High Human T Cell Leukemia Virus Type-1(HTLV-1) Provirus Load in Patients with HTLV-1 Carriers Complicated with HTLV-1-unrelated disorders

Human T-cell leukemia virus type-1 (HTLV-1) is thought to infect mainly CD4 T-cells, and to cause T-cell malignancy adult T-cell leukemia (ATL) after a long latency, a degenerative nervous disorder of HTLV-1-associated myelopathy (HAM), and so on [1, 2]. During the clinically asymptomatic period preceding the diseases, the HTLV-1-infected cell number is low, at about less than 2 - 3% per 100 blood mononuclear cells (MNC). Therefore, infected cells in asymptomatic (healthy) carriers are considered to proliferate polyclonally because the provirus integrates at a random site [3]. Recent work using real-time polymerase chain reaction (PCR) quantification for HTLV-1 provirus (proviral load: VL) and inverse PCR indicates that clonal expansion of HTLV-1-infected cells is important for the maintenance of infection [4‐6]. Interestingly, the proviral integration sites in genomic DNA in asymptomatic and symptomatic carriers without ATL is either random or constant, implying the difference in clonality detected by Southern blotting hybridization (SBH) [7, 8]. Thus, high VL corresponding to an increased number of polyclonal or monoclonal infected cells is one of the key events in HTLV-1-associated pathology. Therefore, a high VL with clonal expansion has potential as a biomarker to predict patients predisposed to ATL or HAM [9, 10]. On the other hand, HTLV-1-infected individuals who are complicated by opportunistic infections, such as parasites, mycosis, viruses and some bacteria, and abnormal immunity due to aging are known to show an increased proviral load with polyclonal expansion [11‐13]. This condition associated with polyclonal expansion of the infected cells was considered to be the intermediate state prior to progression to ATL [14], but the pathological and clinical correlation between clonality and level of VL is not fully understood. Recently, we have had frequent opportunities to see unusual or indeterminate ATL patients or carriers with high VL with discrete band(s) in SBH, but no circulating ATL cells, especially among the elderly.

Accordingly, to address what kind of clonal infected cells contribute to high VL, and clarify unusual ATL or carrier states, we simultaneously analyzed HTLV-1 proviral load and SBH status using the same blood samples. In contrast to the maintenance of stable VL in asymptomatic carriers with no-bands or only faint discrete bands, the VL in symptomatic carriers with complications unrelated HTLV-1 tended to have high VL with dense smears with/without discrete band(s), consisting of mainly polyclonal expansion and partial oligoclonal expansion of the infected cells.

Recent studies including our previous studies [13, 15] suggest that VL in asymptomatic carriers may be approximately one copy per 25 to 1000 MNC. Even in patients with HAM whose VL are known to be high, the VL may be as high as one copy per 10 to 100 MNC. Therefore, we defined a VL of 10% or more per 100 MNC as unusually high.

In the present study, we found that the results of VL and SBH status in healthy carriers were the same as those of the past reports, while there was an extremely high VL with a characteristic band status of high dense smears with or without clonal bands in elderly symptomatic carriers. A VL of 10% or more (range, 10 to 97.4%) was detected in 16 (43.5%) of all samples, 1 (6.3%)/16 asymptomatic healthy carriers, 8 (61.5%)/13 symptomatic carriers unrelated to HTLV-1 and 7(87.5%)/8 patients with lymphoma-type ATL without circulating ATL cells. On the other hand, in SBH analysis, no visible aberrant bands were detectable in low VL samples with less than 10%. All but one of the asymptomatic carriers (mean age; 60) were of this pattern. In contrast, the high VL samples with 10% or more displayed distinctive band patterns accompanied by dense smears with or without discrete clonal band(s), indicating that an increase in polyclonally infected-cells corresponding to dense smears contributed to a high VL. As triggering factors for HTLV-1-infected cells, various microbes and abnormal immunity due to aging in symptomatic carriers were suspected. Furthermore, the observations from sequential samples also support the contribution of dense smears to the elevation of VL. This helps explain the process by which the clonality of HTLV-1-infected cells is established after the disappearance of dense smears.

It is now recognized that clonal expansion of HTLV-1-infected cells is the norm in nonmalignant disease [11]. In the present study, of 13 asymptomatic and 12 symptomatic carriers, the incidence of clonality was 24.0% (6/25 cases), of which 4 cases were accompanied by dense smears and maintained a higher VL. In other word, this suggests that polyclonal expansion, rather than oligoclonal expansion, contributes to a high VL. The contribution of clonal expansion to the elevation of VL in carriers is thought to be small because VL in HAM patients is generally reported to be around 10% on average. In fact, Furukawa et al. [8] reported a high frequency of clonality of 20% in HAM patients and 16% in carriers in families of HAM patients, while the VL was at most 10 to 20% in general. On the other hand, patients co-infected with strongyloidosis and HTLV-1 have been reported to harbor a higher VL of around 50% with a high incidence (39%) of clonality [16, 17]. The relation between VL and clonality is controversial [11, 12] because the decision regarding clonality depends on the sensitivity of the method.

Taken together, our study supports the idea that extremely high VL mainly results from polyclonal expansion of HTLV-1 infected cells at the sensitive level of SBH.

In samples from patients with lymphoma-type ATL without ATL cells, a clonal band(s) was demonstrated in 4 of the 8 patients. Band size between blood and lymph nodes was the same in two of the 4 cases, but no circulating ATL was found. Other aberrant bands, as summarized in Additional file 1, were also observed. Such a band profile in ATL (lymphoma-type) appears to be looks a relic of a symptomatic carriers' past, indicating that high VL with aberrant bands could become a biomarker to predict the development of ATL. Indeed, cases 3 and 5 developed smoldering ATL after 4 years and acute ATL after 3 years, respectively. A conceptual scheme is presented in Figure 5, which shows the biological significance of the fluctuation in a high VL with either dense smears or oligoclonal bands durig multi-step leukomogenesis as a stone corner of dense smear emergence.

Focusing on not only discrete clonal band(s), but also aberrant smears, it is noteworthy that the emergence of dense smears equivalent to polyclonal expansion of infected cells mainly contributed to a high level VL. However, it is reasonable that a high VL does not consist of only polyclonally expanded infected cells, but included abundant small clones in the cell population, because of sensitivity in SBH. SBH for HTLV-1 could evaluate or monitor the clinical clonal status of HTLV-1-infected cells. Clinically, the distinctive profile of high VL and aberrant band status is expected to monitor or predict some events caused by HTLV-1.