymphocytes of a HAM/TSP patient (KODA) by the limiting dilution method. All the clones showed CD3⁺, CD4⁺ and CD25⁺ surface markers and expressed αβ⁺ T-cell receptors to recognize KODA-TV antigens. One of the five T-cell clones (KODA-408) was infected with HTLV-I but the remaining four clones (KODA-400, 404, 405 and 409) were free of HTLV-I infection. KODA-408 recognized both KODA-TV and spinal cord antigens, the latter being extracted from autopsy tissues of a HTLV-I seronegative donor. KODA-408 did not recognize either alloantigens of peripheral blood mononuclear cells extracted from unrelated HTLV-I seronegative donors or purified human myelin basic protein. KODA-408 T-cell clone produced a considerable amount of TNF-aβ, IFN-γ, and IL-6. The CDR3 motif of KODA-408 T-cell receptor showed a unique sequence CASSAGQS of Vβ8-Dβ-Jβ1.5. These results indicated that HAM/TSP CD4⁺ T-cells were polyclonally activated by HTLV-I infection and antigenic stimulation. The T-cell repertoire shaped by HTLV-I infection included T-cells which recognized HTLV-I-infected T-cell antigens as well as spinal cord antigen in particular.     

Cellular immune surveillance against HTLV-I infected T lymphocytes in HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP).

To investigate the cellular immune surveillance against HTLV-I infected T lymphocytes in HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP), we studied the cytotoxic T lymphocytes (CTL) activity against an HTLV-I infected human T cell line (MT-2) and the natural killer (NK) cell activity in 15 HAM patients, 6 HTLV-I carriers, and 15 controls. The activity of CTL against MT-2 cells was found to be significantly elevated in HAM compared with that in the controls. This cytotoxicity in HAM was higher than in HTLV-I carriers, although the difference was not statistically significant. There was an HLA class I restriction in this CTL activity against MT-2 cells in HAM. On the other hand, NK cell activity was significantly lower in HAM than in controls. Cold target inhibition studies suggested that NK cells could not lyse MT-2 cells effectively. There was a positive correlation between the CTL activity against MT-2 cells and the serum antibody titers to HTLV-I in HAM.     

Reduced Foxp3 expression with increased cytomegalovirus-specific CTL in HTLV-I-associated myelopathy

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients show high immune responses to HTLV-I. However, it is unclear whether the cytotoxic T lymphocyte (CTL) responses to other chronic viruses also increase. We investigated the responses in the peripheral blood by using HLA-A*0201/peptide pentamers. The frequency of cytomegalovirus (CMV)-specific CTL tended to be higher in HAM/TSP patients than in healthy controls (HCs). The frequency of CMV-specific CTL positively correlated with that of HTLV-I Tax-specific CTL. The frequency of Foxp3+ cells in CD4+ lymphocytes tended to be higher in HAM/TSP patients than in ACs and HCs. The expression level of Foxp3 was lower in HAM/TSP patients than in HCs and was inversely correlated with the CMV-specific CTL frequency. A percentage of Foxp3+ cells showed a positive correlation with the HTLV-I proviral load. These results suggest that a decrease in the Foxp3 expression may contribute to the high immune response to CMV and that the Foxp3+ regulatory T cells may play a role in the immune surveillance of HTLV-I.     

The effect of TCR Vβ8 peptide protection and therapy on T cell populations isolated from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Vaccination or treatment of Lewis rats with TCR Vβ8 peptides can prevent or reverse the clinical signs of experimental autoimmune encephalomyelitis (EAE) which is mediated predominantly by Vβ8.2⁺ CD4⁺/CD45R lo T cells. However, rats protected or treated with Vβ8 peptides still developed histological lesions in the spinal cord (SC), even though they remained clinical well. We sought to discern phenotypic changes characteristic of these SC infiltrating lymphocytes. In particular, we focused on whether the immunoregulatory mechanism induced by TCR peptides caused a reduction of Vβ8.2⁺ T cells, or induced changes in CD45R lo or hi/CD4⁺ subpopulations that have been associated respectively with EAE induction or recovery. In the Vβ8 peptide vaccinated rats there was a dramatic decrease in the number of Vβ8.2⁺ T cells isolated from the SC early in disease. During the recovery phase, however, the number of Vβ8.2⁺ SC T cells was similar in protected and control groups; in contrast, there was a striking reduction in the number and size of CD45R hi/CD4⁺ T cells in the protected animals. In rats treated with Vβ8.2 peptide, no changes were observed in the number of SC Vβ8.2⁺ T cells or expression of Vβ8.2 message, but similar to vaccinated rats, there was a marked decrease in the number of CD45R hi/CD4⁺ T cells. These data suggest that vaccination with TCR peptides prevented the initial influx of encephalitogenic Vβ8.2⁺ T cells into the central nervous system (CNS), whereas treatment appeared to inactivate Vβ8.2⁺ T cells already present in the CNS. In both cases, TCR peptide-induced inhibition of the encephalitogenic T cells apparently preempted the need for CD45R hi/CD4⁺ T cells that may normally be necessary to resolve the disease.     

Selected cytotoxic T lymphocytes with high specificity for HTLV-I in cerebrospinal fluid from a HAM/TSP patient

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the spinal cord in which HTLV-I Tax-specific cytotoxic T lymphocytes (CTL) have been suggested to be immunopathogenic. However, it is unknown whether the HTLV-I-specific CTL in the central nervous system differ from those in the periphery. We investigated functional T-cell receptor diversity in HTLV-I Tax11-19-specific CTL clones derived from peripheral blood and cerebrospinal fluid (CSF) of a HAM/TSP patient using analogue peptides of the viral antigen. CTL responses to the analogue peptides varied between T-cell clones, however, CTL clones from CSF showed limited recognition of the peptides when compared to those from peripheral blood. This suggests that CTL with highly focused specificity for HTLV-I Tax accumulate in the CSF and may contribute to the pathogenesis of HAM/TSP. Furthermore, this study provides a rationale for analogue peptide-based immunotherapeutic strategies focusing on the immunopathogenic T-cells in HTLV-I-associated neurologic disease.     

HTLV-I specific IFN-gamma+ CD8+ lymphocytes correlate with the proviral load in peripheral blood of infected individuals

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease caused by HTLV-I infection. It has been shown that HAM/TSP patients have high proviral loads and an extraordinarily high frequency of circulating CD8 + cytotoxic T lymphocytes specific for HTLV-I in their peripheral blood when compared to asymptomatic HTLV-I carriers (AC). We have previously described an intracellular cytokine detection assay, in which interferon-gamma (IFN-gamma) + CD8 + lymphocytes are specific for HTLV-I in infected individuals. Here, we have established a competitive polymerase chain reaction assay to measure the proviral load of patients and investigate a potential relationship between proviral load and virus-specific CD8 + lymphocytes. Genomic DNA was extracted from peripheral blood lymphocytes (PBL) from eight HAM/TSP patients and seven AC for the measurement of HTLV-I measuring proviral loads. The same PBL were analyzed for intracellular IFN-gamma expression by flow cytometry. In the HAM/TSP patients and AC, the average proviral loads were 34,482 and 9784 copy/microg DNA (P = 0.021), and the average of IFN-gamma + CD8 + lymphocytes in total PBL were 1.47 and 0.08% (P = 0.001), respectively. It was confirmed that HAM/TSP patients have both high proviral loads and increased HTLV-I-specific CD8 + lymphocytes. Furthermore, we found a positive correlation between both factors in the patients with HAM/TSP (P = 0.044) but not in the AC (P = 0.508). These findings suggest that the high number of HTLV-I-specific lymphocytes may result from the increased proviral load in HAM/TSP patients.     

Reduction in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I–associated myelopathy/tropical spastic paraparesis patients treated with humanized anti-tac

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0,2,6,10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I–infected, activated CD4⁺ lymphocytes.     

Modulation of EAE by vaccination with T cell receptor peptides: Vβ8 T cell receptor peptide-specific CD4 lymphocytes lack direct immunoregulatory activity

Resistance to experimental autoimmune encephalomyelitis (EAE) induced by vaccination with a peptide representing amino acids 39–59 of the rat T cell receptor (TCR) Vβ8 element has been ascribed to the induction of protective antibodies and T lymphocytes, both recognizing the Vβ8 TCR peptide (TCRP) as well as Vβ8 TCR-expressing encephalitogenic lymphocytes. In this study immunization with the Vβ8 TCR peptide conferred partial resistance to active induction of EAE in three of six rats. The immunoregulatory role of TCRP-specific T cells in resistance to EAE was investigated. In vitro, D4⁺ T cell lines reactive with the Vβ8 TCRP did not respond to encephalitogenic Vβ8 TCRP-bearing cell lines nor did they impair their MBP-induced activation. In vivo, activated TCRP-specific line cells did not ameliorate actively induced EAE. The beneficial effect of Vβ8 TCRP-vaccination on the course of EAE may be due to the induction of protective antibodies. Neither before, nor during or after EAE did we observe a cellular response to the Vβ8 TCRP in lymph nodes or spleens of MBP-immunized animals. Moreover, we were not able to established TCRP-specific T cell lines from EAE rats, but from all rats immunized with the TCRP. Our data do not support the assumption that Vβ8 TCRP-reactive CD4⁺ T cells are the population operative in resistance to EAE after recovery from disease.     

Pathogenetic significance of HTLV-I infection and immune surveillance in HAM]

HTLV-I proviral DNA load is significantly increased in HTLV-I associated myelopathy (HAM) compared with asymptomatic HTLV-I seropositive carriers (SPC), and this spread of HTLV-I infection seems to be critically important in the pathogenesis of HAM. Thus, in this report, cellular immune surveillance against HTLV-I was reviewed. (1) MHC class I-restricted cytotoxic T lymphocytes (CTL) activities are detected in peripheral blood mononuclear cells (PBMC) of HAM. CTL release various proinflammatory and cytotoxic cytokines, chemokines, and proteases. Since CTL are also found in the spinal lesions of HAM, CTL may contribute to the tissue damage. In spontaneous proliferation of PBMC in HAM, CD 4/CD 8 is decreased due to the proliferation of CD 8 + CTL. CD 4/CD 8 is inversely correlated with the clinical severity of HAM. Collectively, CTL may be involved in the pathogenesis of HAM. (2) Activity and subsets of natural killer (NK) cells are lower in HTLV-I-seropositive individuals. Moreover, NK have only a weak cytotoxicity against HTLV-I infected cells. (3) Antibody-dependent cell-mediated cytotoxicity (ADCC) are impaired in HAM compared with SPC due to the suppressed effector cell activity. Since ADCC effectively lyse HTLV-I infected cells in vitro, the impaired ADCC may in part allow the spread of HTLV-I infection in HAM, and potentiation of ADCC may have an anti-HTLV-I therapeutic effect.     

Interferon-γ secretion by in vivo activated cytotoxic T lymphocytes from the blood and cerebrospinal fluid during mumps meningitis

Functional studies of cerebrospinal fluid T lymphocytes during acute viral infections of the nervous system are rare. Recently, we had the opportunity to investigate the requirments for interferon-γ (IFN-γ) production of human in vivo activated (primary) cytotoxic T lymphocytes (CTL) generated during acute viral meningitis. Two HLA-B7-restricted, CD4⁻, CD8⁺ CTL clones from cerebrospinal fluid of one patient with mumps meningitis were studied. Although lytic activity was restricted by HLA-B7, the clones produced similar amounts of IFN-γ when stimulated with HLA-matched and mismatched mumps virus-infected target cells. In addition, peripheral blood mononuclear cells of infected patients secreted significant amounts of IFN-γ when incubated with autologous or allogeneic (HLA-A/B-mismatched) mumps virus-infected target cells. T cells capable of lytic activity and IFN-γ secretion could only be isolated from venous blood during the initial phase of the infection. We suggest that the ability of human in vivo activated CTL to secrete INF-γ early during the course of inflammation and in a HLA-unrestricted fashion is important for the elimination of viruses invading the central nervous system.     

Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes

Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4⁺ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8⁺ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8⁺ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4⁺ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord.     

Antiviral CD8<Superscript>+</Superscript> T cells cause an experimental autoimmune encephalomyelitis-like disease in naive mice

Major histocompatibility complex class I-restricted CD8⁺ cytotoxic T lymphocytes are involved in the pathogenesis of multiple sclerosis (MS) and both autoimmune, experimental autoimmune encephalomyelitis, and viral, Theiler’s murine encephalomyelitis virus (TMEV) infection, animal models of MS. Following TMEV infection, certain T cell hybridomas, generated from cloned TMEV-induced CD8⁺ T cells, were able to produce clinical signs of disease (flaccid hind limb paralysis) upon adoptive transfer into naive mice. Dual T cell receptors (TCR) are present on the surface of these cells as both Vβ3 and Vβ6 were detected by polymerase chain reaction (PCR) screening and flow cytometry and multiple Vα mRNAs were detected by PCR screening. This is the first demonstration of antiviral CD8⁺ T cells having more than one TCR initiating an autoimmune disease in the natural host of the virus. We hypothesize that this is a potential mechanism for virus-induced autoimmune disease initiated by CD8⁺ T cells.     

Natural killer (NK) cells in HTLV-I-associated myelopathy/tropical spastic paraparesis-decrease in NK cell subset populations and activity in HTLV-I seropositive individuals

We examined natural killer (NK) cell activity and NK cell subset populations in 18 patients with HTLV-I associated myelopathy (HAM)/tropical spastic paraparesis (TSP), ten HTLV-I seropositive asymptomatic carriers and 20 seronegative healthy controls. The NK cell activity was significantly decreased in HAM/TSP, compared with that in controls. The percentages of NK cell subsets, such as CD16+, CD11b+, CD56+, CD16+ CD56-, CD16-CD56+, CD16+CD8-, or CD16+CD3+ cells were significantly decreased in HAM/TSP patients. Of particular interest is that the percentage of CD16+CD3+ cells, which have a wide spectrum of cytotoxic properties commonly seen in NK, lymphokine activated killer (LAK) and antibody-dependent cellular-cytotoxic (ADCC) effector cells, was significantly decreased in HAM/TSP as compared to asymptomatic carriers as well as controls. The percentage of CD16+CD3+ cells correlated inversely with the value of spontaneous proliferation of peripheral blood lymphocytes (SPP), which is a characteristic change observed in HAM/TSP.     

Activation of macrophages/microglia with the calcium-binding proteins MRP14 and MRP8 is related to the lesional activities in the spinal cord of HTLV-I associated myelopathy

Macrophages and microglia may play an important role in the pathogenesis of chronic inflammatory process in HTLV-I associated myelopathy (HAM) and tropical spastic paraparesis (TSP). However, the etiology and cellular mechanism of chronic inflammation are poorly understood in HAM/TSP. To help to define the roles of macrophages and microglia we analyzed the various patterns of macrophage and microglia activation in the central nervous system (CNS) of HAM/TSP using several monoclonal antibodies recognizing the different states of activation. The results indicate that a large number of macrophages and microglia express both MRP14 and MRP8 in active-chronic inflammatory lesions of the patients with a short duration of illness (2.5 years). In the patient whose duration of illness was 4.5 years, perivascular and parenchymal macrophages and microglia were reactive for MRP8 but not for MRP14. In contrast, MRP14 and MRP8 were negative on the perivascular and parenchymal macrophages and microglia in inactive-chronic lesions and in controls. This study suggests that (a) activated macrophages and microglia as well as CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes are main components of the inflammatory process in the CNS in HAM/TSP, (b) activation of macrophages and microglia is related to the amount of HTLV-I proviral DNA in situ.     

Mediators of central nervous system damage during the progression of human T-cell leukemia type I-associated myelopathy/tropical spastic paraparesis

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) represents one of the most devastating diseases associated with HTLV-I infection. Despite the delineation of clinical features associated with this neurologic disease, more progress needs to be made with respect to understanding the molecular mechanisms relating to the genesis of HAM/TSP. Several factors have been hypothesized to contribute to whether an HTLV-I-infected individual remains asymptomatic, develops adult T-cell leukemia (ATL), or progresses to HAM/TSP. Among the most intriguing of these factors is the immune response mounted by the host against HTLV-I. Several cell populations are crucial with respect to generating an efficient immune response against the virus. This includes CD4(+) T cells, CD8(+) T cells, dendritic cells (DCs), monocytes/macrophages, and HTLV-I-infected cells that interact with immune cells to stimulate their effector functions. Although all of these cell types likely play important roles in the etiology of HAM/TSP, this review focuses specifically on the potential function of the CD8(+) T-cell population during the progression of HTLV-I-induced neurologic disease. The immune response in HAM/TSP patients may transition from a beneficial response aimed at controlling the viral infection, to a detrimental response that ultimately participates in mediating the pathology observed in HAM/TSP. In this respect, the generation of a hyperactive CD8(+) cytotoxic T lymphocyte (CTL) response primarily targeting the HTLV-I Tax protein likely plays a key role in the genesis of pathologic abnormalities associated with HAM/TSP. The efficiency and activity of Tax-specific CD8(+) CTLs may be regulated at a number of levels, and deregulation of Tax-specific CTL activation may contribute to HAM/TSP. This review focuses on potential mechanisms of central nervous system (CNS) damage associated with the genesis of HAM/TSP following HTLV-I infection, focusing on the role of the Tax-specific CTL compartment.     

T cell receptor Vβ gene utilization in myelin basic protein specific clones from CXJ1 recombinant inbred mice

CXJ1 mice are a recombinant inbred strain generated from experimental allergic encephalomyelitis (EAE) resistant BALB/c and EAE susceptible SJL/J progenitors. CXJ1 derive their major histocompatibility complex (MHC) class II and TCR genes from the BALB/c progenitor. However, their susceptibility to EAE is similar to SJL/J. Utilizing myelin basic protein (MBP)-specific CD4⁺ hybridoma clones and a MBP-specific T cell line (TCL) from CXJ1, we found the predominant T cell receptor (TCR) Vβ chain expression to be Vβ8 and Vβ13. Our data support the concept of preferential, but not exclusive, TCR Vβ usage in the MBP-specific response which is independent of MHC class II haplotype or immunodominant peptide.     

Activated T lymphocytes in cerebrospinal fluid of patients with HTLV-I-associated myelopathy (HAM/TSP)

In order to detect activated T lymphocytes in the cerebrospinal fluid (CSF) of patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we studied CSF lymphocytes in untreated patients with HAM/TSP and other neurological diseases (OND). Dual-immunofluorescence staining technique was performed using fluorescence microscopy. No significant difference in the CD4+/CD8+ ratio of CSF lymphocytes was observed between HAM/TSP patients and patients with OND. However, both CD4+ and CD8+ CSF lymphocytes of HAM/TSP patients contained higher percentages of HLA-DR-positive cells than those of patients with OND (P less than 0.05), suggesting that the activated CSF T lymphocytes were composed of both CD4+ and CD8+ subsets in patients with HAM/TSP.     

Diversity of intrathecal antibody synthesis against HTLV-I and its relation to HTLV-I associated myelopathy

The humoral immune response against human T-cell lymphotropic virus type I (HTLV-I) in the central nervous system (CNS) compartment and in the blood was investigated by enzyme immunoassay using 16 synthetic peptides corresponding to HTLV-I core and envelope sequences. We evaluated paired samples of cerebrospinal fluid and serum from HTLV-I seropositive Japanese patients, classified as follows: HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP;n = 39), patients with spinal cord disease ascribed to either HAM/TSP or to some concomitant, HTLV-I-unrelated disease (possible HAM/TSP;n = 6) or carriers without any clinical signs of HAM/TSP (n = 15). HTLV-I-peptide-specific intrathecal antibody synthesis was found in 79% of HAM/TSP patients, but only in 20% of carriers without HAM/TSP. The group of carriers without HAM/TSP showed local synthesis for some peptides (on average 0.3 peptides per patient). In most HAM/TSP patients, however, there was a diverse intrathecal immune response to several HTLV-I synthetic peptides (on average against 3.6 peptides per HAM/TSP patient), most frequently againstgag p19 100–130,env gp21 458–488, andenv gp46 175–199 and 288–317. The intrathecal antibody synthesis against several HTLV-I determinants may represent a pathogenic immune response in HAM/TSP and is possibly related to the infiltration of virus-infected T-cells in the spinal cord.     

HTLV-I alters the multidrug resistance associated protein 1 (ABCC1/MRP1) expression and activity in human T cells

The human T cell lymphotropic/leukaemia virus type I (HTLV-I) causes HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The multidrug resistance associated protein 1 (ABCC1) plays multiple functions in physiopathologic responses. The expression and activity of ABCC1 was studied in T lymphocytes from uninfected and HTLV-I-infected individuals (both asymptomatic and symptomatic/HAM/TSP). ABCC1 expression and activity was reduced to nearly half in T lymphocytes from infected patients compared to control lymphocytes. Only 51.6% of CD4(+) cells from HAM/TSP patients expressed ABCC1 whereas this was seen in 60.3% from asymptomatic individuals, compared to an expression of around 86% in controls. Our results suggest that ABCC1 is negatively regulated in HTVL-I infection, supplying a novel target to investigate the pathogenesis of HTLV-I.