Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity

Inhibition of Akt kinase activity suppresses entry and replication of influenza virus

The possibility of the pandemic spread of influenza viruses highlights the need for an effective cure for this life-threatening disease. Influenza A virus, belonging to a family of orthomyxoviruses, is a negative-strand RNA virus which encodes 11 viral proteins. A numbers of intracellular signaling pathways in the host cells interact with influenza the viral proteins, which affect various stages of viral infection and replication. In this study, we investigated how inhibition of Akt kinase activity impacts on influenza virus infection by using "Akt-in", a peptide Akt inhibitor. In PR8 influenza-infected A549 cells, Akt interacted with the NS1 (Non structural protein 1), and hence increased phosphorylation of Akt kinase activity and NS1. Treatment of cells with either "TCL1- or TCL1b-based Akt-in" efficiently suppressed Akt kinase activity while decreasing the levels of phosphorylated NSI; this, in turn, inhibited viral replication in a dose- and time-dependent manner. The inhibitory effect on viral replication appears to not be due to inhibition of the production of inflammatory cytokines, including IL-6 and IL-8, in the host cells. Inhibition of Akt kinase activity in the host cells inhibited the efficiency of viral entry, which is associated with decreased levels of phosphorylated glycogen synthase kinase 3, a substrate of Ala. Thus inhibition of Akt kinase activity in host cells may have therapeutic advantages for influenza virus infection by inhibiting viral entry and replication. (C) 2014 The Authors. Published by Elsevier Inc

Lifeboat survival and rescue

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Presence of both Akt and Phafin2 are required for induction of autophagy.

<p><b>A–D</b>. Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria (<b>A–B</b>, top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria (<b>A–B</b>, middle panels), but also induction of autophagy (<b>A–B</b>, bottom panels), which is reversible by re-introduction of human Phafin2 (<b>C–D</b>, bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's <i>t</i>-test is shown on the right side. White scale bar represents 10 µm. <b>E–H</b>. Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs (<b>E–F</b>), which is associated with inhibition of autophagy (<b>I–J</b>, lower panels). The observations are reversible by re-introduction of human Phafin2 (<b>G–H</b> and <b>K–L</b>, lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's <i>t</i>-test is shown on the right side. <b>I–L</b>. Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare <b>I</b> and <b>J</b>). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare <b>K</b> and <b>L</b>). Quantification of LC3 puncta per cell with statistical analysis by Student's <i>t</i>-test is shown on the right side. <b>M</b>. LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. <b>N–Q</b>. Akt-siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079795#pone.0079795.s005" target="_blank">fig. S5</a>) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare <b>O</b> and <b>Q</b>). Number of GFP-LC3 puncta per cells with statistical analysis by Student's <i>t</i>-test were shown as a bar graph. <b>R–U</b>. Akt-siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079795#pone.0079795.s005" target="_blank">fig. S5</a>) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells (<b>U</b>). Number of GFP-LC3 puncta per cells with statistical analysis by Student's <i>t</i>-test were shown as a bar graph. <b>V</b>. LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.</p

The interaction of the PtdIns(3)P with Phafin2 determines the lysosomal localization.

<p><b>A</b>. PIP strips were incubated with GST-Phafin2 WT, PH, or FYVE and the binding ability was examined by GST immunoblot. A comparable amount of recombinant wild type, N-term (PH domain), or C-term (FYVE domain) of Phafin2 was used (left panel). <b>B</b>. PtdIns(3)P interacting defective-Phafin2 (R53C, R171A, and R172A) failed to bind to PtdIns(3)P on PIP strip (right panel). <b>C</b>. BiFC analysis, 3-MA or wortmannin treatment abrogated the accumulation of the Akt-Phafin2 protein complexes at perinuclear lysosome after rapamycin treatment. <b>D</b>. Mutant Phafin2 displayed no accumulation of perinuclear lysosome after rapamycin treatment (right panels). <b>E</b>–<b>F</b>. Mutant Phafin2 failed to induce autophagy determined by decreased intensity of LC3-II band (<b>E</b>, lane 6). The percentage of autophagy inhibition was 29.3±7.50% out of three independent experiments. The observation was consistent, as determined by the absence of GFP-LC3 puncta using confocal microscopy (<b>F</b>, lower right side panels). Note that ectopic expression of wild type Phafin2 modestly enhanced autophagy induction determined by LC3-II blot (E, lane4 upper panel) and LC3 puncta (F. lower panels). White scale bar represents 10 µm.</p

Lysosomal interaction of Akt with Phafin2 is a critical step to induce autophagy.

<p>PI3K-Akt pathway that mediates anti-apoptotic signal is suggested to play an important role in the regulation of autophagy in mammalian cells. However, molecular mechanisms by which Akt signal regulates autophagy are largely unknown. In this study, we demonstrated that the presence of both Akt and Phafin2 on the lysosome is critical in the induction of autophagy via interaction of lysosomal PtdIns(3)P in mammalian cells. Akt-Phafin2 functional interaction not only clarifies the molecular basis of the PI3K-Akt signaling pathway in the regulation of autophagy, but also shows how 3-MA (3-methyladenine), a widely used autophagy inhibitor, inhibits autophagy in mammalian cells at molecular levels. It has been suggested that Akt activation prevents induction of autophagy, however, the roles of Akt in the regulation of autophagy induction is not clear. Hence, the current studies add a new twist to the molecular regulation of autophagy via PI3K-Akt signaling pathways in mammalian cells.</p