Figure 4. Biodistribution of HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 fusion proteins.

<p>HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 fusion proteins were purified and labeled with ¹³¹I as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>. SCID mice were s.c. transplanted with CD30⁺ L540cy Hodgkin's lymphoma cells (2×10⁷/animal) and animals with established tumors (>0.5 cm³) were intravenously injected with ¹³¹I -labeled fusion proteins. Animals were sacrificed after 24 h, 48 h and 72 h (HRS3-scFv-IL12: 4 mice/group; HRS3-scFv-Fc-IL12: 3 mice/group), respectively, organs were recovered and tissue retention of radioactivity (%) was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>.</p

Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression

<p>The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair.</p

Galectin-3 prevents TCR synapse formation in late-stage T cells.

<p>(A) CD7⁻ and CD7⁺ T cells were activated by incubation with anti-CD3 mAb plus anti-human CD28 mAb or as control by an isotype-matched IgG1 (medium). Cells were stained for TCR-alpha/beta (green), CD7 (blue), CD3 (red) and for galectin-3 (yellow). Immunofluorescence was visualized by a LSM. Alternatively cells were stained with mAbs specific to CD7 and subsequently recorded for gal-3 expression by staining with the anti-human gal-3mAb or as control by an isotype-matched IgG and analyzed by flow cytometry. A representative experiment out of five is shown. Galectin-3 specific signals were quantitatively recorded by a LSM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells for each data point was recorded. (B) To monitor location of galectin-3 in lipid raft formation, isolated CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine for 24 hrs and then activated for various time intervals (1 min until 20 min) on coverslips coated with the agonistic anti-CD3 mAb plus anti-CD28 mAb. T cell stimulation was stopped with paraformaldehyde and cells were stained with anti-TCR-alpha/betamAb (blue), anti-CD7 mAb (red) and anti-galectin-3 mAb (yellow) together with CtxB (green) for lipid rafts staining. Immunofluorescence was visualized by a LSM and quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. T cell staining after 5 min stimulation of one representative experiment out of five is exemplarily shown. (C) CD7⁻ and CD7⁺ subsets of CD8⁺ CD45RO⁺ T cells were incubated with or without swainsonine and activated as described in (B). Frequencies of cells producing IFN-gamma and showing degranulation indicated by CD107a was monitored by flow cytometry. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#pone-0030713-g003" target="_blank">Fig. 3</a> represent the mean ± SEM of five experiments and were compared using a paired t-test. * p<0.05. FU: fluorescence unit; MFI: mean fluorescence intensity.</p

Figure 8. HRS3-scFv-IL12-Fc-IL2 fusion protein improves lysis of CD30⁺ tumor cells and represses tumor growth in immune-competent mice.

<p>(A) CD30⁺ and CD30⁻ MC38 tumor cells were pre-incubated for 1 h on ice with equimolar amounts of the indicated fusion proteins. Cells were washed extensively and co-cultured (5×10⁴ cells/well) for 48 hrs in round-bottom micro-titer plates together with freshly isolated, non-activated NK cells (each 5×10⁴ cells/well). Co-incubation with NK cells without fusion protein (medium) served as control. Cytotoxicity against CD30⁺ tumor cells was determined by a XTT-based assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>. Data represent the mean of triplicates ± standard error of mean (SEM). Statistical analyses were performed by Student's T test. A representative experiment out of three is shown. (B) Balb/c mice were transplanted with 9G10 hybridoma cells expressing the HRS3-specific anti-idiotypic mAb on the cell surface (n = 19) or for control with C10 hybridoma cells expressing an IgG1 with irrelevant specificity (n = 10) (2.5×10⁶ cells/mouse). At day 7, when the tumor was fairly established, mice received i. v. injections of the purified HRS3-scFv-IL12-Fc-IL2 fusion protein (50 µg/animal) or the same volume PBS for control (5–10 animals/group). Injections were repeated twice every second day. Tumor volume was recorded every 2–3 days. Mean values are shown; p values were determined by Student's T test.</p

TCR synapse formation is impaired in late-stage T cells.

<p>CD8⁺ CD45RO⁺ T lymphocytes were incubated on coverslips coated with the agonistic anti-CD3 mAb (UCHT-1) plus anti-CD28 mAb (CD28.2) or with the anti-CD2 mAbs (L303.1) and (L304.1) plus the anti-CD28 mAb (CD28.2). Cells were activated for various time intervals (1 min until 20 min), incubation was stopped by addition of paraformaldehyde, and cells were stained with TCR-alpha/betamAb (green) plus CD7 mAb (blue) or alternatively with CD2 mAb (blue) plus CD7 mAb (green) together with cholera toxin B (CtxB) (white) for lipid raft staining. Cells were analyzed on a LSM 510 with 630× microscope magnification. A minimum of 100 cells for each data point was recorded. Representative images after 0 min, 1 min, 5 min, 10 min and 20 min stimulation out of five independent experiments are shown. Synapse formation intensity (CTxB intensity) was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030713#s4" target="_blank">Materials and Methods</a>. A minimum of 100 cells of each cell population on each coverslip was recorded. Data represent mean scores from five experiments ± standard error of the mean and were compared using a paired t-test. * p<0.05. FU: fluorescence unit.</p

Biodistribution of HRS3-scFv-Fc-IL12 fusion protein.

1<p>Values represent the mean percentage of injected dose per g tissue (n = 3 mice).</p><p>doi:10.1371/journal.pone.0044482.t002</p

Figure 2. Western blot analysis of the anti-CD30 antibody-cytokine fusion proteins.

<p>(A) Supernatants containing HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 or HRS3-scFv-IL2 and HRS3-scFv-Fc-IL2 fusion proteins, respectively, were separated by SDS-PAGE under reducing (R) and non-reducing (NR) conditions. (B) Supernatants of 293T producer cell lines containing HRS3-scFv-Fc-IL2 and dual cytokine HRS3-scFv-IL12-Fc-IL2 fusion proteins, respectively, were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). Separated proteins were transferred to PVDF membranes and detected by staining the blots with anti-IL12 and anti-IL2 antibodies, respectively.</p

Figure 7. The HRS3-scFv-IL12-Fc-IL2 dual cytokine fusion protein bound to CD30⁺ cells activates resting NK cells to IFN-γ secretion.

<p>CD30⁺ and CD30⁻ MC38 cells (5×10⁴ cells/well) were incubated with equimolar amounts of anti-CD30 fusion proteins for 1 h on ice. Unbound fusion proteins were either present during the assay or washed out. Freshly isolated NK cells (5×10⁴ cells) were co-incubated for 48 hrs. IL2 (500 U/ml) was added for comparison. NK cell activation was recorded by monitoring IFN-γ secreted into the culture medium. Data represent the mean of triplicates ± standard error of mean (SEM). An experiment out of two with similar results is shown.</p

Figure 5. The HRS3-scFv-IL12-Fc-IL2 dual cytokine fusion protein bound to CD30⁺ cells induces T cell amplification and IFN-γ secretion.

<p>(A, B) Pre-activated T cells were incubated for 48 hrs with serial dilutions of equimolar amounts of the anti-CD30 cytokine fusion proteins or the cytokines IL2, IL12 or IL2 plus IL12. Viable cells were counted; dead cells were excluded by trypan blue exclusion. (C, D) CD30⁺ and CD30⁻ MC38 cells (5×10⁴ cells/well) were incubated with equimolar amounts of anti-CD30 fusion proteins for 1 h on ice. Unbound fusion proteins were either present during the assay (C) or washed out (D). Pre-activated T cells (5×10⁴ cells) were co-incubated for 48 hrs. IL2 (500 U/ml) was added for comparison. T cell activation was recorded by monitoring IFN-γ secreted into the culture medium. Data represent the mean of triplicates ± standard error of mean (SEM). An experiment out of two with similar results is shown.</p

Figure 1. Schematic diagram of the anti-CD30 antibody-cytokine fusion proteins used in the study.

<p>IL12 represents the p35–p40 single-chain IL12. hi: IgG1 hinge region; IgG1-Fc: IgG1 CH2CH3.</p