The rockefeller university

Passively administered anti-tumor monoclonal antibodies (mAbs) rapidly kill tumor targets via Fc gamma R-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that Fc gamma R expression by CD11c(+) antigen-presenting cells is required to generate antitumor T cell responses upon ADCC-mediated tumor clearance. Using Fc gamma R-humanized mice, we demonstrate that anti-tumor human (h)IgG1 must engage hFc gamma RIIIA on macrophages to mediate ADCC, but also engage hFc gamma RIIA, the sole hFc gamma R expressed by human dendritic cells (DCs), to generate a potent vaccinal effect. Thus, while next-generation antitumor antibodies with enhanced binding to only hFc gamma RIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFc gamma RIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity.

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility(1-4). Here we show that super-enhancers(5) underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters ('epicentres') of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that beta-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 mu m away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

Properties of the Higgs boson with mass near 125 GeV are measured in proton-proton collisions with the CMS experiment at the LHC. Comprehensive sets of production and decay measurements are combined. The decay channels include gamma gamma, ZZ, WW, tau tau, bb, and mu mu pairs. The data samples were collected in 2011 and 2012 and correspond to integrated luminosities of up to 5.1 fb(-1) at 7 TeV and up to 19.7 fb(-1) at 8 TeV. From the high-resolution gamma gamma and ZZ channels, the mass of the Higgs boson is determined to be 125.02(-0.27)(+0.26) (stat)(-0.15)(+0.14) (syst) GeV. For this mass value, the event yields obtained in the different analyses tagging specific decay channels and production mechanisms are consistent with those expected for the standard model Higgs boson. The combined best-fit signal relative to the standard model expectation is 1.00 +/- 0.09 (stat)(-0.07)(+0.08) (theo) +/- 0.07 (syst) at the measured mass. The couplings of the Higgs boson are probed for deviations in magnitude from the standard model predictions in multiple ways, including searches for invisible and undetected decays. No significant deviations are found.

Herpes simplex virus 1 (HSV-1) is a common virus that can rarely invade the human central nervous system (CNS), causing devastating encephalitis. The permissiveness to HSV-1 of the various relevant cell types of the CNS, neurons, astrocytes, oligodendrocytes, and microglia cells, as well as their response to viral infection, has been extensively studied in humans and other animals. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance, as responses to any given neurotropic virus may differ between humans and other animals. Human CNS neuron cell lines as well as primary human CNS neurons, astrocytes, and microglia cells cultured/isolated from embryos or cadavers, have enabled the study of cell-autonomous anti-HSV-1 immunity in vitro. However, the paucity of biological samples and their lack of purity have hindered progress in the field, which furthermore suffers from the absence of testable primary human oligodendrocytes. Recently, the authors have established a human induced pluripotent stem cells (hiPSCs)-based model of anti-HSV-1 immunity in neurons, oligodendrocyte precursor cells, astrocytes, and neural stem cells, which has widened the scope of possible in vitro studies while permitting in-depth explorations. This mini-review summarizes the available data on human primary and iPSC-derived CNS cells for anti-HSV-1 immunity. The hiPSC-mediated study of anti-viral immunity in both healthy individuals and patients with viral encephalitis will be a powerful tool in dissecting the disease pathogenesis of CNS infections with HSV-1 and other neurotropic viruses.

Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage.

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (alpha-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205(+) APCs and to assess its in vivo effects on protection associated responses (IFN-gamma production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with alpha-DEC-ESAT alone induced ESAT-6-specific IFN-gamma producing CD4(+) T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-gamma(+) production by specific T cells in the lungs, compared to scarce IFN-gamma production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, alpha-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205(+) APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-gamma production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

N-myristoylation is an essential fatty acid modification that governs the localization and activity of cell signaling enzymes, architectural proteins, and immune regulatory factors. Despite its importance in health and disease, there are currently no methods for reversing protein myristoylation in vivo. Recently, the Shigella flexneri protease IpaJ was found to cleave myristoylated glycine of eukaryotic proteins, yet the discriminatory mechanisms of substrate selection required for targeted demyristoylation have not yet been evaluated. Here, we performed global myristoylome profiling of cells treated with IpaJ under distinct physiological conditions. The protease is highly promiscuous among diverse N-myristoylated proteins in vitro but is remarkably specific to Golgi-associated ARF/ARL family GTPases during Shigella infection. Reconstitution studies revealed a mechanistic framework for substrate discrimination based on IpaJ's function as a GTPase "effector'' of bacterial origin. We now propose a concerted model for IpaJ function that highlights its potential for programmable demyristoylation in vivo.