Migration capacity and therapeutic potential as tumor vaccine of <i>cblb</i>−/− BMDCs.

<p>(A) 0.125 mg of TB in FIA was injected in a total volume of 50 µl per hock into wildtype recipients. On day two CFSE labeled wildtype and TAMRA labeled <i>cblb</i>−/− BMDCs were injected in the hock in wildtype control recipients and TB injected recipients. 24 hours thereafter migration of the BMDCs in the draining lymph node and non-draining lymph node (not shown) was measured by flow cytometry. Data represent mean value ± SEM, n = 4 mice per group, two independent experiments. (B) 1×10⁵ B16-OVA cells were injected subcutaneously into the left flank of wildtype recipients. Tumor-bearing mice were subcutaneously vaccinated into the opposite right flank on day five with 2×10⁵ 10 µM SIINFEKL primed semi-mature wildtype <i>versus cblb</i>−/− BMDCs. Tumor growth was monitored thereafter every two/three days. Control animals received PBS. All data points represent tumor volume (mean value ± SEM, n = 4 mice), representative of two independent experiments is shown. (C) Survival of the same animals described in (B) was monitored.</p

Macropinocytosis is not altered by <i>cblb</i>−/− deficiency.

<p>(A) Macropinocytosis was quantified by incubating immature day seven BMDCs with 2 mg/ml FITC-dextran at 37°C or at 4°C (negative control). After 2 and 4 hours, uptake was stopped and analyzed by FACS. Data represent mean value ± SEM; n = 4. (B) Representative dot plot of 4 independent experiments is shown.</p

Cytokine and chemokine production by wildtype <i>versus cblb</i>−/− BMDs after stimulation with different TLR agonists.

<p>Wildtype and <i>cblb</i>−/− BMDCs were stimulated on day seven of culture with 1 µg/mL LPS or 100 nM CpG overnight for, IL-12p70, IL-10, IL-1α, IL-6, TNF-α, IFN-γ, KC, MIP-1α and MCP-1 measurement from cell culture supernatants were performed using Bioplex-Technology. Data represent mean value ± SEM of at least 4 independent experiments, *p<0.05 wildtype BMDCs <i>versus cblb</i>−/− BMDCs.</p

Nitric oxide availability and eNOS expression in ACE2^-/y mice.

<p>Plasma nitrite content <b>(A)</b> and 24-hour urinary excretion (<b>B</b>) of nitrate plus nitrite (NOx) in ACE2^-/y (n = 6) and WT (n = 6) mice. (<b>C)</b> eNOS activity, evaluated by ^•NO release in aorta rings upon application of ACh (DAF-FM bioassay) (ACE2 n = 5; WT n = 6). (<b>D</b>) arginase activity in aorta homogenates of ACE2^-/y (n = 6) and WT (n = 6) mice. Representative western blot and densitometric analysis of eNOS (<b>E</b>) and phosphorylated Ser1177-eNOS (<b>F</b>) protein levels in thoracic aortas from ACE2^-/y (n = 5) and WT (n = 5) mice. (<b>G</b>) Relative expression of phospho-eNOS normalized to the total eNOS levels. (<b>H</b>) eNOS mRNA expression levels (real time PCR analysis). Data are expressed as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i><0.001, Student’s <i>t</i> test.</p

AKT expression in ACE2^-/y mice.

<p>Representative western blots and densitometric analysis of HSP90 (<b>A</b>), AKT (<b>B</b>) and P-AKT (<b>C</b>) aortic protein levels. (<b>D</b>) P-AKT/AKT ratio. Results are representative of four to six separate experiments. Data are expressed as mean ± SEM.</p

Mean tumor volume of B16ova melanoma bearing mice <i>(gp100-pulsed DCs).</i>

<p>Mean tumor volume [mm³] ± SEM.</p

<i>Cblb</i> knockdown in CD8⁺ T cells confers resistance to the inhibitory effects of TGFβ.

<p>Mouse CD8⁺ T cells were nucleofected with <i>cblb</i> siRNA or control siRNA and stimulated with anti-CD3 and anti-CD28 in the presence or absence of TGFβ. The production of IFNγ by CD8⁺ T cells was efficiently suppressed by exposure to TGFβ while <i>cblb</i> knockdown cells still produced high levels of IFNγ. (A) Silencing efficacy of <i>cblb</i> siRNA was analyzed by immunoblotting. (B) Supernatants were taken two days post-transfection and the amount of IFNγ secreted was measured by BioPlex technology. IFNγ levels of each group without TGFβ were set 100%. Means ± SEM of three independent experiments are shown.</p

Oxidative stress and ROS degrading enzymes in ACE2^-/y mice.

<p>Concentration of the TBARS malondialdehyde (MDA) in plasma (<b>A</b>), urine (<b>B</b>), and aorta (<b>C</b>) of ACE2^-/y (n = 5) and WT (n = 5) mice. (<b>D</b>) Representative fluorescent photomicrographs (left) and quantification (right) showing <i>in situ</i> detection of ^⚫O₂^- by confocal microscopy (7 μm sections of thoracic aorta). Aortic sections were labeled with the oxidative dye dihydroethidium (2 μM/L), which is oxidized to EtBr in the presence of ^⚫O₂^- and gives red fluorescence. High EtBr fluorescence was found in aortic walls of ACE2^-/y mice, whereas the signal was almost undetectable in aortas of WT animals. (n = 5–6). (<b>E</b>) H₂O₂ levels in aorta of ACE2^-/y mice. Activity (<b>F, I</b>) and mRNA expression by real time PCR (<b>G, H, J</b>) of ROS degrading enzymes, SOD (<b>F, G, H</b>) and catalase (<b>I, J</b>) in aorta of ACE2^-/y (n = 4) and WT (n = 5) mice. (<b>K</b>) aorta GPx activity. mRNA expression level of UCP1 (<b>L</b>), UCP2 (<b>M</b>), and UCP3 (<b>N</b>) in aorta of ACE2^-/y mice. *<i>P</i><0.05, **<i>P</i><0.01 (Student <i>t</i> test).</p

ACT of polyclonal <i>cblb</i>-silenced T cells reduces tumor growth rates <i>in vivo</i>.

<p>(A) Tumor therapy schedule. Wild type mice s.c. injected with 1×10⁵ B16ova cells were vaccinated with (B-F) SIINFEKL-pulsed DCs or (G) gp100-pulsed DCs on days 6 and 13. Twenty-four hours after DC vaccination, either control or <i>cblb</i> siRNA nucleofected polyclonal CD8⁺ T cells (5×10⁶) were i.v. injected into tumor bearing mice. (B) Tumor growth of untreated mice, (C) DC vaccinated mice, (D) mice treated with DCs plus control-siRNA nucleofected CD8⁺ T cells, and (E) DCs plus <i>cblb</i> siRNA silenced CD8⁺ T cells are shown. Tumor volume was measured every second day. Representative results out of two independent experiments are shown (n = 7 - 12 mice per group). (F) Survival of the animals described in (B–E) was monitored and the respective Kaplan-Maier curves are given. p = 0.03 (DC+CD8⁺<i>cblb</i> siRNA versus DC+CD8⁺ control siRNA). (G) Survival of B16ova-challenged wild type mice treated as in (B-E) but vaccinated with gp100-pulsed DCs. Respective Kaplan-Maier curves are given (n = 3 - 4 mice per group).</p

<i>cblb</i> silencing substantially enhances tumor infiltration of T cells.

<p>Flow cytometric analysis of tumor-infiltrating lymphocytes 7 days (A, B, D, E) or 4 days (C) after a single ACT in combination with DC vaccination into wild type mice was performed. (A) CD8⁺ T cell infiltrates gated on whole tumor, (B) CD8⁺ T cell infiltrates gated on total CD45⁺ leukocytes, (C) SIINFEKL-specific infiltrates gated on CD8⁺ cells (D) CD4⁺CD25^- T cell infiltrates and (E) CD4⁺CD25⁺ T cell infiltrates gated on total CD45⁺ leukocytes are shown. Means ± SEM of two independent experiments are shown (n = 7 - 8 mice per group). (A) p = 0.06 (DC+CD8⁺<i>cblb</i> siRNA versus DC+CD8⁺ control siRNA) (B-E) n.s. (DC+CD8⁺<i>cblb</i> siRNA versus DC+CD8⁺ control siRNA).</p