Leukemic T Cells Are Specifically Enriched In a Unique CD3dimCD7low Subpopulation of CD4+ T Cells In Acute-Type Adult T Cell Leukemia

Abstract

4144 [Background] Adult T-cell leukemia (ATL) is a malignant disorder caused by human T-cell leukemia virus type I (HTLV-I). Morphological discrimination of leukemic cells from non-leukemic T cells is often difficult in ATL since ATL cells reveal morphological diversity except for typical “flower cells”. Although a study using CD3 gating in flow cytometry reported that ATL cells were distinguishable as a CD3low population from normal lymphocytes, these cells were not well characterised as ATL cells. Considering that defective expression of CD7 as well as CD3 is 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. [Methods and Results] (1) In flow cytometry, after dead-cell and monocyte removal, CD4+ T lymphocytes were gated on the CD3 versus CD4 plot. Based on cell density and fluorescence intensity of CD3 and CD7 in this population, we designated three subpopulations on this plot: CD3highCD7high, CD3dimCD7dim and CD3dimCD7low(Results of a representative ATL and a control sample are shown in Figure). The proportion of the CD3dim/CD7low subpopulation was significantly higher in acute-type ATL CD4+ lymphocytes than in normal controls(Figure). (2) To extensively characterise this subpopulation, we next estimated the HTLV-I proviral load by quantitative real-time PCR after FACS sorting based on this CD3 versus CD7 plot. In all patient samples, HTLV-I proviral integration was detected in all subpopulations. However, the proviral load was significantly higher in the CD3dim/CD7low subpopulation compared to the CD3high/CD7high subpopulation. Almost all of the cells in the CD3dim/CD7low subpopulation were HTLV-I infected. (3) We next examined CCR4 and CD25 expression in each subpopulation. Both CCR4 and CD25 expression levels were maintained at very low and similar levels throughout all subpopulations in normal control cells and in the CD3high/CD7high subpopulation of patients with ATL as well. In contrast, CCR4 expression was significantly up-regulated in CD3dim/CD7low subpopulation of patients with ATL compared to the CD3high/CD7high subpopulation (MFI: 36.5±17.2 vs. 3.8±1.1). The expression of CD25 was also up-regulated in the subpopulation (MFI: 7.8±8.0 vs. 2.7±1.6). (4) Monoclonal expansion of HTLV-I-infected cells in the CD3dim/CD7low subpopulation was indicated by the genomic integration site analysis using a long inverse polymerase chain reaction (PCR) method. (5) We reviewed the glass-slide specimens of FACS-sorted samples to evaluate the morphology of each subpopulation on the CD3 versus CD7 plot. Atypical lymphocytes with morphology such as a notch in the nucleus were observed in all subpopulations. The majority of sorted cells from CD3dim/CD7low subpopulation showed “flower cell”-like morphology. (6) We also detected a small CD3dim/CD7dim subpopulation other than the CD3dim/CD7low and CD3high/CD7high subpopulations in all patients with acute-type ATL who were analysed(Figure). This subpopulation contained the same clone as the CD3dim/CD7low subpopulation, although a phenotypical difference existed between these subpopulations. [Conclusion] (1) Above findings indicate that leukemic T cells are specifically enriched in a unique CD3dim/CD7low subpopulation of CD4+ T cells in acute-type ATL. This multi-color FACS system may be useful for precisely monitoring disease during chemotherapy, detecting minimal residual disease and analysing ATL cells. (2) Previous reports have revealed that HTLV-I-infected cells transform through multi-step oncogenesis. Detailed analysis of these three subpopulations (CD3high/CD7high, CD3dim/CD7dim and CD3dim/CD7low) may give some insight into oncogenesis of HTLV-I-infected cells. Disclosures: No relevant conflicts of interest to declare.

Advanced human T‐cell leukemia virus type 1 carriers and early‐stage indolent adult T‐cell leukemia‐lymphoma are indistinguishable based on CADM1 positivity in flow cytometry

“…Peripheral blood samples were collected from inpatients and outpatients at our hospital from June 2011 to September 2014, as described in our previous reports. (8)(9)(10) As shown in Table 1, 60 cases were analyzed (41 AC; 9 cases of smoldering-type ATL; 10 cases of chronic-type ATL). All patients with ATL were categorized into clinical subtypes according to Shimoyama's criteria.…”

“…Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation, as described previously. (8) An unlabeled CADM1 antibody (clone 3E1) and an isotype control chicken IgY antibody were purchased from MBL (Nagoya, Japan). These were biotinylated (by primary amine biotinylation) using biotin N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA).…”

“…Proviral load in FACSsorted PBMC was quantified by real-time quantitative PCR (TaqMan method) using the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA), as described previously. (8,12) Inverse long PCR to assess the clonality of human T-cell leukemia virus type 1-infected cells. For clonality analysis, inverse long PCR was performed as described previously.…”

“…(5)(6)(7) We recently developed a flow cytometry-based method that enables HTLV-1-infected and clonally expanding cells to be purified. (8)(9)(10) In the cell adhesion molecule 1 (CADM1) versus CD7 plot for CD4 + cells in peripheral blood mononuclear cells (PBMC) from HTLV-1-infected patients, three subpopulations (P, CADM1 À CD7 + ; D, CADM1 + CD7 dim ; and N, CADM1 + CD7 À ) are consistently observed. (10) HTLV-1-infected cell clones are enriched in the CADM1 + subpopulations (D and N).…”

Loss of CCR4 antigen expression after mogamulizumab therapy in a case of adult T‐cell leukaemia‐lymphoma

“…We analysed CCR4 expression on patient ATL cells using multi-colour flow cytometry analysis, as described previously (Tian et al, 2011) to reveal the resistance mechanism. After dead cells (propidium iodide positive) and monocytes (CD4 dim CD14 + ) were gated out, a CD3 vs. CD7 plot of CD4 + T cells was constructed, and CCR4 expression on the CD3 dim / CD7 low subpopulation, in which ATL cells were highly enriched, was analysed.…”