In silico tumor growth dynamics in a typical simulation of an untreated mouse.

<p>From upper-left, following clock-wise way, cancer cells dynamics at day 7, 14, 21, 28 following tumor cell inoculation.</p

In silico tumor growth dynamics in a typical simulation of a mouse treated with activated OT-1 cells and anti-CD137.

<p>From upper-left, following clock-wise way, OT-1 cells dynamics at day 7, 14, 16, 20, 24, 28.</p

The conceptual model.

<p>This figure conceptually explains the biological workflow. It represents the first step for successfully modeling the scenario. Arrows represent the logical flow. Labels explain the interactions or the actions (i.e., status change, activities and functions) by the involved entities.</p

Tumor-area curves of virtual mice receiving OT-1 T cells and anti-CD137 mAb.

<p>B16-OVA cells were injected toward the immediate neighborhood in the center of lattice at timestep 0. Immunotherapy on day 3 with 100 µg of rat IgG (control) or anti-CD137 mAb i.p. were simulated injecting the compound around the lattice walls. Virtual mice that also received in the same day activated or resting OT-1 T cells i.v. were simulated in the same way.</p

SimB16 parameters with known values retrieved from immune system specific literature.

<p>CA stands for Chemo-Attractans.</p

B cells are responsible for the reduction of liver gene transfer efficiency.

<p>A) Bioluminescence experiments, assessing <i>in vivo</i> luciferase activity in the liver region of mice injected <i>i.v.</i> with HDA encoding luciferase under a liver specific promoter (5×10¹⁰ viral particles/mouse) 48 h prior the luciferase measurement assay (similar differences were observed on days 4 and 6 -data not shown-). Mice were WT BALB/C mice or belonged to the indicated mutant strains in the background. The table under the graph indicates the immune cells absent in each modified strain. B) Relative plasmid DNA content measured by Quantitative PCR in the liver of mice shown in A excised on day 7 following vector administration. C) <i>In vivo</i> luciferase expression in the C57/BL6 mouse strain including WT, Rag1KO and µMT-KO mice that are selectively deficient in B cells. D) Experiments in mice shown in C performed to quantify viral DNA content in the liver as in B. WT, Wild-type; Rag1KO, BALB/C Rag1^−/− (C.129S7(B6)-Rag1^tm1Mom/J; Rag2KO, BALB/C-Rag^−/−IL-2Rγ^−/−BALB/C; B6µMT, (B6.129S2-Ighm^tm1Cgn/J; B6.Rag1KO, Rag1^−/− B6 (B6.129S7-Rag1^tm1Mom/J, NKs, natural killer; IgG, immunoglobulin G; HDA-<i>luc</i>, helper-dependent adenoviral vectors encoding luciferase. Experiments were performed twice.</p

IgM mediates the mitigation of liver gene transfer by a HDA vector.

<p>A) Rag1KO mice were passively <i>i.v.</i> transferred with serum of non-immunized WT mice or similar serum samples reduced by β-mercaptoethanol. Level of gene transfer of WT mice is provided as a control. Liver gene transfer of luciferase by HDA vector (dose of 5×10¹⁰ viral particles/mouse) was assessed by bioluminescence as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085432#pone-0085432-g001" target="_blank">Figure 1</a>. B) Experiments as in A but passively transferring 2 doses of 130 μg of IgG and IgM (both purified by affinity chromatography from serum) or 400 μl of serum from WT mice. C) B6.µMT-KO mice were passively transferred <i>i.v.</i> with 2 doses of 130 μg of serum IgM purified by affinity chromatography and analyzed for liver gene transduction as in A. Wild-type, WT; Rag1KO, BALB/C Rag1^−/− (C.129S7(B6)-Rag1^tm1Mom/J; B6.µMT, (B6.129S2-Ighm^tm1Cgn/J; B6.Rag1KO, pIgG, polyclonal immunoglobulin G; pIgM, polyclonal immunoglobulin M; HDA-<i>luc</i>, helper-dependent adenoviral vectors encoding luciferase. Experiments were performed twice.</p

IgM antibodies bind adenoviral capsids.

<p>ELISA tests performed on HDA-<i>luciferase</i> coated 96-well plates sequentially incubated with the indicated antibodies and secondary antibodies. Results are presented as absorbance at 450 nm ± standard deviation at the time of stopping the reaction. A) Binding of the indicated polyclonal non-immune IgM and the monoclonal hybridoma-produced IgM and IgG were tested for their ability to bind capsids and were revealed with the proper subclass specific secondary reagents. B) ELISA Experiments as in A extending the developing time to detect potentially weaker interactions and testing WT and Ig-free Rag1KO serum as well as monoclonal IgG and IgM. The background reactivity of secondary antibodies was virtually non-existent. Wild-type, WT; Rag1KO, BALB/C Rag1^−/− (C.129S7(B6)-Rag1^tm1Mom/J; pIgM, polyclonal immunoglobulin M; mIgM, monoclonal immunoglobulin M, mIgG, monoclonal immunoglobulin G. Experiments were repeated twice in replicates and statistical comparisons were performed as described in Materials and Methods section.</p

Monoclonal IgM inhibits gene transfer of Huh7 hepatocellular carcinoma cells.

<p><i>In vitro</i> gene transfer of 2.5×10¹⁰ vp of HDA-<i>luciferase</i> to Huh7 cells (MOI 50.000) in the presence of 2% fetal bovine serum and 5 µg of monoclonal IgM (mIgM) directed to an irrelevant antigen (non crossreactive human AE2 transporter), monoclonal IgG which specifically recognized human interferon alpha-5 or serum polyclonal IgM from adenovirus naïve mice (pIgM) as indicated. Relative content of vector DNA was assessed by real time PCR in quadruplicate samples. pIgM, polyclonal immunoglobulin M; mIgM, monoclonal immunoglobulin M, mIgG, monoclonal immunoglobulin G; HDA-<i>luc</i>, helper-dependent adenoviral vectors encoding luciferase. The experiment was repeated three times.</p

Monoclonal IgM of a specificity unrelated to adenovirus reduces liver gene transfer by HDA vectors.

<p>A) Representative photographs of light emission from the liver region of mice intravenously injected with 5×10¹⁰ vp/mouse 48 h prior to bioluminescence (left panel). When indicated, Rag1KO were pre-injected twice with 130 µg of monoclonal IgM of irrelevant specificity. The dot graph on the right shows quantitative data from the same experiment. Experiments B and C were performed seven days following gene transfer with progressively reduced doses of HDA-<i>luciferase</i> (3.6×10¹⁰ vp/mouse and 2.2×10⁹ vp/mouse, respectively). Gene transfer was assessed by measuring luciferase activity ex-vivo in homogenates from liver samples (left panel). The relative liver content of vector DNA was measured by quantitative PCR (right panel). WT, Wild-type; Rag1KO, BALB/C Rag1^−/− (C.129S7(B6)-Rag1^tm1Mom/J; pIgM, polyclonal immunoglobulin M; mIgM, monoclonal immunoglobulin M, mIgG, monoclonal immunoglobulin G.; HDA-<i>luciferase</i>, helper-dependent adenoviral vectors encoding luciferase; RLU, Relative Light Unit. Experiments were performed twice.</p