Primary human T-cell activation is inhibited upon HTLV FP treatment.

<p>Human peripheral T lymphocytes were isolated from whole blood and activated using CD3 and CD28 antibodies, in the presence of HTLV FP or HTLV Scr at 10μM. Media was collected 48 hours following activation and secretion of IL2, IFN-γ and IL10 was measured by ELISA assay. HTLV FP treatment inhibits IL2 and IFN-γ and elevates IL10 secretion from primary human peripheral T-cells. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05; ***<i>P</i><0.001; ns, not significant.</p

Detection of the immune modulatory region within the HTLV FP.

<p>(<i>A</i>) Sequences of shorter peptides derived from the full length HTLV FP. (<i>B</i>) MOG_35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP derived peptides at 10μM and proliferative responses were assessed as described above. The HTLV FP and HTLV FP_5-13 inhibit T-cell proliferation with higher potency than HTLV FP_9-22 and HTLV FP_14-22. The data is presented as mean inhibition of proliferation. n = 12. (<i>C</i>) MOG_35-55–antigen specific T cells were activated by either (<i>i</i>) irradiated MOG_35-55 presenting APCs (black), (<i>ii</i>) antibodies against CD3 and CD28 (white), or (<i>iii</i>) PMA and Ionomycin (gray) in the presence of HTLV FP_5-13 and HIV FP_5-13 at 10μM. Proliferative responses were assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. (<i>D</i>) Secondary structures of HTLV FP derived peptides as revealed by CD spectroscopy. CD spectra were measured at 25μM in 5mM HEPES buffer containing 1% lyso-phosphatidylcholine (LPC). The HTLV FP and HTLV FP_5-13 exhibit a typical α-helical curve while the HTLV FP_9-22 and HTLV FP_14-22 exhibit a typical random coil. (<i>E</i>) MOG_35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP_5-13 D/L enantiomers at 10μM and proliferative responses were assessed as described above. Both HTLV FP_5-13 enantiomers inhibit T-cells proliferation with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05;<i>**P</i><0.01;***<i>P</i><0.001.</p

Modulation of Th1/Th2 gene expression and cytokine secretion from activated T-cells by the HTLV FP.

<p>MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of the HTLV FP at 10μM. (<i>A</i>) Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces mRNA expression of Th1 specific genes (<i>IFNG</i>, <i>LTA</i>, and <i>STAT4</i>) and elevates mRNA expression of Th2 specific genes (<i>IL4</i> and <i>IL10</i>). The data is presented as arbitrary units. n≥3. (<i>B</i>) Media was collected 24 hours following activation and secretion of cytokines was measured by ELISA assay. IFN-γ secretion is inhibited, IL4 secretion is elevated and TNFα is not affected by the HTLV FP. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05; **<i>P</i><0.01<i>;***P</i><0.001.</p

Administration of HTLV FP alleviates MOG35-55-induced EAE.

<p>(<i>A-B</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of HTLV FP or HTLV Scr at 10μM. (<i>A</i>) The proliferative responses were assessed by H³-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The data is presented as mean inhibition of proliferation. n = 12. (<i>B</i>) IFN-γ was measured by ELISA assay. n = 12. (<i>C-D</i>) EAE was induced in C57BL/6 female mice that were treated with a single dose of HTLV FP, HTLV Scr or vehicle. Two indexes to measure clinical disease severity are displayed. (<i>C</i>) Direct clinical measurement of EAE phenotype in a 5-point scale with increased disease symptoms correlating with higher score value. The data is presented as mean EAE clinical score. (<i>D</i>) Mice were weighed the day before EAE induction, and the change (as a percentage) in weight was recorded. The data is presented as mean change from the initial weight. One-way ANOVA was used for statistical analysis. ^<b>a</b> HTLV FP ≠ Vehicle; ^<b>b</b> HTLV FP ≠ Vehicle & HTLV Scr; ^<b>c</b> HTLV FP ≠ HTLV Scr. ^<b>a,b,c</b><i>P</i><0.05. (<i>E</i>) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. The proliferative responses were assessed by H³-thymidine incorporation assay and normalized to the proliferation of non-activated T-cells. The data is presented as counts per minute (CPM). n = 7. (<i>F-G</i>) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. Media was collected 48 hours following activation and secretion of IFN-γ and IL4 was measured by ELISA assay. HTLV FP treatment inhibits IFN-γ and elevates IL4 secretion from MOG35-55-reactive T-cells. n>8. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05;**<i>P</i><0.01;***<i>P</i><0.001; ns, not significant.</p

CD spectra analysis of the HTLV FP derived peptides by the CDNN secondary structure analysis program.

<p>CD spectra analysis of the HTLV FP derived peptides by the CDNN secondary structure analysis program.</p

Inhibition of T-cell activation by the HTLV FP.

<p>(<i>A</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of several viral derived FPs at 10μM. The proliferative responses were assessed by H³-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The HTLV FP inhibits T-cell proliferation with the same potency as the HIV FP. The data is presented as mean inhibition of proliferation. n = 12. (<i>B</i>) MOG_35-55–antigen specific T cells were activated by either (<i>i</i>) irradiated MOG_35-55 presenting APCs (black), (<i>ii</i>) antibodies against CD3 and CD28 (white), or (<i>iii</i>) PMA and Ionomycin (gray) in the presence of the HTLV and HIV FPs at 10μM. Proliferation was assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. **<i>P</i><0.01;***<i>P</i><0.001.</p

Reduced T-bet expression and elevated Gata3 expression induced by the HTLV FP.

<p>(<i>A-B</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of the HTLV FP at 10μM. Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces <i>TBX21</i> (T-bet) mRNA expression and elevates <i>GATA3</i> mRNA expression. The data is presented as arbitrary units. n≥3. (<i>C-H</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of either HTLV or HIV FP at 10μM. Samples were fixed in 4% PFA and stained with anti T-bet-APC and anti Gata3-FITC antibodies (<i>C-D</i>) 24, (<i>E-F</i>) 48 and (<i>G-H</i>) 72 hours following activation. Each time point is represented as cell count vs. APC or FITC fluorescence histogram. Analysis was performed using LSR-II flow cytometer (BD) and FlowJo cell analysis software (FlowJo, LLC). The HTLV FP reduces T-bet expression and elevates Gata3 expression. n = 3. One-way ANOVA was used for statistical analysis. ns, not significant; *<i>P</i><0.05<i>;** P<0</i>.<i>01</i>.</p

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

<div><p>Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.</p></div

Expression patterns of H1299 in response to co-cultivation with CAFs.

<p>(A) Three principal expression patterns of H1299 cultivated with HK3-T. (B) A heat-map depicting genes that exhibit Pearson correlation of at least 0.9 to representative bait(s) from each pattern shown in A. Beneath is the percentage of IFN targets in each list based on the ‘Interferome database’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061353#pone.0061353-Samarajiwa1" target="_blank">[30]</a>. (C) QRT-PCR analysis of a representative gene from each expression pattern. (D) The H1299 panel was treated with the designated IFNs for 24 h. Shown is a QRT-PCR analysis of MX1 expression.</p

Mutant p53 counteracts IFNβ by SOCS1-mediated attenuation of STAT1 phosphorylation.

<p>(A) Cells were treated with IFNβ for 16 h, fixed and sorted by an “Image stream” FACS. The upper panel depicts representative images from each condition and the graph represents the mean pixel intensity of STAT1 positive cells for the entire population. (B) Same as in A, here the graph shows the similarity between p-STAT and DAPI staining, thereby quantifying both the expression and localization of pSTAT1. (C) Cells were treated with IFNβ for the designated durations, shown is a graph depicting nuclear p-STAT1. (D) The cells were also collected for RNA analysis and a QRT-PCR for SOCS1 expression was performed. (E) H1299¹⁷⁵ cells were introduced with RNAi against LacZ as a control or against SOCS1. p = 0.002 (F) Cells were then treated with IFNβ for 24 h. Shown is a QRT-PCR analysis of MX1 expression. p<0.05. (G) Cells were seeded in trans-wells in serum-free media and treated with IFNβ for 24 h. Migrating cells were collected and counted.</p