Quantification of total T-cell receptor diversity by flow cytometry and spectratyping

BACKGROUND: T-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping. RESULTS: Using flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity. CONCLUSIONS: Using both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire

The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state

Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis

The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis

The Chandra High Energy Transmission Grating: Design, Fabrication, Ground Calibration and Five Years in Flight

Details of the design, fabrication, ground and flight calibration of the High Energy Transmission Grating, HETG, on the Chandra X-ray Observatory are presented after five years of flight experience. Specifics include the theory of phased transmission gratings as applied to the HETG, the Rowland design of the spectrometer, details of the grating fabrication techniques, and the results of ground testing and calibration of the HETG. For nearly six years the HETG has operated essentially as designed, although it has presented some subtle flight calibration effects.Comment: 34 pages (including 30 figures), accepted for publication in PAS

Benchmarking of Mutation Diagnostics in Clinical Lung Cancer Specimens

Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy ‘Sanger’ sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r2 = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation

Isolation of Flow and Nonflow Correlations by Two- and Four-Particle Cumulant Measurements of Azimuthal Harmonics in sNN=\sqrt{s_{_{\rm NN}}} = 200 GeV Au+Au Collisions

A data-driven method was applied to measurements of Au+Au collisions at $\sqrt{s_{_{\rm NN}}} =$ 200 GeV made with the STAR detector at RHIC to isolate pseudorapidity distance $\Delta\eta$-dependent and $\Delta\eta$-independent correlations by using two- and four-particle azimuthal cumulant measurements. We identified a component of the correlation that is $\Delta\eta$-independent, which is likely dominated by anisotropic flow and flow fluctuations. It was also found to be independent of $\eta$ within the measured range of pseudorapidity $|\eta|<1$. The relative flow fluctuation was found to be $34\% \pm 2\% (stat.) \pm 3\% (sys.)$ for particles of transverse momentum $p_{T}$ less than $2$ GeV/$c$. The $\Delta\eta$-dependent part may be attributed to nonflow correlations, and is found to be $5\% \pm 2\% (sys.)$ relative to the flow of the measured second harmonic cumulant at $|\Delta\eta| > 0.7$

Charged-to-neutral correlation at forward rapidity in Au+Au collisions at sNN\sqrt{s_{NN}}=200 GeV

Event-by-event fluctuations of the ratio of inclusive charged to photon multiplicities at forward rapidity in Au+Au collision at $\sqrt{s_{NN}}$=200 GeV have been studied. Dominant contribution to such fluctuations is expected to come from correlated production of charged and neutral pions. We search for evidences of dynamical fluctuations of different physical origins. Observables constructed out of moments of multiplicities are used as measures of fluctuations. Mixed events and model calculations are used as baselines. Results are compared to the dynamical net-charge fluctuations measured in the same acceptance. A non-zero statistically significant signal of dynamical fluctuations is observed in excess to the model prediction when charged particles and photons are measured in the same acceptance. We find that, unlike dynamical net-charge fluctuation, charge-neutral fluctuation is not dominated by correlation due to particle decay. Results are compared to the expectations based on the generic production mechanism of pions due to isospin symmetry, for which no significant (<1%) deviation is observed.Comment: 14 pages, 6 figure

Measurements of Dihadron Correlations Relative to the Event Plane in Au+Au Collisions at sNN=200\sqrt{s_{NN}}=200 GeV

Dihadron azimuthal correlations containing a high transverse momentum (\pt) trigger particle are sensitive to the properties of the nuclear medium created at RHIC through the strong interactions occurring between the traversing parton and the medium, i.e. jet-quenching. Previous measurements revealed a strong modification to dihadron azimuthal correlations in Au+Au collisions with respect to \pp\ and \dAu\ collisions. The modification increases with the collision centrality, suggesting a path-length dependence to the jet-quenching effect. This paper reports STAR measurements of dihadron azimuthal correlations in mid-central (20-60\%) Au+Au collisions at \snn=200~GeV as a function of the trigger particle's azimuthal angle relative to the event plane, \phis=|\phit-\psiEP|. The azimuthal correlation is studied as a function of both the trigger and associated particle \pt. The subtractions of the combinatorial background and anisotropic flow, assuming Zero Yield At Minimum (\zyam), are described. The away-side correlation is strongly modified, and the modification varies with \phis, which is expected to be related to the path-length that the away-side parton traverses. The pseudo-rapidity (\deta) dependence of the near-side correlation, sensitive to long range \deta correlations (the ridge), is also investigated. The ridge and jet-like components of the near-side correlation are studied as a function of \phis. The ridge appears to drop with increasing \phis while the jet-like component remains approximately constant. ...Comment: 50 pages, 39 figures, 6 table

Centrality and transverse momentum dependence of elliptic flow of multi-strange hadrons and ϕ\phi meson in Au+Au collisions at sNN\sqrt{s_{NN}} = 200 GeV

We present high precision measurements of elliptic flow near midrapidity ($|y|<1.0$) for multi-strange hadrons and $\phi$ meson as a function of centrality and transverse momentum in Au+Au collisions at center of mass energy $\sqrt{s_{NN}}=$ 200 GeV. We observe that the transverse momentum dependence of $\phi$ and $\Omega$ $v_{2}$ is similar to that of $\pi$ and $p$, respectively, which may indicate that the heavier strange quark flows as strongly as the lighter up and down quarks. This observation constitutes a clear piece of evidence for the development of partonic collectivity in heavy-ion collisions at the top RHIC energy. Number of constituent quark scaling is found to hold within statistical uncertainty for both 0-30$\%$ and 30-80$\%$ collision centrality. There is an indication of the breakdown of previously observed mass ordering between $\phi$ and proton $v_{2}$ at low transverse momentum in the 0-30$\%$ centrality range, possibly indicating late hadronic interactions affecting the proton $v_{2}$.Comment: 7 pages and 4 figures, Accepted for publication in Physical Review Letter

Observation of charge asymmetry dependence of pion elliptic flow and the possible chiral magnetic wave in heavy-ion collisions

We present measurements of $\pi^-$ and $\pi^+$ elliptic flow, $v_2$, at midrapidity in Au+Au collisions at $\sqrt{s_{_{\rm NN}}} =$ 200, 62.4, 39, 27, 19.6, 11.5 and 7.7 GeV, as a function of event-by-event charge asymmetry, $A_{ch}$, based on data from the STAR experiment at RHIC. We find that $\pi^-$ ($\pi^+$) elliptic flow linearly increases (decreases) with charge asymmetry for most centrality bins at $\sqrt{s_{_{\rm NN}}} = \text{27 GeV}$ and higher. At $\sqrt{s_{_{\rm NN}}} = \text{200 GeV}$, the slope of the difference of $v_2$ between $\pi^-$ and $\pi^+$ as a function of $A_{ch}$ exhibits a centrality dependence, which is qualitatively similar to calculations that incorporate a chiral magnetic wave effect. Similar centrality dependence is also observed at lower energies.Comment: 6 pages, 4 figure