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The morphological discrimination of leukemic from non-leukemic T cells is often difficult in adult T-cell leukemia (ATL) as ATL cells show morphological diversity, with the exception of typical “flower cells.” Because defects in the expression of CD3 as well as CD7 are common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. CD4+CD14− cells subjected to two-color analysis based on a CD3 vs CD7 plot clearly demonstrated the presence of a CD3dimCD7low subpopulation in each of nine patients with acute-type ATL. The majority of sorted cells from this fraction showed a flower cell-like morphology and carried a high proviral load for the human T-cell leukemia virus type 1 (HTLV-I). Genomic integration site analysis (inverse long-range PCR) and analysis of the T cell receptor Vβ repertoire by flow cytometry indicated that the majority of leukemia cells were included in the CD3dimCD7low subpopulation. These results suggest that leukemic T cells are specifically enriched in a unique CD3dimCD7low subpopulation of CD4+ T cells in acute-type ATL. (Cancer Sci 2011; 102: 569–577)
Adult T-cell leukemia (ATL) is a malignant disorder caused by human T-cell leukemia virus type 1 (HTLV-I)(1) and is characterized clinically by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia and a characteristic morphology termed “flower cells.” Importantly, ATL is one of the most incurable lymphoid malignancies. This disease is endemic to several regions in the world, including sub-Saharan Africa, the Caribbean basin, South America and Japan, and 10–20 million people are estimated to be infected by this virus worldwide.(2,3)
Evaluation of the response after chemotherapy for ATL partly depends on the proportion of ATL cells in the peripheral blood. However, the morphological diversity of ATL cells may lead to inaccurate estimations. Accurate estimation of the chemotherapeutic effect is pivotal in clinical practice because ATL cells often become chemoresistant, even during chemotherapy. Methods to detect ATL cells with greater precision than morphological examination are therefore required.
Aberrant expression of cell-surface antigens in myeloid/lymphoid leukemia cells has been studied extensively.(4–6) Using fluorescence-activated cell sorting (FACS) analysis, gating cells with diminished CD45 expression in acute myeloid/lymphoid leukemia is widely used for purifying leukemia cells. However, in ATL there are only limited data regarding the identification of transformed leukemia cells by similar methods. Previous studies indicated that most ATL cells lack CD7 and exhibit diminished CD3 expression.(7–10) Although a study using CD3 gating by FACS analysis has indicated that ATL cells were distinguishable from normal lymphocytes as a CD3low population,(10) these cells were not well characterized as ATL cells.
In the present study, we focused on the enrichment of ATL cells by constructing CD3 vs CD7 plots from multi-color FACS. CD3dimCD7dim and CD3dimCD7low cells were extensively studied and compared with normal control samples. Taken together, our data suggest that ATL cells are purified in CD3dimCD7low subpopulations. The purification of ATL cells by FACS may therefore allow monitoring of disease activity and yield insight into the biology of this disease.
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- Materials and Methods
- Disclosure Statement
To investigate the characteristics of ATL cells, the purification of tumor cells is essential. In the present study, we successfully discriminated the CD3dimCD7low subpopulation in CD4+ T cells in the peripheral blood of patients with acute-type ATL by constructing a CD3 vs CD7 plot of CD4+ T cells from multi-color FACS (Fig. 1). Previously, Yokote et al.(10) reported that CD3low gating facilitated the discrimination of ATL cells by flow cytometry. If we constructed a CD4 vs either CD3 or CD7 plot, in which the downregulated cell subpopulation was not clearly separated, then we could not define distinct cell subpopulations using the CD3 or CD7 marker alone, because the degrees of downregulation of CD3 and CD7 are variable. It should be noted that the combination of CD3 downregulation and diminished expression or absence of CD7 clearly indicates this subpopulation. In addition, gating-out monocytes in the CD4 vs CD14 plot is important for the CD3 vs CD7 plot because monocytes were CD3/CD7 dull-positive based on the nonspecific binding of the antibody.
A substantial subpopulation of T cells has been reported to be CD7-deficient under physiological(16,17) and certain pathological conditions, including autoimmune disorders and viral infection.(18–22) Consistent with these reports, the present study indicated that the proportion of CD4+CD7- T cells in the peripheral blood of healthy adults is up to 10% (Fig. 1B,C). In ATL samples, the CD3 vs CD7 plot revealed various patterns, which may reflect the differences in clinical characteristics of each patient; however, the CD3dimCD7low subpopulation, which was a minor population in the normal controls, was prominent in all ATL samples (Fig. 1B,C). These results prompted us to study this subpopulation in detail. Estimation of the HTLV-I proviral load by quantitative real-time PCR showed that the majority of cells in the CD3dimCD7low subpopulation were infected with HTLV-I (Fig. 2). Immunophenotypic analysis revealed that the expression of CD25, a common ATL marker,(9,23) and CCR4, reported to be expressed in around 90% of cases of ATL,(24,25) were upregulated in the CD3dimCD7low subpopulations of ATL samples in contrast to normal controls in which both markers were weakly expressed in the equivalent subpopulation (Fig. 3A,B). As several studies indicated that ATL cells originate from CD4+CD25+FoxP3+ Treg cells,(26) we next analyzed FoxP3 expression in each subpopulation. In the CD3dimCD7low subpopulation, the FoxP3 expression levels were variable, consistent with previous reports.(27) In one case, FoxP3 expression was upregulated in the CD3highCD7high and CD3dimCD7dim subpopulations suggesting that they were normal Treg cells.
The analysis of clonality is extremely important for determining whether cells are transformed and Southern blot analysis is usually used to confirm clonality. However, in the present study, the cell number following cell sorting was not sufficient for Southern blotting, and thus inverse long PCR for clonality analysis of HTLV-I-infected cells was used.(25) Studies of four ATL samples revealed clonal expansion of ATL cells in the CD3dimCD7low subpopulations, although minor clones may exist in the population (Fig. 4). When PCR was performed in duplicate, we found that the major bands were consistently detected in all cases. However, the detection of multiple minor bands was not consistent. As reported previously, the inverse long PCR method stochastically amplifies the template originating from small clones.(28,29) The minor bands observed in the present study will contain small clones. However, the presence of nonspecific bands cannot be eliminated.
The inverse long PCR method is commonly used for clonality analysis; however, it cannot quantify the size of major/minor clones and the degree of enrichment in each subpopulation. Therefore, we tested the FACS-based TCR-Vβ repertoire analysis combined with our multi-color FACS system (Fig. 5). In ATL patient no. 3, almost all cells in the CD3dimCD7low subpopulations were clonal cells with TCR-Vβ9. Inverse long PCR analysis in the same patient showed multiple minor bands in the CD3dimCD7low subpopulations (Fig. 4D). These results did not conflict with those of the TCR-Vβ repertoire analysis, as the inverse long PCR method is a more sensitive method for detecting small clones compared with flow cytometry. Taken together, the series of analyses in the present study indicated that the CD3dimCD7low subpopulations consist of highly purified ATL cells in patients with acute-type ATL.
A substantial proportion of cells in the CD3dimCD7dim subpopulation consisted of morphologically abnormal lymphocytes (Fig. 6) that exhibited upregulation of CD25 and CCR4 expression (Fig. 3A,B). Using the inverse long PCR method, a similar band pattern between CD3dimCD7dim and CD3dimCD7low subpopulations was observed in patients no. 6 and 8, suggesting that these cells belonged to the same clone (Fig. 4A,B). However, not all of the cells in this subpopulation were infected with HTLV-I because the HTLV-I proviral load was less than that of the CD3dimCD7low subpopulation (Fig. 2). Thus, at least a small number of the CD3dimCD7dim cells were expected to be ATL cells. Those cells observed in the CD3dimCD7dim subpopulation that were phenotypically different from the CD3dimCD7low subpopulations were of particular interest. We detected a band of the same size on inverse long PCR in the CD3dimCD7dim subpopulations as in the CD3dimCD7low subpopulation. This may have been because the two subpopulations originated from the same clone that evolved from a CD3dimCD7dim to a CD3dimCD7low phenotype. Further studies are required to determine the characteristics of the CD3dimCD7dim subpopulation in greater detail.
The results of the present study indicated that HTLV-I-infected cells distribute from a CD7high to a CD7low subpopulation, although the proportion of HTLV-I-infected cells was remarkably low in the CD3highCD7high subpopulation (Fig. 2). A considerable proportion of cells in the CD3highCD7high subpopulation consisted of morphologically atypical lymphocytes (Fig. 6), but the CD25 and CCR4 levels were not upregulated (Fig. 3A,B). When analyzing the pattern of the inverse long PCR of the CD3highCD7high subpopulations, we observed a difference from those of the CD3dimCD7dim and CD3dimCD7low subpopulations (Fig. 4). In patients no. 6 (Fig. 4B) and 7 (Fig. 4C), the band detected in the CD3highCD7high subpopulation may represent an expanded clone that was not transformed. Most likely, these cells do not represent ATL cells, but oligoclonal HTLV-I-infected lymphocytes. Previous studies indicated that HTLV-I-infected cells undergo transformation through multi-step oncogenesis.(30) A detailed analysis of these three subpopulations may therefore provide some insight into the oncogenesis of HTLV-I-infected cells.
Accurate determination of ATL cells in peripheral blood is critical for estimating the response to chemotherapy. However, as discussed above, morphological studies (Fig. 6) have limitations in their ability to discriminate ATL from non-ATL cells.(31,32). Recently, hematopoietic stem cell transplantation has been explored as a promising treatment to overcome the poor prognosis of this most incurable lymphoid malignancy,(33,34) and monitoring minimal residual disease following hematopoietic stem cell transplantation is more important. Our method of analyzing ATL cells may be particularly useful for monitoring minimal residual disease. Although the CD3dimCD7dim subpopulation in our analysis may have included some ATL cells, this is a minor population in the peripheral blood of patients with acute-type ATL, and it is sufficient for practical use to monitor the CD3dimCD7low subpopulation. Another possible use of our procedure is for the definitive classification of ATL subtypes according to Shimoyama’s criteria.(8) A proportion of abnormal lymphocytes in peripheral blood comprise part of the criteria for ATL-subtype classification but it is sometimes confusing. Our multi-color FACS system may clearly quantify this proportion.
In conclusion, we have constructed a multi-color FACS system to purify ATL cells in the peripheral blood of patients with acute-type ATL. This system may be useful for precisely monitoring the disease during chemotherapy, detecting minimal residual disease and analyzing ATL cells. This system may be of great benefit in investigating oncogenesis in HTLV-I-infected cells.