HTLV-I specific IFN-γ+ CD8+ lymphocytes correlate with the proviral load in peripheral blood of infected individuals
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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2014, Journal of Theoretical BiologyCitation Excerpt :Intuitively, one may expect that the more CTLs there are, the stronger the immune response, and thus the lower the proviral load. However, the notion that large numbers of CTLs can be associated with large numbers of virus-infected cells is not new, and has been observed in clinical data (Kubota et al., 2000; Nagai et al., 2001; Wodarz et al., 2001). Moreover, experiments have demonstrated that a fast rate of clearance of virus-expressing infected cells is associated with a reduced proviral load (Asquith et al., 2005).