c-FLIP_L protects BMDE from TNF-α-induced apoptosis.

<p>A-B: BMDE from c-FLIP^f/f Lysm-Cre, c-FLIP^f/f Lysm-Cre S Tg⁺, or c-FLIP^f/f Lysm-Cre L Tg⁺ mice were cultured with 4-OHT or EtOH for 4 days and then stimulated or not with 50 ng/ml TNF-α for 24 h. Apoptosis rates were determined by measuring the percent of Annexin V⁺/7AAD⁺ cells within the CD11b^int CCR3⁺ population by flow cytometry. Representative FACS plots are shown in A. Numbers indicate the frequency of Annexin V⁺/7AAD⁺ eosinophils in each sample. B: The effect of TNF-α on apoptosis was determined by dividing the percent Annexin V⁺/7AAD⁺ cells in each TNF-α-treated sample by that in the paired control sample. The data were obtained in three independent experiments. Error bars represent standard deviations. *, p<0.05; **, p<0.01 (Student's <i>t</i>-test). C: Frequency of Annexin V⁺ BMDE with (right) or without (left) treatment with anti-Fas antibody. Filled red histograms represent 4-OHT-treated c-FLIP^f/f cells, and open blue histograms represent 4-OHT-treated c-FLIP^f/f ER-Cre cells.</p

Summary of mouse genetic models used for <i>in vitro</i> experiments.

<p>Summary of mouse genetic models used for <i>in vitro</i> experiments.</p

A model for c-FLIP as a molecular switch between pro- and anti-apoptotic TNF-α-mediated signaling in eosinophils.

<p>A: Upon TNFRI signaling, c-FLIP_L inhibits caspase 8 cleavage. The c-FLIP_L cleavage product p43FLIP mediates TNFRI-mediated activation of NF-κB, which upregulates c-FLIP, creating a positive feedback loop that protects eosinophils from TNF-α-mediated apoptosis. B: In the absence of c-FLIP, TNFRI signaling results in caspase 8 cleavage and apoptosis. TNFRI-mediated activation of NF-κB fails to protect eosinophils from apoptosis because NF-κB cannot upregulate c-FLIP.</p

c-FLIP_L is required to protect eosinophils from TNF-α-mediated apoptosis <i>in vivo</i>.

<p>A–B: Frequency of eosinophils in day 3 thioglycollate-elicited PEC from c-FLIP^f/f, c-FLIP^f/f Lysm-Cre, c-FLIP^f/f Lysm-Cre S Tg⁺, c-FLIP^f/f Lysm-Cre L Tg⁺, c-FLIP^f/f Lysm-Cre TNF-α^−/− mice. Representative FACS plots are shown in A. Numbers indicate the frequency of CD11b^int CCR3⁺ eosinophils in each sample. Each data point in B represents an individual mouse (n = 7 for c-FLIP^f/f, n = 12 for c-FLIP^f/f Lysm-Cre, n = 5 for c-FLIP^f/f Lysm-Cre S Tg⁺, n = 5 for c-FLIP^f/f Lysm-Cre L Tg⁺, and n = 4 for c-FLIP^f/f Lysm-Cre TNF-α^−/−). Horizontal lines indicate geometric means, and error bars represent 95% CI. **, p<0.01 vs. c-FLIP^f/f Lysm-Cre (Mann-Whitney).</p

Summary of mouse genetic models used for <i>in vivo</i> experiments.

<p>Summary of mouse genetic models used for <i>in vivo</i> experiments.</p

TNF-α upregulates c-FLIP in BMDE.

<p>A: Representative cytospin of BMDE. B: c-FLIP expression was measured by qPCR in WT BMDE before and after stimulation with 50 ng/ml TNF-α for 24 h. The data were obtained in four independent experiments. **, p<0.01 (Student's <i>t</i>-test).</p

c-FLIP is required for the survival of thioglycollate-elicited eosinophils <i>in vivo</i>.

<p>A–B: Frequency of CD11b^int CCR3⁺ eosinophils in peripheral blood from c-FLIP^f/f and c-FLIP^f/f Lysm-Cre mice. Representative FACS plots are shown in A. The absolute number of eosinophils/µl peripheral blood is plotted in B. Bars represent geometric means, and error bars represent 95% CI. NS, not significant (Mann-Whitney). C: Absolute numbers of eosinophils in day 3 thioglycollate-elicited PEC samples as determined by differential counting or flow cytometry (CD11b^int CCR3⁺). Bars represent geometric means, and error bars represent 95% CI. n = 10 (c-FLIP^f/f), n = 8 (c-FLIP^f/f Lysm-Cre). *, p<0.05; **, p<0.01 (Mann-Whitney). D: Experimental setup for bone marrow chimera experiments. E: Absolute numbers of eosinophils in day 3 thioglycollate-elicited PEC samples in bone marrow chimeric mice as determined by differential counting. Each data point represents an individual mouse (n = 2 KO chimeras, n = 6 mixed chimeras). For mixed chimeras, gray squares represent the total number of PEC eosinophils, black circles represent the number of PEC eosinophils from the c-FLIP^f/f donor, and open circles represent the number of PEC eosinophils from the c-FLIP^f/f Lysm-Cre donor. Horizontal lines represent geometric means, and error bars represent 95% CI. **, p<0.01 vs. total PEC eosinophils and vs. WT PEC eosinophils in mixed chimeric mice (Mann-Whitney).</p

Data_Sheet_1_Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases.docx

Autophagy, a highly conserved intracellular process, has been identified as a novel mechanism regulating T lymphocyte homeostasis. Herein, we demonstrate that both starvation- and T cell receptor-mediated autophagy induction requires class I phosphatidylinositol-3 kinases to produce PI(3)P. In contrast, common gamma chain cytokines are suppressors of autophagy despite their ability to activate the PI3K pathway. T cells lacking the PI3KI regulatory subunits, p85 and p55, were almost completely unable to activate TCR-mediated autophagy and had concurrent defects in PI(3)P production. Additionally, T lymphocytes upregulate polyinositol phosphatases in response to autophagic stimuli, and the activity of the inositol phosphatases Inpp4 and SHIP are required for TCR-mediated autophagy induction. Addition of exogenous PI(3,4)P2 can supplement cellular PI(3)P and accelerate the outcome of activation-induced autophagy. TCR-mediated autophagy also requires internalization of the TCR complex, suggesting that this kinase/phosphatase activity is localized in internalized vesicles. Finally, HIV-induced bystander CD4+ T cell autophagy is dependent upon PI3KI. Overall, our data elucidate an important pathway linking TCR activation to autophagy, via induction of PI3KI activity and inositol phosphatase upregulation to produce PI(3)P.</p

Pin1 modulates PU.1 protein stability.

<p>(A) Immunoblot analysis of PU.1 protein expression in WT and Pin1-null FL-cultured bone marrow-derived DC (FLDC) and primary MEF. For FLDC, the immunoblot shown is representative of cells derived from 3 different mice. For MEF, the immunoblot shown is representative of 3 different experiments. (B) Quantitation of PU.1 mRNA expression from WT and Pin1-null FLDC (n = 5) and MEF (n = 3). (C) GST-Pin1 pull down in WT and Pin1-null MEF lysates. 1 mg of total lysate was incubated with GST alone, WT GST-Pin1, or WW GST-Pin1 for 2 hours. After binding, beads were washed, resuspended in SDS-Page sample buffer, boiled, and then analyzed by immunoblot. Membranes were probed for expression of PU.1. (D) PU.1 protein expression in WT and Pin1-null MEFs after being treated with 150 µg/ml cycloheximide (CHX) to inhibit protein synthesis for 2, 4, 6, 8, or 10 hours. The immunoblot shown is representative of two independent experiments. PU.1 protein expression is plotted in the bottom graph as a percentage of total PU.1 protein at time zero, and reflects the values obtained from immunoblots shown directly above.</p

Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1

<p>The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that <i>Atg3-</i>, <i>Atg5</i>-, or <i>Pik3c3/Vps34</i>-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of <i>Atg7</i>-deficient T cells is defective. By using an adoptive transfer and <i>Listeria monocytogenes</i> (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8⁺ T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.</p