Stochastic expansions maintain the clonal stability of CD8+ T cell populations undergoing memory inflation driven by murine cytomegalovirus

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV

Early Priming Minimizes the Age-Related Immune Compromise of CD8+ T Cell Diversity and Function

The elderly are particularly susceptible to influenza A virus infections, with increased occurrence, disease severity and reduced vaccine efficacy attributed to declining immunity. Experimentally, the age-dependent decline in influenza-specific CD8+ T cell responsiveness reflects both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. In this study, we asked whether early priming limits the time-related attrition of immune competence. Though primary responses in aged mice were compromised, animals vaccinated at 6 weeks then challenged >20 months later had T-cell responses that were normal in magnitude. Both functional quality and the persistence of ‘preferred’ TCR clonotypes that expand in a characteristic immunodominance hierarchy were maintained following early priming. Similar to the early priming, vaccination at 22 months followed by challenge retained a response magnitude equivalent to young mice. However, late priming resulted in reduced TCRβ diversity in comparison with vaccination earlier in life. Thus, early priming was critical to maintaining individual and population-wide TCRβ diversity. In summary, early exposure leads to the long-term maintenance of memory T cells and thus preserves optimal, influenza-specific CD8+ T-cell responsiveness and protects against the age-related attrition of naïve T-cell precursors. Our study supports development of vaccines that prime CD8+ T-cells early in life to elicit the broadest possible spectrum of CD8+ T-cell memory and preserve the magnitude, functionality and TCR usage of responding populations. In addition, our study provides the most comprehensive analysis of the aged (primary, secondary primed-early and secondary primed-late) TCR repertoires published to date

Preservation of microvascular barrier function requires CD31 receptor-induced metabolic reprogramming

Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. The molecular mechanisms of EB recovery are poorly understood. Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. Using this model, we show that EB recovery requires a CD31 receptor-induced, robust glycolytic response sustaining junction re-annealing. Mechanistically, this response involves src-homology phosphatase activation leading to Akt-mediated nuclear exclusion of FoxO1 and concomitant \u3b2-catenin translocation to the nucleus, collectively leading to cMyc transcription. CD31 signals also sustain mitochondrial respiration, however this pathway does not contribute to junction remodeling. We further show that pathologic microvascular leakage in CD31-deficient mice can be corrected by enhancing the glycolytic flux via pharmacological Akt or AMPK activation, thus providing a molecular platform for the therapeutic control of EB response

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Active learning for software engineering

© 2019 Copyright held by the owner/author(s). Publication rights licensed to ACM. Software applications have grown increasingly complex to deliver the features desired by users. Software modularity has been used as a way to mitigate the costs of developing such complex software. Active learning-based program inference provides an elegant framework that exploits this modularity to tackle development correctness, performance and cost in large applications. Inferred programs can be used for many purposes, including generation of secure code, code re-use through automatic encapsulation, adaptation to new platforms or languages, and optimization. We show through detailed examples how our approach can infer three modules in a representative application. Finally, we outline the broader paradigm and open research questions

Symbolic Software Model Validation

Abstract—Modeling is the crucial first step in formal verification. Some models are constructed by humans from source code, while others are extracted automatically by tools. Regardless of how a model is constructed, verification is only as good as the model; therefore, it is essential to validate the model against the implementation it represents. In this paper we present two complementary approaches to software model validation. The first, data-centric model validation, checks that, for data structures relevant to the property being verified, all operations that update these data structures are captured in the model. The second, operation-centric model validation, checks that each operation being modeled is correctly simulated by the model. Both techniques are based on a combination of symbolic execution and satisfiability modulo theories (SMT) solving. We demonstrate the application of our methods on several case studies, including the address translation logic in the Bochs x86 emulator, the Berkeley Packet Filter, a TCAS benchmark suite, the FTP server from GNU Inetutils, and a component of the XMHF hypervisor. I

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals

Comparison of TCRβ diversity, and inter-individual sharing and similarity for the D^bNP₃₆₆⁺Vβ8.3⁺CD8⁺ and D^bPA₂₂₄⁺Vβ7⁺CD8⁺ repertoires.

<p>Shown are the relative measures of TCR<b>β</b> repertoire diversity, (A–D) the number of different clonotypes and (E–H) Simpson's diversity index, and (I–L) % of repertoire comprised of shared clonotypes, and (M–P) inter-individual TCR repertoire similarity, as measured by the Morisita-Horn similarity index. The Simpson's diversity and Morisita-Horn similarity indices account for the clonal dominance hierarchy among the different clonotypes and vary between 0 (minimum diversity/similarity) and 1 (maximum diversity/similarity). Each of the diversity, inter-individual sharing and similarity measures were estimated for a standard sample size of 22 TCR sequences per individual mouse repertoire. The repertoire diversities were calculated for each mouse per age/priming group for primary (A, E) and secondary (B, F) D^bNP₃₆₆⁺Vβ8.3⁺CD8⁺ TCR repertoires and for primary (C, G) and secondary (D, H) D^bPA₂₂₄⁺Vβ7⁺CD8⁺ TCR repertoires. The repertoire similarities were assessed between pairs of primary (M) and secondary (N) D^bNP₃₆₆⁺Vβ8.3⁺CD8⁺ TCR repertoires and between pairs of primary (O) and secondary (P) D^bPA₂₂₄⁺Vβ7⁺CD8⁺ TCR repertoires within the same age/priming group. To evaluate TCR sharing, clonotypes were first defined as shared or non-shared across all D^bNP₃₆₆-specific or D^bPA₂₂₄-specific TCRβ repertoires. The proportions of the 22 TCRβ sequences per D^bNP₃₆₆⁺Vβ8.3⁺CD8⁺ TCR repertoire (I, J) or D^bPA₂₂₄⁺Vβ7⁺CD8⁺ TCR repertoire (K, L) that were comprised of shared clonotypes were then estimated. A Mann-Whitney test was used to compare between young and aged mice for the primary responses and between young mice, aged mice primed young and aged mice primed old for the secondary responses. For the comparison between age/priming groups for the secondary responses, the statistical significance for each pairwise comparison was determined at p<0.0167 (*), using Bonferroni correction for multiple pairwise comparisons.</p