Elsevier

Journal of Neuroimmunology

Volume 102, Issue 2, 24 January 2000, Pages 208-215
Journal of Neuroimmunology

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

https://doi.org/10.1016/S0165-5728(99)00175-7Get rights and content

Abstract

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-γ (IFN-γ)+ 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-γ expression by flow cytometry. In the HAM/TSP patients and AC, the average proviral loads were 34,482 and 9784 copy/μg DNA (P=0.021), and the average of IFN-γ+ 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.

Introduction

Human T lymphotropic virus type I (HTLV-I) is a human retrovirus well known as the causative agent for adult T-cell leukemia/lymphoma (ATL) (Uchiyama et al., 1997) and a slowly progressive neurological disorder, termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al., 1986). HAM/TSP patients have upper motor neuron signs and mild sensory and sphincter dysfunction (Osame et al., 1987). Pathologically, HAM/TSP is characterized by perivascular infiltration of inflammatory cells with demyelination; lesions are most prominent in the thoracic spinal cord (Akizuki et al., 1987). Certain immunological parameters have been shown to be abnormal in HAM/TSP patients in comparison with HTLV-I-infected asymptomatic carriers (AC) and healthy controls (HC). These include high HTLV-I proviral loads in peripheral blood lymphocytes (PBL) (Yoshida et al., 1989; Kubota et al., 1993; Nagai et al., 1998), high antibody titers to HTLV-I in both sera and cerebrospinal fluid (CSF) (Osame et al., 1987) and increased spontaneous lymphoproliferation in vitro (Itoyama et al., 1988; Jacobson et al., 1988; Usuku et al., 1988). In addition, HAM/TSP patients show extraordinarily high levels of circulating HTLV-I-specific CD8+ cytotoxic T lymphocytes (CTL), which are specific for the HTLV-I Tax 11–19 peptide in human leukocyte antigen (HLA)-A2 patients (Jacobson et al., 1990; Kannagi et al., 1991; Koenig et al., 1993). The frequency of HTLV-I Tax-specific CD8+ CTL is as high as 1 in 75 to 1 in 320 CD8+ cells in PBL of HAM/TSP patients (Elovaara et al., 1993). The frequency of these lymphocytes in CSF cells from a patient with HAM/TSP is similar in magnitude to those in PBL (Jacobson et al., 1992). Immunohistochemical analysis of the spinal cord lesions reveals the expression of MHC class I molecules and an accumulation of infiltrating CD4+ and CD8+ lymphocytes. These CD8+ lymphocytes predominate with duration of illness (Moore et al., 1989; Umehara et al., 1993). Molecular biological studies have shown the presence of the HTLV-I genome and its expression in the affected lesions of HAM/TSP patients (Kira et al., 1992; Hara et al., 1994; Kubota et al., 1994; Lehky et al., 1995; Moritoyo et al., 1996). Therefore, we have hypothesized that the increased number of the virus-specific CD8+ cells might play a critical role in the inflammatory responses in HAM/TSP patients (Jacobson, 1996).

We have previously reported that HAM/TSP patients have increased numbers of IFN-γ+ CD8+ cells in the PBL than AC by a flow cytometric assay combined with intracellular cytokine staining (Kubota et al., 1998). The IFN-γ production from CD8+ cells was blocked by an addition of anti-major histocompatibility complex (MHC) class I antibody and was observed when antigen-presenting cells prepulsed with HTLV-I peptide or autologous CD4+ cells were added. Since CD4+ cells are the main reservoir for HTLV-I in vivo (Richardson et al., 1990), these data suggest that these CD8+ cells produce the cytokine through recognition of HTLV-I antigens bound to MHC class I molecules on the infected CD4+ cells. Thus, we can detect HTLV-I-specific IFN-γ+ CD8+ cells in the PBL of the infected individuals using this assay.

In patients with human immunodeficiency virus type 1 (HIV-1), another chronic retroviral infection, it has been demonstrated that the frequency of HIV-1-specific lymphocytes inversely correlates with the viral burden (Greenough et al., 1997; Ogg et al., 1998). However, in HTLV-I infection, it is still uncertain if the virus-specific CD8+ lymphocytes have any relationship to the proviral load in HTLV-I-infected individuals. To address this issue, we employed a competitive polymerase chain reaction (PCR) technique for measuring the proviral load and a flow cytometric analysis combined with intracellular IFN-γ staining for detecting HTLV-I-specific CD8+ lymphocytes in the same series of samples from HTLV-I-infected individuals, which included eight HAM/TSP patients and seven AC. We have found that HAM/TSP patients, when compared to AC, have both high proviral loads and increased HTLV-I-specific CD8+ lymphocytes in the PBL. Moreover, we showed that the virus-specific CD8+ lymphocytes positively correlated with the proviral loads in PBL of the HAM/TSP patients. These data suggests that the high number of HTLV-I-specific lymphocytes in the HAM/TSP patients may result from the increased proviral load.

Section snippets

Subjects

PBL were collected by gradient centrifugation from eight HAM/TSP patients and seven AC. HTLV-I infection was confirmed by Western blot of sera from these cases. The diagnosis of HAM/TSP was made according to neurological symptoms and serological testing for HTLV-I in CSF (Osame et al., 1987). The clinical data of the HAM/TSP patients are summarized in Table 1. Patient 5 had abnormal responses in the lower extremities and extensor plantar responses indicative of corticospinal tract lesion(s) as

Validation of quantitative PCR

To generate a standard curve, we amplified a constant number of competitor and a known number of wild-type HTLV-I pX with the same primers in each tube. We made a standard curve in each experiment. Fig. 1A represents a PCR done for a standard curve. A plateau effect of PCR was observed over 3 of LOG (wild-type pX), when plotted LOG (wild-type pX/competitor) to LOG (wild-type pX) (data not shown). When we plotted them between 1 and 3 of LOG (wild-type pX) (Fig. 1B), we could achieve linearity.

Discussion

In this study, we demonstrated that HAM/TSP patients have both high proviral loads and increased IFN-γ+ CD8+ lymphocytes specific for HTLV-I in the same samples, when compared to AC. The proportion of HTLV-I-specific IFN-γ+ CD8+ lymphocytes positively correlate with the proviral load in PBL of the HAM/TSP patients but not in the AC. Moreover, in patient 3, high number of IFN-γ+ CD8+ lymphocytes was persistent over 5 years. These data suggest that high levels of both HTLV-I-specific lymphocytes

Acknowledgements

We thank Samantha Soldan, Mashiro Nagai, and Allen Waziri for critical comments of this manuscript.

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