TCR Signal Strength Controls Dynamic NFAT Activation Threshold and Graded IRF4 Expression in CD8+ T Cells

TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy

Dual cathode system for electron beam instruments

An electron beam source having a single electron optical axis is provided with two coplanar cathodes equally spaced on opposite sides from the electron optical axis. A switch permits selecting either cathode, and a deflection system comprised of electromagnets, each with separate pole pieces equally spaced from the plane of the cathodes and electron optical axis, first deflects the electron beam from a selected cathode toward the electron optical axis, and then in an opposite direction into convergence with the electron optical axis. The result is that the electron beam from one selected cathode undergoes a sigmoid deflection in two opposite directions, like the letter S, with the sigmoid deflection of each being a mirror image of the other

The Peculiar Velocities of Local Type Ia Supernovae and their Impact on Cosmology

We quantify the effect of supernova Type Ia peculiar velocities on the derivation of cosmological parameters. The published distant and local Ia SNe used for the Supernova Legacy Survey first-year cosmology report form the sample for this study. While previous work has assumed that the local SNe are at rest in the CMB frame (the No Flow assumption), we test this assumption by applying peculiar velocity corrections to the local SNe using three different flow models. The models are based on the IRAS PSCz galaxy redshift survey, have varying beta = Omega_m^0.6/b, and reproduce the Local Group motion in the CMB frame. These datasets are then fit for w, Omega_m, and Omega_Lambda using flatness or LambdaCDM and a BAO prior. The chi^2 statistic is used to examine the effect of the velocity corrections on the quality of the fits. The most favored model is the beta=0.5 model, which produces a fit significantly better than the No Flow assumption, consistent with previous peculiar velocity studies. By comparing the No Flow assumption with the favored models we derive the largest potential systematic error in w caused by ignoring peculiar velocities to be Delta w = +0.04. For Omega_Lambda, the potential error is Delta Omega_Lambda = -0.04 and for Omega_m, the potential error is Delta Omega_m < +0.01. The favored flow model (beta=0.5) produces the following cosmological parameters: w = -1.08 (+0.09,-0.08), Omega_m = 0.27 (+0.02,-0.02) assuming a flat cosmology, and Omega_Lambda = 0.80 (+0.08,-0.07) and Omega_m = 0.27 (+0.02,-0.02) for a w = -1 (LambdaCDM) cosmology.Comment: 4 pages, 2 figures, 1 table, accepted for publication in ApJ Letter

Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8(+) T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei

Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8(+) T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes

GENOMICS OF ENDOGLIN PATHWAY IN PREECLAMPSIA

THE GENOMICS OF ENDOGLIN PATHWAY IN PREECLAMPSIA Mandy J. Bell, PhD, RN University of Pittsburgh, 2012 Preeclampsia is a pregnancy disorder that greatly impacts maternal and fetal/neonatal health and wellbeing. This case-control candidate gene association study investigated endoglin pathway genetic variation and its association with preeclampsia. Tagging single nucleotide polymorphisms (tSNPs) in ENG, TGFβ1, TGFβR1, ALK1, and TGFβR2 were genotyped with iPLEX® and TaqMan® in maternal/fetal dyads. The Prenatal Exposures and Preeclampsia Prevention study provided maternal DNA extracted from peripherally collected white blood cell pellets, along with umbilical cord serum we used for fetal DNA extraction. Data on 355 white (181 cases/174 controls) and 60 black (30 cases/30 controls) women matched on ancestry, age, and parity were analyzed. Separate subgroup allele/genotype/haplotype tests were conducted with Chi-square or Fisher’s exact tests. Binary logistic regression provided odds ratios for tSNPs with significant genotype tests. Analysis of maternal/fetal dyads was not conducted, because unlike the maternal samples, the fetal samples did not provide a quality template suitable for iPLEX® data collection. In white women, variation in ENG (rs11792480, rs10121110) and TGFβR2 (rs6550005) was associated with preeclampsia. Allelic frequency distributions in rs11792480, rs10121110, and rs6550005 were significantly different among cases and controls while genotype distributions of rs10121110 and rs6550005 were further associated with preeclampsia (p-values < .05). For rs10121110, women with the AA genotype were 2.290 times more likely to develop preeclampsia compared to the GG genotype (99% CI [1.022, 5.133], p = .008). ENG haplotype TACGA, which contains rs11792480 and rs10121110 risk alleles, was also over-represented in cases (p = .022). In black women, variation in TGFβ1 (rs4803455, rs4803457), TGFβR1 (rs10739778), and TGFβR2 (rs6550005, rs1346907, rs877572) was associated with preeclampsia. Allelic frequency distributions in rs10739778, rs6550005, rs1346907, and rs877572 were significantly different among cases and controls while genotype distributions of rs10739778, rs4803455, and rs4803457 were further associated with preeclampsia (p-values < .05). For rs4803457, women with the CT genotype were 7.437 more times likely to develop preeclampsia compared to the CC genotype (99% CI [1.192, 46.408], p = .005). These results demonstrate that variation in ENG pathway genes is associated with preeclampsia, with different genes from the same pathway contributing to preeclampsia in white compared to black women

The Tec kinase ITK differentially optimizes NFAT, NF-κB, and MAPK signaling during early T cell activation to regulate graded gene induction [preprint]

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal kinase ITK simultaneously trigger many biochemically separate TCR signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, we examined Erk1/2 activation and NFAT, NF-κB translocation in naive OT-I CD8+ cell nuclei. We observed consistent digital activation of NFAT1 and Erk-MAPK, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength and was tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis also revealed genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells

Type Ia supernova parameter estimation: a comparison of two approaches using current datasets

By using the Sloan Digital Sky Survey (SDSS) first year type Ia supernova (SN Ia) compilation, we compare two different approaches (traditional \chi^2 and complete likelihood) to determine parameter constraints when the magnitude dispersion is to be estimated as well. We consider cosmological constant + Cold Dark Matter (\Lambda CDM) and spatially flat, constant w Dark Energy + Cold Dark Matter (FwCDM) cosmological models and show that, for current data, there is a small difference in the best fit values and $\sim$ 30% difference in confidence contour areas in case the MLCS2k2 light-curve fitter is adopted. For the SALT2 light-curve fitter the differences are less significant ($\lesssim$ 13% difference in areas). In both cases the likelihood approach gives more restrictive constraints. We argue for the importance of using the complete likelihood instead of the \chi^2 approach when dealing with parameters in the expression for the variance.Comment: 16 pages, 5 figures. More complete analysis by including peculiar velocities and correlations among SALT2 parameters. Use of 2D contours instead of 1D intervals for comparison. There can be now a significant difference between the approaches, around 30% in contour area for MLCS2k2 and up to 13% for SALT2. Generic streamlining of text and suppression of section on model selectio

Criteria for the use of omics-based predictors in clinical trials.

The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to 'omics'-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI-sponsored clinical trials in which omics tests will be used to guide therapy

The next controversy in genetic testing: clinical data as trade secrets?

Sole-source business models for genetic testing can create private databases containing information vital to interpreting the clinical significance of human genetic variations. But incomplete access to those databases threatens to impede the clinical interpretation of genomic medicine. National health systems and insurers, regulators, researchers, providers and patients all have a strong interest in ensuring broad access to information about the clinical significance of variants discovered through genetic testing. They can create incentives for sharing data and interpretive algorithms in several ways, including: promoting voluntary sharing; requiring laboratories to share as a condition of payment for or regulatory approval of laboratory services; establishing – and compelling participation in – resources that capture the information needed to interpret the data independent of company policies; and paying for sharing and interpretation in addition to paying for the test itself. US policies have failed to address the data-sharing issue. The entry of new and established firms into the European genetic testing market presents an opportunity to correct this failure