SAP expression on CD8+ T cells and HTLV-1 specific CD8+T cells in HTLV-1 infection.

<p>In A) Summary of data for percentage SAP expression on total CD8+ T cells in HTLV-1 infected and healthy individuals. In B) Representative dot plots showing gating strategy for HTLV-1 tetramer positive CD8+T cells, isotype control, and SAP expression on HTLV-1 specific CD8+ T cells. In C) Summary data of SAP expression on subjects for whom HTLV-1 specific cluster could be demonstrated matched with SAP expression on CD8+ T cells in HTLV-1 infected subjects (ATLL (9), AC (8)) (p-values based on Mann-Whitney and Wilcoxon matched-pairs signed rank test).</p

Expression patterns of 2B4 on virus-specific CD8+T lymphocytes.

<p>A), Representative dot plots showing gating strategy for 2B4 and percentage of 2B4 expression on virus-specific CD8+ T lymphocytes, HTLV-1, CMV and EBV. B) Summary data of expression of 2B4 on HTLV-1 infected (ATLL, AC); CMV− and EBV-specific CD8+ T cells in ATLL, AC and HD. Horizontal line represents the mean. (p-value based on by Mann-Whitney test).</p

Detectable Tax-specific CD8+ T cells in all samples used in this study.

a<p>human leucocyte antigen.</p>b<p>cytomegalovirus.</p>c<p>epstein-barr virus.</p

Improved perforin expression on HTLV-1 specific CD8+ T cells on ligand blockade in ATLL and AC subjects.

<p>PBMC from 6 ATLL and 5 AC subjects were stimulated for 6 hours with and without HTLV-1 peptide in the presence or absence of CD48 in 7 independent experiments. In a) Representative dot plots showing perforin release in peptide only stimulated and peptide stimulated samples to which anti-CD48 was added. In b) Summary of data for all 6 ATLL subjects in 7 independent experiments. In c) summary of data for all 5 asymptomatic carriers in 7 independent experiments (p-value based on Wilcoxon matched-pairs signed rank test).</p

Characterization of memory population of 2B4+CD8+ T cells by CCR7, CD45RA, CD62L and CD57 expression.

<p>2B4+CD8+ T cells tended towards effector and terminally differentiated memory phenotype relative to 2B4-CD8+ T cells. A, B) Summary of frequency of CD45RA and CCR7 coexpression on 2B4+CD8+ and 2B4-CD8+ T cells in HTLV-1 infected (6 ATL and 5 AC) and 5 healthy donors (HD). 2B4+CD8+ T cells expressing CD45RA and CCR7 showed a mature effector phenotype in the effector memory and terminally differentiated compartments compared to 2B4-CD8+ T lymphocytes. C) Summary of frequency of CD57 on CD8+ T cells were significantly higher in 2B4+ compared to 2B4− populations in both ATLL and AC D) Lower CD62L expression on 2B4+CD8+ compared to 2B4-CD8+ T cells. Data for CD57 and CD62L was obtained from 6 ATL and 5 AC. Phenotypic patterns are indicated by legends. Horizontal lines represent mean ± S.D; statistical comparisons, p- value based on Mann-Whitney and Wilcoxon matched-pairs signed rank test.</p

Characteristics of the Study Groups.

a<p>M/F = male/female,</p>b<p>ATLL = adult T-cell leukemia/lymphoma,</p>c<p>AC = asymptomatic carrier.</p>d<p>HD = healthy donor.</p

CD48 MFI (mean fluorescence intensity) on CD4 T cells.

<p>In A) Representative histograms of CD48 MFI on CD4+ T cell; ATLL (opened histogram), AC (grayed histogram), HD (black histogram) B) summary data comparing CD48 MFI (mean fluorescence intensity) on total CD4+ T cells in HTLV-1 infected and uninfected subjects (13 ATLL, 13 AC and 12HD). CD48 MFI was visibly lower in ATLL and AC relative to HD. The horizontal line represents the mean ± S.D (p-value based on Mann-Whitney test).</p

Increased CD107a degranulation of HTLV-1 specific CD8+ T cells on ligand blockade in both ATLL and AC.

<p>In A), Representative dot plots show CD107a surface degranulation on HTLV-1 specific CD8+ T cells in varying conditions. In B) Summary plot of CD107a surface degranulation in tax-stimulated samples in the presence and absence of CD48 antibody addition in ATLL (11 independent experiments in 7 patients) and AC (12 independent experiments in 8 subjects). Experiments were done in duplicates (p-value based on Wilcoxon matched-pairs signed rank test).</p

2B4 expression on total CD8+Tcells in HTLV-1 infection.

<p>Appropriate isotype controls were used to set quadrant gates, A) Dot plot of isotype and positive 2B4 staining in a representative sample. Numbers indicate frequency of 2B4 expression on total CD8+ T cells of an HTLV-1 infected subject B),Summary data showing 2B4+ expression on total CD8+ lymphocytes in 52 HTLV-1 infected subjects (21 ATL and 31 AC) and 12 uninfected healthy donors (HD). Each dot represents a single individual. Horizontal bars represent mean values (p-value based on Mann-Whitney test).</p

Additional file 1: Figure S1. of Efficient elimination of pancreatic cancer stem cells by hedgehog/GLI inhibitor GANT61 in combination with mTOR inhibition

Staining of Capan-1 M9 cells without first antibodies as negative controls (A) and with antibody against β-catenin or Notch 1 (intra cellular domain: NICD) (B). Scale bar: 100 μm. Similar results were obtained in the staining of PANC-1, MIA PaCa-2 and Capan-1 cells (data not shown). The experiment was repeated three times and the representative results were indicated. Figure S2. The effects of signaling inhibitors on the frequency of CD133+ cells and the viability of pancreatic cancer cells. We treated Capan-1 M9 cells with the inhibitors at the indicated concentration for 3 days, then determined the frequency of CD133+ cells and their cell viability by flow cytometry and the MTT assay, respectively. The results are presented as percentages of control values in untreated cells, showing the mean and standard deviation (s.d.) of 4 replicates obtained in 1 representative experiment. Figure S3. The effects of GANT61 on the surface markers and the shape of Capan-1 M9 pancreatic cancer cells. (A) Flow cytometric analysis of CD133 and CD44 expression on Capan-1 M9 cells treated with or without GANT61. We treated the Capan-1 M9 cells for 3 days, then analyzed them. (B) Representative photographs of Capan-1 M9 cells untreated and treated with GANT61. GANT61 treatment induced a morphologic change of the Capan-1 M9 cells. This may correlate with the increased baseline level in the flow cytometric analysis of GANT61 treated cells shown in (A), which reflect the changes in the structure of the cell surface. Scale bar: 100 μm. Figure S4. The SMO-agonist SAG does not stimulate the sphere formation in Capam1M9 cells. We treated Capan-1 M9 cells with indicate4d concentration of SAG during sphere formation and determined the number of spheres formed per 96-well plate in triplicate. All data are mean and s.d. in 1 representative experiment out of 2. Table S1.Tumorigenesis of pancreatic　cancer cell lines in NOD/SCID mice. Figure S5. The limiting dilution method is necessary to analyze the sphere-forming ability of PANC-1 cells. (A) PANC-1 cells form large aggregates in the multicellular sphere formation assay. We plated single-cell suspensions (103 cells in 0.5 ml) in 24 wells and cultured them. Representative photographs from day 2. (B) Representative photographs of PANC-1 spheres in the single-cell sphere formation assay. We plated single-cell suspensions (1.2 cells in 100 μl) in 96 wells and cultured them for 8 days. Scale bar: 100 μm. Figure S6. A heatmap of the DNA microarray demonstrates upregulation of GLI1 expression in spheres of Capan-1 M9 cells under stem cell culture condition. M9: Capan-1 M9 cells in two-dimensional culture with 10 % FCS, M9 Sphere: Capan-1 M9 spheres formed in a stem cell culture medium. Figure S7. (A) The hedgehog (Hh)/GLI inhibitor GANT61 (10 μM) in combination with the mTOR inhibitor rapamycin (10 nM) effectively reduced the sphere formation of Capan-1 M9 cells. The results are presented as in Fig. 3c. (B) The hedgehog (Hh)/GLI inhibitor GANT61 in combination with the mTOR inhibitor rapamycin impairs in vivo growth of xenografted pancreatic carcinoma in nude mice. Tumor growth of Capan-1 M9 xenografts which were treated with vehicle, 40 mg/kg of body weight, 1 mg/kg of body weight, or combination. Tumor volume was shown as the mean and SD from 5 mice (10 tumors) from each group. * P < 0.05. Figure S8. A schematic representation of the possible mechanism(s) underlying the synergistic effect of double blockage of Hh and mTOR signaling. The decoupling of GLI activation from SHH-PTCH-SMO signaling and/or the redundancy of canonical and noncanonical GLI activation may underlie the ineffectiveness of Hh/SMO inhibitors. This makes GLI as the key molecule in Hh pathway. The difference in the effect of Hh/GLI inhibitors (which reduces CD133+ cell content) and the mTOR inhibitor (which does not reduce CD133+ cell content) suggests that the mTOR function is not mediated only by S6K. Assuming the specific function of mTOR (*) will explain the different effect. (PDF 466 kb