Findings in our previous analysis of acute-type ATL samples prompted our analysis of various clinical subtypes of patients infected with HTLV-I to examine whether the CD3 versus CD7 profile reflects the progression of oncogenesis in HTLV-I-infected cells 
. Representative flow cytometric data shown in suggested that the CD3 versus CD7 profile changed during disease progression. As the disease stage progressed, the D and L subpopulations increased with concomitant decreases in the CD3high
(H) subpopulation. , a summary of the flow cytometric data of all cases analyzed, reveals that the two-dimensional plot of the proportions of the D versus L subpopulations could divide all cases into three groups. Group 1, the area under the diagonal line, equivalent to 55% of the H subpopulation in which all normal controls were included (Figure S2
), contained the majority of HTLV-I ACs. Group 3 was the area beyond 80% of the L subpopulation, and the majority of acute-type cases were included in this group. Group 2, located between Groups 1 and 3 (i.e.
, less than 55% of the H subpopulation and 80% of the L subpopulation), included indolent-type (smoldering and chronic) cases and some AC cases. These results suggest that the CD3 versus CD7 expression profile reflects disease stage. Initially, both the D and L subpopulations gradually and simultaneously increased. However, at the clinically advanced stage, the increase in the L subpopulation was prominent. The change is considered to reflect the biological difference between the D and L subpopulations, which needs to be clarified.
In HTLV-I infection, the small clones of infected cells are considered to coexist from the AC stage 
. A selected clone from the multiple small clones then grows and progresses to the malignant state, and the emergence of a dominant clone indicates disease progression in ATL 
. As shown in , major bands suggesting dominant clones were evident in patients with progressed clinical subtypes or those in the advanced group in the CD3 versus CD7 profile, and major bands existed exclusively in the D and L subpopulations. These data also support the idea that increases in the D and L subpopulations correlate with the progression of disease stage. AC cases in Group 2 had high HTLV-I proviral loads (>4%; ) and clear major bands were observed by inverse long PCR in these cases (, right). Sasaki et al.
reported that two cases of HTVL-I AC with oligoclonal bands on Southern blots and high VLs (20%) had progressed to ATL by 4 and 3.5 years later 
. The two cases may correspond to HTLV-I AC in Group 2 proposed in our study. In fact, two cases of ACs in our series that were included in Group 2 progressed to smoldering ATL (). AC cases in Group 2 could be regarded as advanced carriers. Our flow cytometric analysis could apparently discriminate high-risk AC cases from stable ones. Follow-up analysis of these cases is warranted to determine whether AC cases included in Group 2 progress to ATL. Flow cytometric data for these AC cases included in Group 2 () were similar to those for indolent ATL cases in Group 2. These ACs in Group 2 can be considered essentially the same as smoldering ATL cases. Some of the ACs categorized according to Shimoyama's criteria should in fact be separated and regarded as a subtype together with at least some of the smoldering ATL cases.
Iwanaga et al.
reported that high HTLV-I proviral load (>4%) in whole PBMCs was a risk factor for progression to ATL 
. In , the ACs with VLs>4% were distributed between Groups 1 and 2. These findings suggest that not all ACs with high VLs are currently in an advanced stage, although they may have the potential to develop ATL in the future.
In general, the categorization by flow cytometric profile correlated well with the current classification of clinical subtypes, with some exceptional cases of acute-type and smoldering-type disease (). The only manifestation of three smoldering cases categorized in Group 1 was skin lesions; they fell into Group 1 because they showed minimal abnormalities in peripheral blood 
. Three acute-type ATL cases categorized in Group 2 had indolent clinical courses. A diagnosis of acute-type disease is made when the indolent-type and lymphoma-type are excluded, according to Shimoyama's criteria. The CD3 versus CD7 plot may discriminate the cases that will follow an indolent clinical course from the aggressive acute-type ATL.
The VL in each subpopulation indicated that HTLV-I-infected cells were relatively concentrated in the D and L subpopulations (representative data are shown in ). These data are consistent with downregulation of CD3 and CD7 being relevant to HTLV-I infection, although cells without HTLV-I infection may also contribute to this change to some extent, as a substantial subpopulation of T cells has been reported to be CD7-deficient under physiological 
and certain pathological conditions, including autoimmune disorders and viral infection 
. To more precisely analyze phenotypic changes in HTLV-I-infected cells, markers that indicate HTLV-I infection should be incorporated in future studies.
A summary of this study is shown in . In the CD3 versus CD7 profile, most AC cases were included in Group 1, in which the D and L subpopulations were relatively small. Consistent with disease progression to smoldering- and chronic-type ATL, a decrease in the H subpopulation and increases in the D and L subpopulations occur (Group 2). In this step, increases in the sizes of clones in the D and L subpopulations are observed. Further expansion of the leukemic clone results in progression to acute-type ATL in which the L subpopulation has expanded (Group 3). According to a study by Yamaguchi et al.
, the natural course of ATL is to progress from the HTLV-I carrier state through the intermediate state, smoldering ATL, and chronic ATL, and finally to the acute ATL, indicating a process of multistage leukemogenesis 
. We consider this study to successfully link the progressive clinical status and phenotypic changes in HTLV-I-infected cells. However, the way in which this profile reflects multistep oncogenesis in HTVL-I infection at the molecular level remains unclear. Further molecular analyses of the three subpopulations will help in understanding the mechanism(s).
Summary of the study: the CD3 versus CD7 profile reflects progression of disease stage in patients infected with HTLV-I.