Fig 1.
IL-10 promoted cell growth of IL-2-dependent HTLV-1-infected T-cell lines (ILTs).
ILTs were established by long-term culture of PBMCs from patients with ATL (#22, #227, and H2) and HAM/TSP (#294, #439, and #441) in the presence of rhIL-2. A. Cell surface expression of CD4 (solid line) and CD8 (dotted line) (top), and intracellular expression of HTLV-1 Tax (bottom) with (blue) or without (red) stimulation with 50 ng/ml PMA for 18 h were analyzed by flow cytometry. Closed histograms indicate staining with control antibodies. B. NF-κB activities in ILTs were measured by a transient reporter assay. The luciferase activities were standardized by TK-RL activities and indicated as relative values against Jurkat. C. HTLV-1 proviral loads in ILTs were measured by qPCR. The results were standardized against TL-OmI (1.8 copies/cell) [78]. N.D., not detected. D. The cytokine profiles in the supernatants of ILTs were analyzed with a multiplex bead-based immunoassay, following incubation at a cell concentration of 106/ml for 24 h in the presence of rhIL-2. The results are presented as the means and SD of duplicate samples. E. The growth of ILT cells in the presence (○) or absence (●) of rhIL-10 (20 ng/mL) was assessed by the trypan blue exclusion method. Cell cultures were diluted with fresh medium every 2 days, regardless of the cell growth, and the viable cell number was counted. The total live cell numbers in culture are presented as relative values compared with the cell number at day 0. All culture medium contained rhIL-2. F. Intracellular Tax expression in ILT cells cultured with or without rhIL-10 for at least 7 days, with the medium being replaced 2 days before harvest, was analyzed by flow cytometry, and the proportion of Tax-expressing cells (%) is indicated in the bar graph. In A, B, and D, HAM/TSP-derived ILT cells were cultured in the medium without rhIL-10 at least for 1 week before use. Similar results were obtained in two independent experiments.
Fig 2.
IL-10 enhanced expression of Ki67 and survivin in HAM/TSP-derived ILTs.
ILTs were cultured with or without IL-10 for 7–11 days, and subjected to flow cytometry (A, B) and immunoblot assays (C, D). A. Permeabilized cells were stained with anti-Ki67 (solid line) or control (closed histogram) antibodies. Numbers indicate MFI of Ki-67 expression. B. Cells were stained with Annexin V, and the proportion of apoptotic cells (%) was indicated as the mean and SD of 2–3 independent experiments. The representative flow cytometry data are shown in S2 Fig. C. ILTs cultured with or without IL-10 were treated with MG132 (10 μM) for 3 h, then subjected to immunoblot assays probed with antibodies to caspase-3, cleaved caspase-3, and β-actin. Approximate sizes (kDa) of caspases are indicated. The results of a similar experiment without MG132-treatment is shown in S3 Fig. D. ILTs cultured with or without IL-10 were subjected to immunoblot assays to detect survivin. The number under each band represents the relative value of survivin expression normalized against β-actin. Similar results were obtained in two independent experiments.
Fig 3.
IL-10 induced STAT3-phosphorylation and mild suppression of NF-κB activity in ILTs.
A. Luciferase activities in ILT-H2 and ILT-294 cells containing NF-κB-luc or STAT3-luc and TK-RL reporter genes were measured following incubation with (closed bar) or without (open bar) IL-10 (20 ng/ml) for 48 h. The NF-κB and STAT3 activities were standardized with TK promoter activities, and the relative values against the sample without IL-10 are presented as the mean and SD of duplicate samples. B. The surface IL-10Rα expression (open histogram) on various ILTs was assessed by flow cytometry. The numbers represent relative MFI of IL-10Rα compared with isotype controls (closed histogram). C. ILT cells were cultured with or without IL-10 for two (ILT-294 and -441) or one (ILT-439, -22, -227, and -H2) weeks, and cell lysates were probed with antibodies to phospho-STAT3 (p-STAT3), STAT3, and β-actin in an immunoblotting assay. The numbers under each band represent the relative values of intensity of the band against β-actin. The ratio of the value of p-STAT3 normalized against total STAT3 was also indicated in the bottom of the panel. Representative results of two independent experiments are shown. * p<0.05, ** p<0.01.
Fig 4.
STAT3 knockdown inhibited expression of IL10, BIRC5 (survivin), MYC, and IRF4.
A. ILT cells were transfected with control siRNA (si-CTRL) (open bar) or si-STAT3 (black bar), and subjected to qRT-PCR 48 h after electroporation. Indicated gene expression levels were measured and standardized with ACTB (β-actin) mRNA levels in each sample. The relative values against the si-CTRL samples are presented as means and SD of duplicate samples. Representative results of two independent experiments are shown. * p<0.05, ** p<0.01. B. ILT-22 and ILT-294 cells cultured with (■) or without (□) rhIL-10 for at least 1 week, and IL10 mRNA levels were measured. The normalized values against ACTB were indicated as means and SD of duplicate samples. N.D., not detected. C. ILT-H2 (●), ILT-22 (▲), and Jurkat (◯) cells were incubated with indicated concentrations of AS101 or vehicle control (0.13% ethanol) for 5 days and the viable cell number was assessed using Cell Counting Kit-8. Relative values against vehicle control were plotted as means and SD of duplicate samples. D. ILT-H2 (▲), ILT-22 (●), ILT-294 (+) (cultured with IL-10) (■), and ILT-294 (-) (cultured without IL-10) (□) cells were incubated with indicated concentrations of Cucurbitacin I or vehicle control (0.1% DMSO) for 2 days, and the cell number was evaluated by Cell Counting Kit-8. The relative values against vehicle controls indicate means and SD of duplicate samples. E. Cell lysates of ILTs were harvested 48 h (ILT-294, ILT-22) or 72 h (ILT-H2) after transfection with si-CTRL or si-STAT3, probed with antibodies to caspase-3, cleaved caspase-3, survivin, and IRF4 in immunoblotting assay, and presented together with the β-actin immunoblots of corresponding membranes. Approximate sizes of caspases are indicated. a, b, denote the same images for β-actin because the same membranes were used for the detection of caspases and IRF4, respectively. ILT-294 cells were cultured in the presence of IL-10 in A and E.
Fig 5.
Critical roles of IRF4 in expansion of ILTs.
A. Intracellular IRF4 expression (solid line) was analyzed in various ILT cells following incubation with or without IL-10 for 7–11 days. Closed histograms represent staining with control antibody. The numbers indicate the proportions (%) of cells with high IRF4 expression. B. ILT-294 (cultured with rhIL-10), ILT-22, and ILT-H2 cells were transfected with si-CTRL and si-IRF4, and the cell lysates were subjected to immunoblot assays for cleaved caspase-3, caspase-3, survivin, and β-actin 48 hours after electroporation. Approximate sizes of caspases are indicated. a, b, c, denote the same images for β-actin because the same membranes were used, respectively. C. Indicated ILTs and Jurkat cells were transfected with si-CTRL, si-STAT3, or si-IRF4, and the total viable cell count were evaluated 3 days after electroporation by trypan blue exclusion test. The data represent the mean and SD of duplicate or triplicate samples. The immunoblot confirming the knockdown efficiency of STAT3 in Jurkat cells is also indicated. IRF4 was not detectable in Jurkat. D. Intracellular IRF4 and Ki67 expression of the ILTs prepared in C were analyzed by flow cytometry. ILT-294 cells were cultured in the presence of IL-10 in B, C, D. Numbers in D indicate the proportion (%) of cells in each quadrant. Similar results were obtained in two independent experiments. * p<0.05, ** p<0.01.