The Teaching of Singing in Meiji Period : Mainly on the contents of teaching

Additional file 3: Fig. S4. Spontaneous gp120 shedding from cell surface. The susceptibility of gp41 mutants to spontaneously shed gp120 was determined by flow cytometry and ELISA as described previously [79]. Briefly, culture medium of transiently transfected envelope expressing cells was exchanged for fresh medium containing Brefeldin A (BioLegend) and 0.2 % Sodium azide. Cells were then incubated for 15 h at 37˚C, 5 % CO2. (a) Level of envelope expression before and after incubation was compared by staining with 2G12. (b) Amount of gp120 released during incubation was determined by gp120 capture ELISA. As a positive control, cells expressing WT envelope was incubated with 20 µg/ml sCD4, which trigger gp120 shedding. Cells expressing SIV Env (SIV) and no Env (No Env) were used as negative control. The results are shown as the means ± standard errors of four replicas

TRAF6 is required for NF-κB activation in HTLV-1 transformed T cells.

<p>(<b>A</b>) Western blots were performed with the indicated antibodies using whole cell lysates from C8166, MT-2 and MT-4 cells transduced with control or TRAF6 shRNA. (<b>B</b>) qRT-PCR of TRAF6, CD25 and cIAP2 mRNAs in C8166, MT-2 and MT-4 cells transduced with lentiviruses expressing control or TRAF6 shRNA.</p

Tax requires IL-17RB for NF-κB activation in T cells.

<p>(<b>A</b>) NF-κB and HTLV-1 LTR luciferase assays and qRT-PCR of IL-17RB mRNA in Jurkat Tax Tet-On cells transduced with lentiviruses expressing control or IL-17RB shRNA. Cells were also transiently transfected with NF-κB and HTLV-1 LTR luciferase reporters and treated with Dox (1 µg/ml) for 24 h. (<b>B</b>) qRT-PCR of Tax, CD25 and cIAP2 mRNAs in Jurkat Tax Tet-On cells from panel (A).</p

Essential roles for IKK and Tax in the induction of IL-17RB.

<p>(<b>A</b>) qRT-PCR of IL-17RB and CD25 mRNAs in C8166, MT-2 and Jurkat cells treated with the IKKβ inhibitor sc-514 (10 µM) for 4 h. (<b>B</b>) qRT-PCR of IL-17RB, IKKα and IKKβ mRNAs in C8166 cells transduced with lentiviruses expressing control, IKKα or IKKβ shRNAs. (<b>C</b>) qRT-PCR of IL-17RB and Tax mRNAs in Jurkat Tax Tet-On cells treated with Dox (1 µg/ml) for the indicated times. (<b>D</b>) qRT-PCR of IL-17RB and Tax mRNAs in C8166 cells transduced with lentiviruses expressing control or Tax shRNAs. (<b>E</b>) Western blot of IL-17RB, Tax and β-actin using lysates from C8166 cells transduced with lentiviruses expressing control or the indicated Tax shRNAs. (<b>F</b>) qRT-PCR of IL-17RB and Tax mRNAs in Jurkat cells transduced with lentiviruses expressing empty vector (CTR), Tax, Tax M22 or Tax M47. (<b>G</b>) qRT-PCR of IL-17RB or Tax mRNAs in 293 cells transduced with lentiviruses expressing empty vector (CTR) or Tax. (<b>H</b>) NF-κB luciferase assay with lysates from 293 cells transduced with lentiviruses expressing control (CTR) or IL-17RB shRNA, and then transfected with empty vector (EV) or pCMV4-Tax, together with an NF-κB luciferase reporter and pRL-TK. IL-17RB mRNA and 18S rRNA were detected by RT-PCR (bottom panel). (<b>I</b>) qRT-PCR of IL-25 mRNA in Jurkat cells transduced with lentiviruses expressing empty vector (CTR) or Tax at multiplicities of infection (MOIs) of 5 and 10.</p

IL-17RB and IL-25 are essential for NF-κB activation and survival of HTLV-1 transformed cells.

<p>(<b>A</b>) Proliferation/viability assay of MT-2 and C8166 cells transduced with lentiviruses expressing control or IL-17RB shRNA using CellTiter-Glo (left). Cell counts of MT-2, C8166 and HUT-102 cells transduced with control or IL-17RB shRNA. (<b>B</b>) qRT-PCR of indicated mRNAs in HTLV-1 transformed cell lines transduced with lentiviruses expressing control or IL-17RB shRNA. (<b>C</b>) NF-κB EMSA using nuclear extracts from MT-2, C8166 and HUT-102 cells transduced with control, IL-17RB or IL-25 shRNAs (left). Western blots were performed with the indicated antibodies using whole-cell lysates from MT-2 and C8166 cells transduced with control, IL-17RB or IL-25 shRNAs (center and right). (<b>D</b>) qRT-PCR of CD25 and IL-25 mRNAs in MT-2 cells transduced with lentiviruses expressing control or IL-25 shRNA. (<b>E</b>) Western blots were performed with the indicated antibodies using whole cell lysates from MT-2 and MT-4 cells transduced with lentiviruses expressing control or IL-25 shRNAs. (<b>F</b>) qRT-PCR of CD40 and OX40 mRNAs in C8166 cells expressing CD40 or OX40 shRNAs (top). Proliferation/viability assay of C8166 cells expressing control, CD40 or OX40 shRNAs.</p

IL-17RB is overexpressed in HTLV-1 immortalized T cell clones and transformed cell lines.

<p>(<b>A</b>) Flow cytometric analysis of CD3/CD4/CD8/CD25 markers with T-MT-2 clone 2. (<b>B</b>) Differential gene expression of T cells at week 1 (top) and week 12 (bottom) compared to week 0 (parental T cells) analyzed using RNA-Seq and “DESeq” R package and plotted as an MA plot. DESeq plotMA displays differential expression (log-fold changes) versus expression strength (log average read count). (<b>C–E</b>) qRT-PCR of indicated mRNAs in T cell clones. (<b>F</b>) Flow cytometric analysis of IL-17RB was performed on the indicated immortalized HTLV-1 immortalized T-cell clones and HTLV-1+ cell lines (top). qRT-PCR of IL-17RB mRNA in HTLV-1+ and ATL cell lines (bottom). (<b>G, H</b>) qRT-PCR of IL-17RA and IL-25 mRNAs in HTLV-1+ and ATL cell lines.</p

A Critical Role for IL-17RB Signaling in HTLV-1 Tax-Induced NF-κB Activation and T-Cell Transformation

<div><p>Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis.</p></div

IL-17RB and IL-25 are essential for the HTLV-1-induced immortalization of T cells.

<p>(<b>A</b>) qRT-PCR of IL-17RB and IL-25 mRNAs in primary T cells co-cultured with lethally irradiated MT-2 cells after 0 (W0), 1 (W1) and 2 (W2) weeks. (<b>B</b>) <i>In vitro</i> immortalization assay using PBMCs transduced with lentiviruses expressing control (CTR), IL-17RB or IL-25 shRNAs and co-cultured with lethally irradiated MT-2 cells. (<b>C</b>) Flow cytometric analysis of CD4 and CD8 markers using immortalized PBMCs expressing control shRNA.</p

Expression and function of CXCR3 on CD4⁺ T cells in HBZ-Tg and HBZ-Tg/IFN-γ KO mice.

<p>(A) Splenocytes were obtained from mice, and the CXCR3 expression level in CD4⁺ T cells was evaluated by flow cytometry. Three mice of each strain were analyzed and the result was summarized in the graph. (B) The migration activity of CD4⁺ T cells towards CXCL10. CD4⁺ T cells were isolated from splenocytes using magnet beads. Murine recombinant CXCL10 was added at concentrations of 0, 200, or 500 ng/mL. Migrating cells were counted by flow cytometry.</p

CXCL10 is not associated with systemic inflammation in HBZ-Tg mice.

<p>(A) HBZ-Tg and CXCL10 KO mice were crossed to establish HBZ-Tg/CXCL10 KO mice. (B) HBZ-Tg/CXCL10 KO mice developed dermatitis. (C) At 24 weeks old, more than 50% of HBZ-Tg/CXCL10 KO mice developed dermatitis. (D) Splenocytes were obtained from CXCL10 KO mice and HBZ-Tg/CXCL10 KO mice. Cells were stained with anti-CD4, anti-CD44, anti-CD62L, and anti-Foxp3, and analyzed by flow cytometry. Among CD4⁺ T cells of HBZ-Tg/CXCL10 KO mice there were increased numbers of increased effector/memory and Foxp3 expressing cells.</p