CD8+ T Cell and NK Responses to a Novel Dengue Epitope: A Possible Role for KIR3DL1 in Dengue Pathogenesis: A Dissertation

Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS126-34. We predicted higher frequencies of NS126-34-specific CD8+ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8+T cells. We generated a tetramer against this epitope (B57-NS126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34-specific, other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8+ T cells. In vitro stimulation of CD8+ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation. During the course of our analysis we found substantial binding of B57-NS126-34 TET to CD8- cells. We demonstrated that the B57-NS126-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS126-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1+ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness. Our data identified CD71 as biologically useful marker to study DENV-specific CD8+ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection

GW Librae: Still Hot Eight Years Post-Outburst

We report continued Hubble Space Telescope (HST) ultraviolet spectra and ground-based optical photometry and spectroscopy of GW Librae eight years after its largest known dwarf nova outburst in 2007. This represents the longest cooling timescale measured for any dwarf nova. The spectra reveal that the white dwarf still remains about 3000 K hotter than its quiescent value. Both ultraviolet and optical light curves show a short period of 364-373 s, similar to one of the non-radial pulsation periods present for years prior to the outburst, and with a similar large UV/optical amplitude ratio. A large modulation at a period of 2 h (also similar to that observed prior to outburst) is present in the optical data preceding and during the HST observations, but the satellite observation intervals did not cover the peaks of the optical modulation so it is not possible to determine its corresponding UV amplitude. The similarity of the short and long periods to quiescent values implies the pulsating, fast spinning white dwarf in GW Lib may finally be nearing its quiescent configuration.Comment: 6 figures, accepted in A

A Chandra Study of the Rosette Star-Forming Complex. III. The NGC 2237 Cluster and the Region's Star Formation History

We present Chandra X-ray images of the NGC 2237 young star cluster on the periphery of the Rosette Nebula. We detect 168 X-ray sources, 80% of which have counterparts in USNO, 2MASS, and deep FLAMINGOS images. These constitute the first census of the cluster members with 0.2<~M<~2 Msun. Star locations in near-infrared color-magnitude diagrams indicate a cluster age around 2 Myr with a visual extinction of 1<Av<3 at 1.4 kpc, the distance of the Rosette Nebula's main cluster NGC 2244. We derive the K-band luminosity function and the X-ray luminosity function of the cluster, which indicate a population ~400-600 stars. The X-ray-selected sample shows a K-excess disk frequency of 13%. The young Class II counterparts are aligned in an arc ~3 pc long suggestive of a triggered formation process induced by the O stars in NGC 2244. The diskless Class III sources are more dispersed. Several X-ray emitting stars are located inside the molecular cloud and around gaseous pillars projecting from the cloud. These stars, together with a previously unreported optical outflow originating inside the cloud, indicate that star formation is continuing at a low level and the cluster is still growing. This X-ray view of young stars on the western side of the Rosette Nebula complements our earlier studies of the central cluster NGC 2244 and the embedded clusters on the eastern side of the Nebula. The large scale distribution of the clusters and molecular material is consistent with a scenario in which the rich central NGC 2244 cluster formed first, and its expanding HII region triggered the formation of the now-unobscured clusters RMC XA and NGC 2237. A large swept-up shell material around the HII region is now in a second phase of collect-and-collapse fragmentation, leading to the recent formation of subclusters. Other clusters deeper in the molecular cloud appear unaffected by the Nebula expansion.Comment: Accepted to ApJ. 49 pages, 16 figures, and 4 tables

A Chandra Study of the Rosette Star-Forming Complex. II. Clusters in the Rosette Molecular Cloud

We explore here the young stellar populations in the Rosette Molecular Cloud (RMC) region with high spatial resolution X-ray images from the Chandra X-ray Observatory, which are effective in locating weak-lined T Tauri stars as well as disk-bearing young stars. A total of 395 X-ray point sources are detected, 299 of which (76%) have an optical or near-infrared (NIR) counterpart identified from deep FLAMINGOS images. From X-ray and mass sensitivity limits, we infer a total population of about 1700 young stars in the survey region. Based on smoothed stellar surface density maps, we investigate the spatial distribution of the X-ray sources and define three distinctive structures and substructures within them. Structures B and C are associated with previously known embedded IR clusters, while structure A is a new X-ray-identified unobscured cluster. A high mass protostar RMCX #89 = IRAS 06306+0437 and its associated sparse cluster is studied. The different subregions are not coeval but do not show a simple spatial-age pattern. Disk fractions vary between subregions and are generally 20% of the total stellar population inferred from the X-ray survey. The data are consistent with speculations that triggered star formation around the HII region is present in the RMC, but do not support a simple sequential triggering process through the cloud interior. While a significant fraction of young stars are located in a distributed population throughout the RMC region, it is not clear they originated in clustered environments.Comment: Accepted to the Astrophysical Journal. 49 pages, 12 figures. For a version with high-quality figures, see http://www.astro.psu.edu/users/edf/RMC_accepted.pd

Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes

A Chandra Study of the Rosette Star-Forming Complex. I. The Stellar Population and Structure of the Young Open Cluster NGC 2244

We present the first high spatial resolution Chandra X-ray study of NGC 2244, the 2 Myr old stellar cluster immersed in the Rosette Nebula. Over 900 X-ray sources are detected; 77% have optical or FLAMINGOS near-infrared (NIR) stellar counterparts and are mostly previously uncatalogued young stellar cluster members. All known OB stars with spectral type earlier than B1 are detected and the X-ray selected stellar population is estimated to be nearly complete between 0.5 and 3 Msun. The X-ray luminosity function (XLF) ranges from 29.4<logLx<32 ergs/s in the hard (2-8keV) band. By comparing the NGC 2244 and Orion Nebula Cluster XLFs, we estimate a total population of 2000 stars in NGC 2244. A number of further results emerge from our analysis: The XLF and the associated K-band luminosity function indicate a normal Salpeter initial mass function (IMF) for NGC 2244. This is inconsistent with the top-heavy IMF reported from earlier optical studies that lacked a good census of <4Msun stars. The spatial distribution of X-ray stars is strongly concentrated around the central O5 star, HD 46150. The other early O star, HD 46223, has few companions. The cluster's stellar radial density profile shows two distinctive structures. This double structure, combined with the absence of mass segregation, indicates that this cluster is not in dynamical equilibrium. The spatial distribution of X-ray selected K-excess disk stars and embedded stars is asymmetric with an apparent deficit towards the north. The fraction of X-ray-selected cluster members with K-band excesses caused by inner protoplanetary disks is 6%, slightly lower than the 10% disk fraction estimated from the FLAMINGOS study based on the NIR-selected sample. This is due to the high efficiency of X-ray surveys in locating disk-free T Tauri stars.[Abridged]Comment: Accepted for publication in ApJ (March 1, 2008 v675 issue). 61 pages, 20 figures, 7 tables. Updated a statement on NGC 2244-334 and added a referenc

Covert Genetic Selections to Optimize Phenotypes

In many high complexity systems (cells, organisms, institutions, societies, economies, etc.), it is unclear which components should be regulated to affect overall performance. To identify and prioritize molecular targets which impact cellular phenotypes, we have developed a selection procedure (“SPI”–single promoting/inhibiting target identification) which monitors the abundance of ectopic cDNAs. We have used this approach to identify growth regulators. For this purpose, complex pools of S. cerevisiae cDNA transformants were established and we quantitated the evolution of the spectrum of cDNAs which was initially present. These data emphasized the importance of translation initiation and ER-Golgi traffic for growth. SPI provides functional insight into the stability of cellular phenotypes under circumstances in which established genetic approaches cannot be implemented. It provides a functional “synthetic genetic signature” for each state of the cell (i.e. genotype and environment) by surveying complex genetic libraries, and does not require specialized arrays of cDNAs/shRNAs, deletion strains, direct assessment of clonal growth or even a conditional phenotype. Moreover, it establishes a hierarchy of importance of those targets which can contribute, either positively or negatively, to modify the prevailing phenotype. Extensions of these proof-of-principle experiments to other cell types should provide a novel and powerful approach to analyze multiple aspects of the basic biology of yeast and animal cells as well as clinically-relevant issues

Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE, and Enhances Virus Infectivity and Stability

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines

Elucidating the role of T cells in protection against and pathogenesis of dengue virus infections

Dengue viruses (DENV) cause significantly more human disease than any other arbovirus, with hundreds of thousands of cases leading to severe disease in thousands annually. Antibodies and T cells induced by primary infection with DENV have the potential for both positive (protective) and negative (pathological) effects during subsequent DENV infections. In this review, we summarize studies that have examined T-cell responses in humans following natural infection and vaccination. We discuss studies that support a role for T cells in protection against and those that support a role for the involvement of T cells in the pathogenesis of severe disease. The mechanisms that lead to severe disease are complex, and T-cell responses are an important component that needs to be further evaluated for the development of safe and efficacious DENV vaccines