The importance of autophagy in memory CD8 T cell differentiation in vivo is not well defined. We show here that autophagy is dynamically regulated in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection. Autophagy decreased in activated proliferating T cells, and was then upregulated at the peak of the effector T cell response. Consistent with this model, deletion of the key autophagy genes Atg7 or Atg5 in virus-specific CD8 T cells had minimal effect on generating effector cells but greatly enhanced their death during the contraction phase resulting in compromised memory formation. These findings provide insight into when autophagy is needed during effector and memory T cell differentiation in vivo and also warrant a re-examination of our current concepts about the relationship between T cell activation and autophagy.

SUMMARY Host resistance to viral infection requires Type-I (α/β) and -II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8 processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-␤ (TGF-␤) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-␤ type III receptor (T␤RIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-␤ and inhibin receptor. Here we demonstrate that T␤RIII functions as a BMP cell surface receptor. T␤RIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-␤. T␤RIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous T␤RIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for T␤RIII function, we demonstrate that BMP-2 specifically stimulates T␤RIII-mediated epithelial to mesenchymal cell transformation. The ability of T␤RIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-␤ signaling suggests a broader role for T␤RIII in orchestrating TGF-␤ superfamily signaling.

Autophagy regulates cell differentiation, proliferation and survival in multiple cell types, including cells of the immune system. Herein we examined the effects of a disruption of autophagy on the differentiation of invariant natural killer T cells (iNKT cells). Using mice with a T lymphocyte specific deletion of Atg5 or Atg7,two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell derived IFNγ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel set of reversible, multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discreet pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a novel candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.