Thymic alteration in LynΔN mice is dependent on TNFR1 signaling.

<p><b>A)</b> Total thymocyte numbers from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Total thymocytes from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were analyzed by flow cytometry for CD4 and CD8 expression, as shown in this representative flow cytometry profile. <b>C)</b> Proportion of all thymocyte subsets was quantified for LynΔN/TNFR1+/− and LynΔN/TNFR1−/− mice; each dot represents one individual mice. For these experiments, n = 5 for TNFR1+/−, TNFR1−/−, LynΔN/TNFR1+/− and n = 10 for LynΔN/TNFR1−/−. P value, Student <i>t</i> test for unpaired data.</p

TNF specific cell death is increased in LynΔN thymocytes.

<p><b>A)</b> Semi-quantitative RT-PCR analysis of TNFα and actin using RNA isolated from 2-week-old LynΔN and control thymi. <b>B)</b> Freshly isolated thymocytes from control or LynΔN mice were left untreated or incubated with either TNFα (50 ng/ml) for 18 h. Then, AnnexinV positive cells were determined by flow cytometry. Results are expressed as the percentage of TNF specific cell death (percentage of AnV+ cells in TNF stimulated condition – percentage of AnV+ cells in control condition). For this experiment, n = 3 for WT and n = 4 for LynΔN mice. P value, Student <i>t</i> test for unpaired data. <b>C)</b> Freshly isolated thymocytes from control or LynΔN mice were left untreated or incubated with either TNFα (50 ng/ml) for 10 and 30 minutes. Degradation and phosphorylation of IκBα was assayed by western blot analysis of total cell extracts. Hsp60 was used as a loading control.</p

Normal splenic T cell but abnormal B cell distribution in LynΔN transgenic mice.

<p><b>A)</b> Total splenocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Splenic T cells from LynΔN and control mice were analyzed by flow cytometry (CD4 and CD8 expression), as shown in this representative flow cytometry profile. <b>C)</b> Proportion and <b>D)</b> cell numbers of CD4+ and CD8+ splenic T cell of 2-week old control and LynΔN mice were quantified; Lines indicated the mean, and each dot represents one individual mice. For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data. <b>E)</b> Splenic B cells from LynΔN and control mice were analyzed by flow cytometry (CD19 and B220 expression), as shown in this representative flow cytometry profile. <b>F)</b> Proportion and <b>G)</b> number of CD19+/B220+ splenic B cell of 2-week old control and LynΔN mice were quantified; Lines indicated the mean, and each dot represents one individual mice. For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data.</p

Abnormal thymocyte maturation in LynΔN transgenic mice.

<p><b>A)</b> Representative thymi from 2-week old control and LynΔN mice. Bare scale : 0.5 cm. <b>B)</b> Total thymocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>C)</b> Total thymocytes from LynΔN and control mice were analyzed by flow cytometry for CD4 and CD8 expression, as shown in this representative flow cytometry profile. <b>D)</b> Proportion and <b>E)</b> number of all thymocyte subsets of 2-week old control and LynΔN mice were quantified; Lines indicate the mean, and each symbol represents one individual mouse<b>.</b> For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data.</p

Splenic alterations in LynΔN mice are dependent on TNFR1 signaling.

<p><b>A)</b> Total splenocyte numbers from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Splenic B cells from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were analyzed by flow cytometry (CD19 and B220 expression), as shown in this representative flow cytometry profile. <b>C)</b> Proportion of CD19/B220 splenic B cell was quantified for LynΔN/TNFR1+/− and LynΔN/TNFR1−/− mice; each dot represents one individual mice. For these experiments, n = 5 for TNFR1+/−, TNFR1−/−, LynΔN/TNFR1+/− and n = 10 for LynΔN/TNFR1−/−. P value, Student <i>t</i> test for unpaired data.</p

TCR dependent proliferation and apoptosis are unaltered in total thymocytes of LynΔN mice.

<p><b>A)</b> Apoptosis index determined by TUNEL staining (green staining) was analyzed onto frozen thymi sections of 2-week old LynΔN and control mice. <b>B)</b> AnnexinV positive cells were analyzed onto freshly isolated thymocytes from control and LynΔN transgenic mice (n = 6 in each group) by flow cytometry. <b>C)</b> Total thymocytes from LynΔN and control mice were stimulated or not with 10 µg/ml of plate-coated anti-CD3 for the indicated time periods and AnnexinV+/PI+ dead cells were determined by flow cytometry. <b>D)</b> Total thymocytes from LynΔN and control mice were stimulated with 10 µg/ml of plate-coated anti-CD3 plus soluble anti-CD28 (2 µg/ml) mAbs for 72 h. 10 µM BrdU was added for 16 hours, and proliferation was measured by BrdU incorporation. Results are expressed as mean ± SD. Data are representative of at least 3 independent experiments.</p

Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux

<p>Metastatic renal cell carcinomas (mRCC) are highly vascularized tumors that are a paradigm for the treatment with antiangiogenesis drugs targeting the vascular endothelial growth factor (VEGF) pathway. The available drugs increase the time to progression but are not curative and the patients eventually relapse. In this study we have focused our attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives only suboptimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neoadjuvant setting, we exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which downregulated the activity of the lysosomal protease CTSB (cathepsin B) and led to incomplete autophagic flux. Amino acid deprivation initiates autophagy enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib stimulated the expression of ABCB1 (ATP-binding cassette, sub-family B [MDR/TAP], member 1), which participates in the accumulation of the drug in autolysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar or the permeabilization of lysosome membranes with Leu-Leu-O-methyl (LLOM) resensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50. Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on our results we propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.</p