Increased HTLV type 1 tax specific CD8+ cells in HTLV type 1-asociated myelopathy/tropical spastic paraparesis: correlation with HTLV type 1 proviral load

Less than 1% of individuals infected with the human T lymphotropic virus type 1 (HTLV-1) develop an inflammatory neurological disorder, termed HTLV-1-associated myelopathy/tropical spastic (HAM/TSP), while the vast majority of those infected remain asymptomatic HTLV-1 carriers (ACs). The fundamental viroimmunological differences between these groups are not well understood. To address this issue, we have investigated HTLV-1-specific T cell responses and measured the proviral load in these groups. Frequencies of HTLV-1-specific CD8(+) cells were demonstrated to be significantly higher in HAM/TSP patients than in ACs by using intracellular cytokine staining and soluble divalent HLA-A2/Ig fusion protein loaded with HTLV-1 Tax 11-19 peptide. It is consistent with the observed increase in HTLV-1-specific cytotoxic T lymphocytes in HAM/TSP patients. These CD8(+) cells produced interferon (IFN)-gamma in recognition of HTLV-1 antigens bound to HLAs on the infected CD4(+) cells. Using phenotypic markers indicative for T cell differentiation, memory and/or effector HTLV-1 Tax-specific CD8(+) cells were found to be increased in HLA-A2 HAM/TSP patients. HTLV-1 proviral load was elevated in HAM/TSP patients when compared to ACs. In addition, the proviral load in HAM/TSP patients correlated with the frequency of HTLV-1-specific IFN-gamma(+)CD8(+) cells or Tax-HLA-A2/Ig(+)CD8(+) cells, especially with the effector cells. In contrast, the proviral load inversely correlated with memory cells. These results suggest that HTLV-1 antigens may continuously stimulate HTLV-1-specific CD8(+) cells and differentiate them from memory cells into effector cells in vivo. These differentiated HTLV-1-specific CD8(+) cells may play a role in the pathogenesis of HAM/TSP.     

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.     

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.     

Human T cell leukemia virus Type I (HTLV-I) infection induces greater expansions of CD8 T lymphocytes in persons with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers

A quantitative study of the T cell receptor repertoire was performed ex vivo on CD4 and CD8 T cell subsets of human T cell leukemia virus type I (HTLV-I)-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Indexes of oligoclonality that compiled all repertoire modifications were calculated for peripheral blood mononuclear cells and for CD4 and CD8 T cell subsets. Both patients with HAM/TSP and asymptomatic carriers had greater T lymphocyte expansions than did uninfected donors, which was independent of age and at least twice higher in the CD8 than in the CD4 cell compartment. Some expanded CD8 T cells corresponded to cytotoxic T lymphocytes directed against various epitopes of the immunodominant Tax protein. Patients with HAM/TSP had significantly higher CD8 cell expansions than did asymptomatic carriers. These results highlight the prognostic value of measuring CD8 T cell expansions during follow-up of HTLV-I infection.     

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.     

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.     

Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells

Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)-unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling.     

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.     

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.     

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.     

Molecular analysis of T cell clonotypes in muscle-infiltrating lymphocytes from patients with human T lymphotropic virus type 1 polymyositis

Epidemiological studies have shown that a correlation may exist between human T cell lymphotropic virus type 1 (HTLV-1) infection and a form of polymyositis (PM). To characterize muscle-infiltrating lymphocytes (MILs) from patients with HTLV-1 PM, we examined the T cell receptor (TCR) beta-chain variable region repertoire and clonotype of MILs and peripheral blood mononuclear cells (PBMC) from 3 patients, using TCR complementarity-determining region 3 (CDR3) length spectratyping and DNA sequencing. Immunohistochemical studies showed that MILs from patients with HTLV-1 PM contain both CD4(+) and CD8(+) T cells. Although some clonotypes observed in PBMC were also found in MILs in all patients examined, MILs consisted predominantly of locally expanded clones. One clonotype in MILs was derived from human leukocyte antigen (HLA)-A*02/Tax11-19 tetramer-positive cells, the CDR3 motif of which contains amino acid residues for HLA-A*02/Tax peptide-TCR interaction. We conclude that certain T cell clones proliferate in the muscle lesions of HTLV-1 PM and may contribute to the pathogenesis of the disease.     

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.     

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.     

HLA-A*26, HLA-B*4002, HLA-B*4006, and HLA-B*4801 alleles predispose to adult T cell leukemia: the limited recognition of HTLV type 1 tax peptide anchor motifs and epitopes to generate anti-HTLV type 1 tax CD8(+) cytotoxic T lymphocytes

Genetic risk for adult T cell leukemia (ATL) has been implicated by ethnic and familial segregation of ATL patients from HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the genetic risk for ATL, we characterized HLA class I alleles of ATL patients and analyzed the anchor motifs of HTLV-1 peptides binding to HLA class I molecules, using 291 lines of anti-HTLV-1 CD8(+) cytotoxic T lymphocytes (CTLs) generated in vitro with a total of 165 synthetic peptides for HTLV-1 Tax and Env proteins. Allele frequencies of HLA-A*26, B*4002, B*4006, and B*4801 were significantly higher in ATL patients than in HAM/TSP patients and asymptomatic HTLV-1 carriers in southern Japan. CD8(+) CTL analysis revealed the HTLV-1 Tax peptide sequence to completely lack anchor motifs of peptides binding to HLA-A*26,B*4002, and B*4006 molecules but to possess one anchor for HLA-B*4801, while the HTLV-1 Env peptide sequence had many anchor motifs for HLA-A*26, B*4002, B*4006, and B*4801 molecules. Most ATL patients featured heterozygous HLA class I alleles composed of HLA-A*26, B*4002, B*4006, and B*4801, with a lower number of HTLV-1 Tax peptide anchor motifs and epitopes generating anti-HTLV-1 Tax CD8(+) CTLs than individuals possessing other HLA alleles. The relationship between Tax epitope and ATL incidence was verified by the significantly decreased number of HTLV-1 Tax epitopes in ATL patients compared with asymptomatic HTLV-1 carriers (p < 0.01) as well as late onset ATL patients (p < 0.001). These results indicate that HLA-A*26, B*4002, B*4006, and B*4801 alleles predispose to ATL because of the limited recognition of HTLV-1 Tax peptide anchor motifs and epitopes capable of generating anti-HTLV-1 Tax CD8(+) CTLs.     

Short communication an interferon-γ ELISPOT assay with two cytotoxic T cell epitopes derived from HTLV-1 tax region 161-233 discriminates HTLV-1-associated myelopathy/tropical spastic paraparesis patients from asymptomatic HTLV-1 carriers in a Peruvian population

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic and progressive disorder caused by the human T-lymphotropic virus type 1 (HTLV-1). In HTLV-1 infection, a strong cytotoxic T cell (CTL) response is mounted against the immunodominant protein Tax. Previous studies carried out by our group reported that increased IFN-γ enzyme-linked immunospot (ELISPOT) responses against the region spanning amino acids 161 to 233 of the Tax protein were associated with HAM/TSP and increased HTLV-1 proviral load (PVL). An exploratory study was conducted on 16 subjects with HAM/TSP, 13 asymptomatic carriers (AC), and 10 HTLV-1-seronegative controls (SC) to map the HAM/TSP-associated CTL epitopes within Tax region 161-233. The PVL of the infected subjects was determined and the specific CTL response was evaluated with a 6-h incubation IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs) stimulated with 16 individual overlapping peptides covering the Tax region 161-233. Other proinflammatory and Th1/Th2 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. In addition, a set of human leukocyte antigen (HLA) class I alleles that bind with high affinity to the CTL epitopes of interest was determined using computational tools. Univariate analyses identified an association between ELISPOT responses to two new CTL epitopes, Tax 173-185 and Tax 181-193, and the presence of HAM/TSP as well as an increased PVL. The HLA-A*6801 allele, which is predicted to bind to the Tax 181-193 peptide, was overpresented in the HAM/TSP patients tested.     

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.     

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.     

Retrovirally induced CTL degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-I-associated neurologic disease

CD8(+) T cells contribute to central nervous system inflammation in human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We analyzed CD8(+) T-cell dysfunction (degranulation and IFN-gamma production) and have demonstrated that CD8(+) T cells of patients with HAM/TSP (HAM/TSP patients) spontaneously degranulate and express IFN-gamma in ex vivo unstimulated culture. CD8(+) T cells of HTLV-I asymptomatic carriers and healthy donors did not. Spontaneous degranulation was detected in Tax11-19/HLA-A*201 tetramer(+) cells, but not in CMV pp65 tetramer(+) cells. Interestingly, degranulation and IFN-gamma production in CD8(+) T cells was induced by coculture with autologous CD14(+) cells, but not CD4(+) T cells, of HAM/TSP patients, which correlated with proviral DNA load in CD14(+) cells of infected patients. Moreover, the expression of IL-15, which induced degranulation and IFN-gamma production in infected patients, was enhanced on surface of CD14(+) cells in HAM/TSP patients. Blockade of MHC class I and IL-15 confirmed these results. Thus, CD8(+) T-cell dysregulation was mediated by both virus infection and enhanced IL-15 on CD14(+) cells in HAM/TSP patients. Despite lower viral expression than in CD4(+) T cells, HTLV-I-infected or -activated CD14(+) cells may be a heretofore important but under recognized reservoir particularly in HAM/TSP patients.